US011179337B2

( 12 ) Eber
United
et al .
States Patent ( 10 ) Patent No.: US 11,179,337 B2
(45 ) Date of Patent : Nov. 23 , 2021
(54) DRY POWDER COMPOSITION ( 58 ) Field of Classification Search
COMPRISING LONG-CHAIN RNA None
See application file for complete search history .
( 71 ) Applicant: CureVac AG , Tübingen (DE ) (56) References Cited
U.S. PATENT DOCUMENTS
( 72 ) Inventors: Fabian Johannes Eber , Stuttgart (DE ) ; 2002/0150626 A1 10/2002 Kohane et al.
Benyamin Yazdan Panah , Tübingen 2005/0032730 A1 2/2005 Von der Mulbe et al .
( DE ) ; Stefanie Sewing , Tübingen (DE ) ; 2005/0059624
2005/0250723
Al
Al
3/2005 Hoerr et al.
11/2005 Hoerr et al.
Thomas Ketterer , Gomaringen (DE ); 2006/0188490 A1 8/2006 Hoerr et al.
Thorsten Mutzke, Reutlingen (DE ) ; 2007/0172430 A1 7/2007 Brito et al .
Tilmann Roos , Kusterdingen ( DE ) ; 2008/0025944 Al 1/2008 Hoerr et al .
Michael Sonntag , Tübingen (DE ) ; 2008/0267873
2009/0324584
Al
Al
10/2008
12/2009
Hoerr et al.
Hoerr et al.
Michael Wiggenhorn , Munich (DE ) ; 2010/0048883 Al 2/2010 Ketterer et al .
Katharina Kolland , Augsburg ( DE ) 2010/0189729 A1 7/2010 Hoerr et al.
2010/0203076 A1 8/2010 Fotin - Mleczek et al .
2010/0291156 A1 11/2010 Barner et al .
( 73 ) Assignee : CureVac AG , Tübingen (DE ) 2010/0305196 Al 12/2010 Probst et al .
2011/0053829 A1 3/2011 Baumhof et al .
2011/02 50225 Al 10/2011 Fotin - Mleczek et al .
( * ) Notice: Subject to any disclaimer, the term of this 2012/0021043 A1 1/2012 Kramps et al .
patent is extended or adjusted under 35 2012/0258046 A1 10/2012 Mutzke
U.S.C. 154 ( b ) by 0 days. 2013/0129754 Al 5/2013 Thess et al.
2013/0142818 Al 6/2013 Baumhof et al .
2013/0259879 Al 10/2013 Baumhof et al .
( 21 ) Appl . No .: 16 /679,536 2013/0280283 Al 10/2013 Lorenz et al .
(22) Filed : Nov. 11 , 2019 (Continued )
FOREIGN PATENT DOCUMENTS
( 65 ) Prior Publication Data
US 2020/0060971 A1 WO WO 2011/069528 6/2011
Feb. 27 , 2020 WO WO 2011/069529 6/2011
(Continued )
Related U.S. Application Data OTHER PUBLICATIONS
( 62 ) Division of application No. 15 / 575,284 , filed as
application No. PCT/ EP2016 / 000842 on May 20 , “ Mini -Buchi Spray Dryer, ” technical specification sheet down
2016 , now Pat . No. 10,517,827 . loaded from Buchi.com on May 20 , 2019 .
Foreign Application Priority Data ( Continued )
( 30 )
May 20 , 2015 ( EP ) 15001517
Primary Examiner - Kimberly Chong
(74 ) Attorney , Agent, or Firm - Parker Highlander PLLC
( 51 ) Int . Cl . ( 57 ) ABSTRACT
A61K 48/00 ( 2006.01 )
A61K 9/16 ( 2006.01 ) The present invention is directed to a storage - stable formu
A61P 31/00 ( 2006.01 ) lation of long - chain RNA . In particular, the invention con
A61P 31/12 ( 2006.01 ) cerns a dry powder composition comprising a long -chain
A61P 37/06 ( 2006.01 ) RNA molecule . The present invention is furthermore
A61P 37/08 ( 2006.01 )
A61P 31/04 ( 2006.01 ) directed to methods for preparing a dry powder composition
A61P 33/02 (2006.01 ) comprising a long - chain RNA molecule by spray -drying.
A61P 35/00 ( 2006.01 ) The invention further concerns the use of such a dry powder
A61K 39/145 ( 2006.01 ) composition comprising a long -chain RNA molecule in the
A61K 39/00 ( 2006.01 ) preparation of pharmaceutical compositions and vaccines , to
( 52 ) U.S. CI . a method of treating or preventing a disorder or a disease, to
CPC A61K 9/16 ( 2013.01 ) ; A61K 9/1682 first and second medical uses of such a dry powder compo
(2013.01 ) ; A61K 39/145 ( 2013.01 ) ; A61K sition comprising a long - chain RNA molecule and to kits ,
48/005 (2013.01 ) ; A61K 48/0091 ( 2013.01 ) ; particularly to kits of parts, comprising such a dry powder
A61P 31/00 (2018.01 ) ; A61P 31/04 ( 2018.01 ) ; composition comprising a long - chain RNA molecule .
A61P 31/12 (2018.01 ) ; A61P 33/02 ( 2018.01 ) ;
A61P 35/00 (2018.01 ) ; A61P 37/06 ( 2018.01 ) ; 28 Claims , 12 Drawing Sheets
A61P 37/08 (2018.01 ) ; A61K 48/00 ( 2013.01 ) ;
A61K 2039/53 (2013.01 ) Specification includes a Sequence Listing .
 US 11,179,337 B2
Page 2

( 56 ) References Cited FOREIGN PATENT DOCUMENTS

U.S. PATENT DOCUMENTS WO WO 2016/165825 10/2016
WO WO 2016/165831 10/2016
2013/0295043 A1 11/2013 Kallen et al . WO WO 2016/174227 11/2016
2013/0336998 Al 12/2013 Kallen et al . WO WO 2016/184576 11/2016
2015/0037326 A1 2/2015 Butler-Ransohoff et al . WO WO 2016/193206 12/2016
2015/0050302 Al 2/2015 Thess WO WO 2016/193226 12/2016
2015/0057340 A1 2/2015 Thess et al . WO WO 2016/203025 12/2016
2015/0093413 A1 4/2015 Thess et al . WO WO 2017/001058 1/2017
2015/0118183 A1 4/2015 Baumhof WO WO 2017/009376 1/2017
2015/0118264 A1 4/2015 Baumhof et al . WO WO 2017/021546 2/2017
2015/0165006 A1 6/2015 Thess et al . WO WO 2017/025120 2/2017
2015/0184195 Al 7/2015 Thess WO WO 2017/025447 2/2017
2015/0218554 A1 8/2015 Thess WO WO 2017/036580 3/2017
2015/0306249 Al 10/2015 Baumhof et al . WO WO 2017/064146 4/2017
2015/0320847 Al 11/2015 Thess et al . WO WO 2017/081110 5/2017
2016/0130345 Al 5/2016 Fotin -Mleczek et al . WO WO 2017/108087 6/2017
2016/0166668 A1 6/2016 Kallen et al . WO WO 2017/109134 6/2017
2016/0166678 A1 6/2016 Kallen et al . WO WO 2017/109161 6/2017
WO WO 2020-023533 1/2020
2016/0166710 A1 6/2016 Baumhof
2016/0166711 A1 6/2016 Schnee et al .
2016/0168207 A1 6/2016 Kramps et al. OTHER PUBLICATIONS
2016/0168227 A1 6/2016 Kallen et al .
2016/0235864 A1 8/2016 Schlake International Preliminary Report Patentability issued in correspond
2016/0304883 A1 10/2016 Grund ing PCT Application No. PCT/EP2016 /000843 , dated Nov. 15 ,
2016/0304938 A1 10/2016 Wochner 2016 .
2016/0326575 Al 11/2016 Von Der Mulbe International Search Report and Written Opinion issued in corre
2016/0331844 Al 11/2016 Fotin -Mleczek et al . sponding PCT Application No. PCT/EP2016 /000843 , dated Nov.
2017/0014496 A1 1/2017 Fotin -Mleczek et al . 15 , 2016
2017/0029847 Al 2/2017 Thess Jensen et al., “ Spray drying of siRNA -containing PLGA nanoparticles
2017/0114378 A1 4/2017 Wocner et al . intended for inhalation ,” J. Control. Release, 142 ( 1 ) : 138-145 , 2010 .
2017/0252430 A1 9/2017 Fotin -Mleczek Office Action issued in U.S. Appl. No. 15 / 575,284 , dated Aug. 28 ,
2017/0304459 Al 10/2017 Jadhav et al. 2018 .
2017/0326225 A1 11/2017 Rauch Office Action issued in U.S. Appl. No. 15 / 575,284 , dated May 23 ,
2018/0044687 A1 2/2018 Thess et al . 2019 .
2018/0125952 Al 5/2018 Fotin -Mleczek Office Action issued in U.S. Appl. No. 15 / 575,284 , dated Nov. 16 ,
2018/0126003 A1 5/2018 Hoerr 2018 .
2018/0142275 Al 5/2018 Roos et al . Qiu et al . , “ Effective mRNA pulmonary delivery by dry powder
2018/0147146 A1 5/2018 Eber et al . formulation of PEGylated synthetic KL4 peptide , ” Journal of
>>

2018/0148727 A1 5/2018 Grund Controlled Release, 314 : 102-115 , 2019 .

U.S. Patent Nov. 23 , 2021 Sheet 1 of 12 US 11,179,337 B2

3
Orion

444

Fig. 1
U.S. Patent Nov. 23 , 2021 Sheet 2 of 12 US 11,179,337 B2

R2564 ( SEQ ID NO : 1 )
GGGGCGCUGCCUACGGAGGUGGCAGCCAUCUCO GCAUCAAGCUUACCAUGAAGG
CCAUCCI GUGGUCC CUGUACA GACAC GUGCAUCG
GCUACCI CCAACAACAGCACCGA IGGACA CUCGAGAAGAACGUCACGG
UGACCCACUCCGUGAACCUGCUGGAGGACAAGCACAACGGGAAGCUCUGCAAGCUGCGGG
GCGUCGCCCCGCUGCACCUCGGGAAGUGCAACAUCGCCGGCUGGAUCCUGGGGAACCCGG
AGUGCGAGAGCCUGUCCACCGCGAGCUCCUGGAGCUACAUCGUGGAGACCUCCAGCUCCG
ACAACGGCACGUGCUACCCCGGCGACUUCAUCGACUACGAGGAGCUCCGCGAGCAGCUGA
GCUCCGUGAGCUCCUUCGAGCGG ICGAGAUCUUCCCCAAGACCAGCUCCUGGCCCAACC
ACGACAGCAACAAGGGGGUCACCGCCGCCUGCCCGCAC GGCGCGAAGUCCUUCUACA
AGAACCUGAUCUGGCUCGUGAAGAAGGGGAACAGCUACCCCAAGCUGUCCAAGAGCUACA
UCAACGACAAGGGCAAGGAGGUGCUGGUCCUCUGGGGGAUCCACCACCCCAGCACCUCCG
CAGCAGAGCCUGUACCA ACGC CCGCU
ACUCCAAGAAGUUCAAGCCCGAGAUCÍ AUCO GAAGGUCCGCGACCA AGGGCC
GGAUGAACUACUACUGGACGCUGGUGGAGCCCGGGGACAAGAUCACCUUCGAGGCGACCG
GCAACCU GCUACGCC AUGGAGCGG CGO GGGA GCAUCA
UCAUCUCCGACA GUGCACGACUGCAACACGACCUGCCAGACCCCGAAGGGCGCCA
UCAACACCAGCCUGCCCUUCCAGAACAUCCACCCCAUCACGAUCGGGAAGUGCCCCAAGU
ACGUGAAGUCCACCAAGCUGCGCCUCGCGACCGGCCUGCGGAACGUCCCGAGCAUCCAGU
CCCGCGGGCUGUUCGGCGCCAUCGCCGGGUUCAUCGAGGGCGGCUGGACCGGGAUGGUGG
ACGGCUGGUACGGGUACCACCACCAGAACGAGCAGGGCAGCGGGUACGCCGCCGACCUCA
AGUCCACGCAGAACGCGAUCGACGAGAUCACCAACAAGGUGAACAGCGUCAUCGAGAAGA
UGAACA CCAGOUCA BAGGAGUUCAACCACCUGGAGAA GAUCGAGA
ACCUGAACAAGAAGGUCGACGACGGCUUCCUCGACAUCUGGACGUACAACGCCGAGCUGC
UGGUGCUCCUGGAGAACGA UGGACUAC ACUCCAACGUGAAGAACCUCU
ACGAGAAGGUCCGGAGCCAGCUGAAGAACAACGCCAAGGAGAUCGGGAACGG GCUOCG
AGUUCUACCACAAGUGCGACAACACCUGCAUGGAGUCCGUGAAGAACGGGACCUACGACU
ACCCCAAGUACAGCGAGGAGGCCAAGCUGAACCGCGAGGAGAUCGACGGCGUGAAGCUCG
AGUCCACGCGGAUCUACCAGAUCCUGGCGAUCUACAGCACCGUCGCCAGCUCCCUGGUGC
UCGUGGUCAGCCUGGGGGCCAUCUCCUUCUGGAUGUGCAGCAACGGCUCCCUGCAGUGCC
GCAUCUGCAUCUGACCACUAGUGCAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAU
AAGAGAAAGAAAAUGAAGAUCAAUAGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAA
AGCCAACACCCUGUCUAAAAAACAUAAAUUUCUUUAAUCAUUUUGCCUCUUUUCUCUGUG
CUUCAAUUAAUAAAAAAUGGAAAGAACCUAGAUCUAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUGCAUCCCCC CCC
Cco CCAAAGGCI UUUUCAGAGCCACCAGAAUU

Fig . 2
U.S. Patent Nov. 23 , 2021 Sheet 3 of 12 US 11,179,337 B2

con ansia
soival 3 RECOLL_PB RC0011 phs

Fig . 3
U.S. Patent Nov. 23 , 2021 Sheet 4 of 12 US 11,179,337 B2

.................
90

Fig . 4
U.S. Patent Nov. 23 , 2021 Sheet 5 of 12 US 11,179,337 B2
11
2Theta
()

40
Y. . .

Year WA

30
*

WWW, 25
5
.
Fig

20

***
DBAT-SD21LBR20C15013 -SDPR2LB0C1h5081A3
2RT-SP0C1h508.1A38 T-SPR20CN135L0B1AB3 ??
???
??
????
???
?
??
???
????

50 0 40 0 30 0 20 0 10 0
) counts( Intensity
U.S. Patent Nov. 23 , 2021 Sheet 6 of 12 US 11,179,337 B2

TOS

(um)dpiarmteiclre
6
.
Fig
moment

7 5 N

I] % [ amount particle normalized

U.S. Patent Nov. 23 , 2021 Sheet 7 of 12 US 11,179,337 B2

A.

23
SEM **

Fig . 7A
U.S. Patent Nov. 23 , 2021 Sheet 8 of 12 US 11,179,337 B2

B.

content

Fig . 7B
U.S. Patent Nov. 23 , 2021 Sheet 9 of 12 US 11,179,337 B2

C.

Fig . 70
U.S. Patent Nov. 23 , 2021 Sheet 10 of 12 US 11,179,337 B2

D.

Fig . 7D
U.S. Patent Nov. 23 , 2021 Sheet 11 of 12 US 11,179,337 B2

D(nn)iameter
8
.
Fig

OI

SO2 SD3
-
T
-OL
-
T
w
^ mo

35,0 25,0 20,0

30,0)% ( Intensity 15,0 10,0 5,0
U.S. Patent Nov. 23 , 2021 Sheet 12 of 12 US 11,179,337 B2

01
-
d
...
L
-
OS
€

D(nm)iameter
9
.
Fig

6 0 0
( 9vor ] ju sod sapped
20 0
 US 11,179,337 B2
1 2
DRY POWDER COMPOSITION system , is typically not stable and therefore may have little
COMPRISING LONG-CHAIN RNA chance of affecting most disease processes ( see e.g. Poxon
et al . , Pharmaceutical development and Technology , 5 ( 1 ) ,
This application is a divisional of U.S. application Ser. 115-122 (2000 ) ) , the problem of stability becomes even
No. 15 / 575,284 , filed Nov. 17 , 2017 , now U.S. Pat. No. 5 more prominent in the case of RNA . It is generally known
10,517,827 , which is a national phase application under 35 that the physico - chemical stability of RNA molecules in
U.S.C. § 371 of International Application No. PCT/EP2016 / solution is extremely low. RNA is susceptible to hydrolysis
000842 , filed May 20 , 2016 , which claims benefit of Euro- by ubiquitous ribonucleases or by divalent cations and is
pean Application No. 15001517.0 , filed May 20 , 2015 , and typically rapidly degraded , e.g. already after a few hours or
.

International Application No. PCT/EP2015 / 002019 , filed 10 days in solution . Rapid degradation occurs even in the
Oct. 13 , 2015 , the entire contents of each of which are absence of RNases, e.g. when RNA is stored in solution at
hereby incorporated by reference. room temperature for a few hours or days .
The sequence listing that is contained in the file named To avoid such rapid degradation, RNA in solution ) is
“ CRVCP0189USC1.txt ” , which is 4 KB (as measured in typically stored at -20 ° C. or even -80 ° C. and under RNAse
Microsoft Windows® ) and was created on Nov. 8 , 2019 , is 15 free conditions to prevent degradation of the RNA . Such
filed herewith by electronic submission and is incorporated storage conditions, however, do not sufficiently prevent a
by reference herein . loss of function over time . Additionally, applying such
The present invention is directed to a storage - stable conditions is very cost -intensive, especially for shipping and
formulation of long -chain RNA . In particular, the invention storage, e.g. whenever such low temperatures have to be
concerns a dry powder composition comprising a long - chain 20 guaranteed .
RNA molecule . The present invention is furthermore The only method for stabilization of long-chain RNA ,
directed to methods for preparing a dry powder composition which is known and applied, comprises lyophilization or
comprising a long-chain RNA molecule by spray -drying. freeze - drying of the RNA ( see e.g. WO2011 /069587 and
The invention further concerns the use of such a dry powder WO2011 /069586 ) . Lyophilization is a method , which is
composition comprising a long -chain RNA molecule in the 25 known and recognized in the art to enhance storage stability
preparation of pharmaceutical compositions and vaccines, to of temperature sensitive biomolecules , such as nucleic acids .
a method of treating or preventing a disorder or a disease , to During lyophilization , water is typically removed from a
first and second medical uses of such a dry powder compo- frozen sample containing nucleic acids via sublimation . The
sition comprising a long - chain RNA molecule and to kits , process of lyophilization is usually characterized by a pri
particularly to kits of parts, comprising such a dry powder 30 mary and a secondary drying step . In the primary drying
composition comprising a long - chain RNA molecule. step , free, i.e. unbound , water surrounding the nucleic acid
In gene therapy and many other therapeutically relevant ( sequence ) and optionally further components, evaporates
biochemical and biotechnological applications , the use of from the frozen solution . Subsequently, water that is bound
nucleic acid molecules for therapeutic and diagnostic pur- by the nucleic acids on a molecular basis may be removed
poses is of major importance. For example, rapid progress 35 in a secondary drying step by adding thermal energy .
has occurred in recent years in the field of gene therapy, Thereby, the hydration sphere surrounding the nucleic acids
where promising results have been achieved . Nucleic acids is lost . Lyophilization is the most common processing
are therefore regarded as important tools for gene therapy method for removing moisture from biopharmaceuticals,
and prophylactic and therapeutic vaccination against infec- and it can increase stability, temperature tolerance, and shelf
tious and malignant diseases . 40 life of these products. However, lyphilization does have its
Other than DNA , application of RNA also represents a limitations, especially if scale - up is needed . One major
favored tool in modern molecular medicine . It also exhibits disadvantage of lyophilization is that every single vial
some superior properties over DNA cell transfection . As containing a sample has to be lyophilized separately . The
generally known, transfection of DNA molecules may lead lyophilized product cannot be separated into distinct charges
to serious complications. For example, application of DNA 45 or aliquots, as the lyophilized product is not provided e.g. in
molecules bears the risk that the DNA integrates into the powder form . Instead , a cake -like product is obtained by
host genome. Integration of foreign DNA into the host lyophilization , which cannot be divided into distinct charges
genome can have an influence on the expression of host or aliquots. Therefore, if a powder - like product is desired, a
genes and can trigger the expression of an oncogene or the further step of granulation must be carried out . At present,
inactivation of a tumor suppressor gene. Furthermore , an 50 lyophilization of samples for a scaled- up production
essential gene — and, as a consequence , the product of such involves cost -intensive equipment, since, for example, a lot
an essential gene may also be inactivated by the integra- of lyophilizers are needed for market production , requiring
tion of the foreign DNA into the coding region of the gene. large production facilities . Together with the time required
The result of such an event may be particularly dangerous if for lyophilization and the additional requirement of a granu
the DNA is integrated into a gene, which is involved in 55 lation step that renders lyophilization a technique, which is
regulation of cell growth . Notwithstanding the risks associ- often not suitable for industrial scale production . Especially
ated with its application, DNA still represents an important in an environment where budgets are tightening, and where
tool . However, these risks do not occur if RNA, particularly time and facility space are at a premium , lyophilization may
mRNA , is used instead of DNA . An advantage of using not be considered, e.g. by the pharmaceutical industry, as a
RNA rather than DNA is that no virus -derived promoter 60 competitive process .
element has to be administered in vivo and no integration A minor number of case reports refer to spray -dried
into the genome may occur. Furthermore, the RNA , in order ribonucleotides. Double -stranded short interfering RNAs
to exert its function , does not need to overcome the barrier ( siRNA ) for inhalation were dried by using a spray -drying
to the nucleus. technology. Jensen et al . (2010) studied parameters to be
However, a main disadvantage of the use of RNA is its 65 applied in spray drying of siRNA - loaded poly (D ,L -lactide
instability . Even though it is understood that DNA , such as co - glycolide) (PLGA ) nanoparticles ( NPs ) for providing
naked DNA, when introduced into a patient circulatory nano - composite micro -particles for inhalation. As siRNA
 US 11,179,337 B2
3 4
was believed to denature at temperatures above 55 ° C. , that
? composition according to the invention is characterized by
study applied relatively low drying temperatures ( Tinter = 45 ° superior handling properties. For example, the inventive dry
C .; Toutlet = 30 ° C. ) and high excipient concentrations (10- powder composition can be packaged in any quantity or in
30 % ) to achieve a dry powder with a residual humidity of any container or dosage form , respectively. Handling of the
less than 4 % . 5 dry powder composition according to the invention is further
US2011 / 077284 discloses the provision of dry powders of improved by its free-flowing properties. In particular, the
therapeutic and inhalable short siRNAs against influenza inventive dry powder composition does not form agglom
virus. Therein , drying temperatures were determined experi erates or aggregates that would inhibit packaging and / or
mentally . Generally, the inlet temperature was from about dosage. Due to its flowability, the dry powder composition
65 ° C. to about 125 ° C. , while the outlet temperature ranged 10 according to the invention can be readily further processed .
from about 30 ° C. to about 70 ° C. In the examples presented
therein , siRNAs are dried at a Toutlet of 55 ° C. The powders For e.g.
instance, the dry powder composition can be transferred,
from one vessel to another or from a larger vessel into
generally had a moisture content of typically less than 10 %
by weight, or less than 5 % by weight, or less than 3 % by a plurality of smaller vessels , simply by pouring . The
weight. Also ,the study characterized the chemical stability 15 inventive dry powder composition can readily be packaged
in a variety of packages and final dosage forms according to
of the dry powder. Less than 10 % by weight of the active the actual requirements. Advantageously, the dry powder
siRNA were degraded upon storage of the dry powder
composition under ambient conditions for a period of 18 composition according to the invention provides excellent
months. However, the biological activity of the siRNA storage stability.
stored as a dry powder was not determined . 20 In particular, the invention provides a dry powder com
Summarizing the above, there is a long - lasting and urgent position comprising a long - chain RNA molecule , wherein
need in the art to provide means , which allow ( a skilled the long - chain RNA molecule preferably comprises at least
person ) to store long - chain RNA without loss of activity, an 30 nucleotides. Preferably, the long - chain RNA molecule is
effect, which is commonly observed , particularly in in vivo a molecule as defined herein . More preferably, the long
applications. In this context, a challenging problem resulting 25 chain RNA molecule is not an RNA molecule selected from
from prior art approaches is to ensure stability of nucleic the group consisting of a small interfering RNA ( siRNA ), a
acids , particularly stability upon storage and delivery of microRNA , a small nuclear RNA ( SARNA ), a small -hairpin
longer single -stranded RNA . Another problem of the prior ( sh) RNA , a riboswitch, a ribozyme or an aptamer. Even
art is the loss of biological activity of nucleic acids subse- more preferably, the long -chain RNA is not an siRNA, most
quent to storage . Finally, by using prior art methods, only 30 preferably not a double - stranded siRNA .
small amounts of nucleic acid are obtained . The provision of In a preferred embodiment, the dry powder composition
a suitable form for delivering these nucleic acids but also the as described herein does not comprise an RNA molecule
production, transport and storage thereof, especially trans- comprising less than 30 nucleotides, less than 200 nucleo
port of RNA, is an issue due to the conditions to ensure tides or less than 250 nucleotides. The dry powder compo
temperatures of -20 ° C. and less for shipment. Furthermore , 35 Sition does preferably not comprise an RNA molecule
lyophilization of long - chain RNA , especially for the use as selected from the group consisting of a small interfering
medicament, bears the problem that it is very cost- and RNA ( siRNA ), preferably a single - stranded or a double
time -intensive, particularly if commercial production in a stranded siRNA, a microRNA, a small nuclear RNA
scaled -up process is envisaged. ( snRNA ), a small -hairpin ( sh) RNA, a riboswitch, a
The underlying object is therefore to provide a nucleic 40 ribozyme or an aptamer. Even more preferably, the dry
acid molecule, in particular a long -chain RNA , exhibiting no powder composition does not comprise an siRNA, most
loss of activity when stored prior to its use and being preferably not a double - stranded siRNA .
available by cost-avoiding production process. In particular, With respect to the following description of the inventive
it is an object of the present invention to provide a long- dry powder composition it is noted that the definitions and
chain RNA molecule in a storage - stable formulation . One 45 specifications provided therein may also be applied to the
object of the invention is to provide a dry powder compo- inventive method, which is subsequently described as
sition comprising a long -chain RNA molecule . In addition , another aspect of the invention .
it is an objective of the invention to provide a method for In the context of the present invention , the term “ dry
preparing a dry powder comprising a long -chain RNA powder" ( or " dry powder composition " ) typically refers to a
molecule , wherein the RNA molecule retains its chemical 50 composition that is — amongst other features — characterized
integrity and its biological activity. It is another object of the by its residual moisture content, which is preferably low
present invention that such methods are applicable under enough in order to prevent the formation of aggregates that
industrial large -scale production conditions , preferably by a would reduce or inhibit the flowability of the powder. As
continuous process. It is a particular object to provide a used herein , the term “ residual moisture content ” (or
method that allows drying a liquid comprising long - chain 55 “ residual moisture ” ) typically refers to the total amount of
RNA molecules . solvent present in the dry powder composition. Said total
The object underlying the present invention is solved by amount of residual solvents in the dry powder composition
the claimed subject -matter. is determined using any suitable method known in the art.
In aa first aspect , the invention relates to a long -chain RNA For example, methods for determining the residual moisture
molecule in a particulate formulation. In particular, the 60 content comprise the Karl -Fischer -titrimetric technique or
invention concerns a dry powder composition comprising a the thermal gravimetric analysis ( TGA ) method . In a pre
long -chain RNA molecule . Prior to the invention described ferred embodiment, the residual solvent comprised in the
herein , long -chain RNA ( in contrast to shorter double- dry powder composition is water or an essentially aqueous
stranded RNAs) was never provided as a dry powder com- solution and the residual moisture content corresponds to the
position . Advantageously, the dry powder composition 65 residual water content of the dry powder composition, which
according to the invention provides a storage - stable form of is determined by the Karl -Fischer -titrimetric technique.
a long - chain RNA molecule . In addition, the dry powder Without being bound by any theory, the low residual mois
 US 11,179,337 B2
5 6
ture content of the inventive dry powder composition is according to the invention is preferably characterized by
expected to contribute to its excellent storage stability. parameters such as , for example, Dv10 , Dv50 , Dv90 or the
Preferably, the residual moisture content of the dry pow- mass median aerodynamic diameter (MMAD ).
der composition according to the invention is 15 % ( w / w ) or The parameter “ Dv50 ” ( or “ volume D50 ” or “ volume
less , more preferably 10 % ( w / w ) or less , even more prefer- 5 weighted D50 " ) relates to the median particle size based on
ably 9 % ( w / w ), 8 % ( w / w ), 7 % ( w / w ), 6 % ( w / w ) or 5 % a volume weighted particle size distribution . Dv50 thus
( w / w ). In a preferred embodiment, the residual moisture typically describes the particle size ( based on a volume
content of the dry powder composition is 5 % ( w / w ) or less , weighted distribution ), preferably the diameter of a particle
preferably 4 % ( w / w ) or less . In a particularly preferred in micrometers ( um ), with 50 % of the particles in the
embodiment, the residual moisture content is 7 % ( w / w ) or 10 distribution having a larger size and 50% of the particles in
less . In a further preferred embodiment, the residual mois- the distribution having a smaller size than Dv50 . In a volume
ture content of the dry powder composition in the range from weighted distribution , the parameter “ Dv50 " typically
0 % to 15 % ( w / w ), from 0 % to 10 % ( w / w ), from 0% to 7 % relates to the diameter (e.g. in micrometers) of aa hypotheti
( w / w ), from 0 % to 5 % ( w / w ), from 0 % to 4 % ( w / w ), from cal spherical particle, which has the volume of the corre
3 % to 6 % ( w / w ) or from 2 % to 5 % ( w / w ). 15 sponding actual particle in the distribution (which may or
In a further preferred embodiment, the residual moisture may not be spherical).
content of the dry powder composition as described herein Analogously, the parameter “ Dv10 ” corresponds to the
is 5 % ( w / w ) or less , more preferably 4 % ( w / w ) or less , even cut -off size ( preferably in um ) of the particles in a volume
more preferably 3 % ( w / w ) or less , 2 % ( w / w ) or less , or 1 % weighted distribution , which represent 10% of the total
( w / w ) or less . Alternatively, the residual moisture content of 20 volume of the sample, and which have a particle size equal
the dry powder composition as described herein is prefer- to or smaller than the Dv10 value . Preferably, the Dv10 of
ably in the range from from 0% to 5 % ( w / w ), from 0 % to 4 % the dry powder composition according to the invention is at
( w / w ), from 0 % to 3 % ( w / w ), from 0% to 2 % ( w / w ) or from least 0.2 , 0.3 , 0.4 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 um . More
0% to 1 % ( w / w ). preferably, the Dv10 of the dry powder composition is equal
In aa further preferred embodiment, the present invention 25 to or lower than 5 , equal to or lower than 10 , or equal to or
provides a dry powder composition having a residual water lower than 20 um . Most preferably, the Dv10 is in a range
content of 15 % ( w / w ) or less , more preferably 10 % ( w / w ) or from 1 um to 10 um or from about 3 um to about 5 um .
less , even more preferably 9 % ( w / w ), 8 % ( w / w ), 7 % ( w / w ), The parameter “ Dv90 ” corresponds to the cut -off size
6 % ( w / w ) or 5 % ( w / w ). According to a preferred embodi- ( preferably in um ) of the particles in a volume weighted
ment, the residual water content of the dry powder compo- 30 distribution , which represent 90 % of the total volume of the
sition is 5 % ( w / w ) or less , preferably 4 % ( w / w ) or less . In sample, and which have a particle size equal to or smaller
a particularly preferred embodiment, the residual water is than the Dv90 value . Preferably, the Dv90 of the dry powder
7 % ( w / w ) or less . In a further preferred embo nt, the composition according to the invention is at least 1 , 2 , 3 , 4 ,
residual water content of the dry powder composition in the 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 or 50 um .
range from 0% to 15 % (w /w ), from 0% to 10% (w /w ), from 35 Preferably, the Dv90 of the dry powder composition accord
0 % to 7 % ( w / w ), from 0% to 5 % ( w / w ), from 0 % to 4 % ing to the invention is equal to or lower than 1.500 , 1.250 ,
( w / w ), from 3 % to 6 % ( w / w ) or from 2 % to 5 % ( w / w ). 1.000 , 750 , 600 , 500 , 400 , 300 , 200 or 100 um . More
Preferably , the dry powder composition comprising a preferably, the Dv90 may be in a range from 0.3 um to 2.000
long -chain RNA molecule comprises a plurality of particles. um , from 1 um to 1.000 um , from 2 um to 500 um or from
Therein , the term “particle ” typically refers to an individual 40 2 um to 200 um . In a preferred embodiment, the Dv90 of the
solid particle of the dry powder composition . The individual dry powder composition is at least 1 um or in the range from
particles of the dry powder composition according to the 1 to 200 um . In a particularly preferred embodiment, the
invention are preferably physically separated from each Dv90 of the dry powder composition is at least 3 um , at least
other, i.e. the individual particles that constitute the dry 5 um or at least 20 um .
powder may be in lose and reversible contact with each other 45 The mass median aerodynamic diameter (MMAD )
( as opposed to an irreversible link between individual par- describes the particle size based on the aerodynamic prop
ticles ) . Preferably, the term “ particle ” refers to the smallest erties of the respective particle, in particular its settling
physical entity of the inventive dry powder composition . behaviour. The MMAD is preferably determined using any
The particles of the inventive dry powder composition do suitable instrument known in the art, such as , for instance ,
preferably not stick to each other. The particulate nature of 50 an APSTM spectrometer (TSI Inc. ) . The MMAD relates to the
the formulation contributes to the superior characteristics of median aerodynamic diameter of the particle distribution
the inventive dry powder composition, e.g. its free flowabil- and is the diameter of a unit density sphere having the same
ity. settling velocity, in air, as the particle . The MMAD thus
In a preferred embodiment, the plurality of individual typically describes the particle size , preferably in microm
particles of the dry powder composition according to the 55 eters ( um ), with 50% of the particles in the distribution
invention is characterized by a size distribution, wherein the having a larger size and 50% of the particles in the distri
size of individual particles may be the same or different from bution having a smaller size than the MMAD value . Pref
each other. Typically, the size of the particles of a dry erably, the dry powder composition according to the inven
powder composition is characterized by a Gaussian distri- tion is characterized by a MMAD of at least 0.3 um .
bution or a quasi-Gaussian distribution . Preferably, the dry 60 Alternatively, the MMAD of the dry powder composition
powder composition according to the invention is charac- according to the invention is at least 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ,
2

terized by a volume weighted particle size distribution as 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 or 50 um . Preferably, the

determined , for instance , by static light scattering tech- MMAD of the dry powder composition according to the
niques , such as laser diffraction , or by using a cascade invention is equal to or less than 1.500 , 1.250 , 1.000 , 750 ,
impactor. In a volume weighted distribution, the contribu- 65 600 , 500 , 400 , 300 , 200 or 100 um . Further preferably , the
tion of each particle in the dry powder composition relates MMAD may be in a range from 0.3 um to 2.000 um , from
to the volume of that particle. The dry powder composition 1 um to 1.000 um , from 2 um to 500 um or from 2 um to 200
 US 11,179,337 B2
7 8
um . In a preferred embodiment, the MMAD of the dry consists of particles with a sphericity in the range from 0.7
powder composition is at least 1 um or in the range from 1 to 1 , more preferably in the range from 0.8 to 1 , from 0.85
to 200 um . In a particularly preferred embodiment, the to 1 , or from 0.9 to 1 .
MMAD of the dry powder composition is at least 3 um , at Alternatively, the sphericity of those particles of the dry
least 5 um or at least 20 um . 5 powder composition that have a particle size equal to Dv50
The dry powder composition is preferably characterized as defined herein is at least 0.7 , preferably at least 0.75 , at
by using the span of the particle size distribution as a least 0.8 , at least 0.85 , at least 0.9 , at least 0.95 or 1 .
parameter. Therein , the span ( for a volume weighted distri- Preferably, the sphericity of those particles of the dry
bution) is defined according to the following formula powder composition that have a particle size equal to Dv50
10 as defined herein is in the range from 0.7 to 1 , more
preferably in the range from 0.8 to 1 , from 0.85 to 1 , or from
Span Dv90 - Dv10 0.9 to 1. Even more preferably, those particles of the dry
Dv50 powder composition that have a particle size equal to Dv90
as defined herein have a sphericity of at least 0.7 , preferably
For distributions, which are not volume weighted, the Dv 15 0.95
of at orleast1. 0.75, at least
Preferably , the0.8sphericity
, at least 0.85 , at least
of those 0.9, atofleast
particles the
values in the formula above are respectively replaced by the dry powder composition that have a particle size equal to
corresponding Dx values , e.g. Dn90 for a number weighted
distribution . In a preferred embodiment, the inventive dry Dv90 as defined herein is in the range from 0.7 to 1 , more
powder composition is characterized by a low span value , preferably in the range from 0.8 to 1 , from 0.85 to 1 , or from 2

which indicates a narrow (or more homogeneous) particle 20 0.9Further to 1.

size distribution . Typically, a narrow distribution enhances ticles of thepreferably , the average sphericity of those par
dry powder composition that have a particle size
the flowability of the dry powder composition. Preferably,
the span of the dry powder composition according to the equal to or lower than Dv50 as defined herein is at least 0.7 ,
present invention is equal to or less than 5 , more preferably preferably at least 0.75 , at least 0.8 , at least 0.85 , at least 0.9 ,
2

equal to or less than 4 , and even more preferably equal to or 25 atparticles

least 0.95 or 1. Preferably , the averagethat
sphericity
have a ofparticle
those
less than 3. In a particularly preferred embodiment, the size equaloftotheordry powder composition
lower than Dv50 as defined herein is in the
particle size distribution of the dry powder composition
according to the invention is characterized by a span of equal range from 0.7 to 1 , more preferably in the range from 0.8
to or less than about 2 or a span of equal to or less than about 30 tothe1 average
, from 0.85 to 1 , or from 0.9 to 1. Even more preferably ,
sphericity of those particles of the dry powder
1.5 .
In a preferred embodiment, the dry powder composition composition that have a particle size equal to or lower than
comprises a plurality of spherical particles. As used herein , Dv90, atas least defined herein is at least 0.7 , preferably of at least
the term “ spherical ” comprises not only geometrically per 0.75 0.8 , least 0.85 , least 0.9 , at least 0.95 or
fect spheres, but also more irregular shapes, such as sphe 1. Preferably, the average sphericity of those particles of the
roidal , elipsoid , oval or rounded particles . The shape of an 35 dry powder composition that have a particle size equal to or
individual particle can be determined by known methods tolower than Dv90 as defined herein is in the range from 0.7
and by using instruments, which are commercially available , to 11 ,, more or
preferably in the range from 0.8 to 1 , from 0.85
from 0.9 to 1 .
such as LasentecTM ( particle chord length FBRM) , In the context of the present invention , the term “ RNA ”
MalvernTM (Fraunhofer diffraction) or Coulter CounterTM
( electric zone sensing ). Typically, the volume and the sur- 40 is used as abbreviation for ribonucleic acid. RNA is aa nucleic
face area of an individual particle are determined . By using These acid molecule , i.e. a polymer consisting of nucleotides.
such parameters, Waddell's sphericity 4 ( herein also uridine -nucleotides are usually adenosine-monophosphate,
monophosphate, guanosine-monophosphate and
referred to as “ sphericity ” or “ circularity ” ) may be calcu
lated , e.g. by using the following formula cytidine -monophosphate monomers, which are connected to
45 each other along a so - called backbone. The backbone is
formed by phosphodiester bonds between the sugar , i.e.
surface area of sphere of equal ribose, of a first and a phosphate moiety of a second ,
volume to the particle adjacent monomer. The specific succession of the monomers
Y=
surface area of the particle is called the RNA sequence . As used herein , the term “ RNA
50 molecule ” is not limited to any particular type of RNA .
For example, RNA may be obtainable by transcription of
It is preferred that the average sphericity of the particles, a DNA -sequence, e.g. , inside a cell . In eukaryotic cells ,
which are contained in the inventive dry powder composi- transcription is typically performed inside the nucleus or the
tion, is at least 0.7 , preferably at least 0.75 , at least 0.8 , at mitochondria . In vivo , transcription of DNA usually results
least 0.85 , at least 0.9, at least 0.95 or 1. Preferably, the 55 in the so - called premature RNA, which has to be processed
average sphericity of the particles, which are contained in into so - called messenger -RNA , usually abbreviated as
the dry powder composition , is in the range from 0.7 to 1 , mRNA . Processing of the premature RNA , e.g. in eukaryotic
more preferably in the range from 0.8 to 1 , from 0.85 to 1 , organisms, comprises a variety of different posttranscrip
or from 0.9 to 1. In a particularly preferred embodiment, the tional-modifications such as splicing, 5 '-capping, polyade
average sphericity of the particles, which are contained in 60 nylation, export from the nucleus or the mitochondria and
the inventive dry powder composition , is in the range from the like . The sum of these processes is also called maturation
0.7 to 1 . of RNA . The mature messenger RNA usually provides the
In another preferred embodiment, the dry powder com- nucleotide sequence that may be translated into an amino
position according to the invention consists of particles , acid sequence of a particular peptide or protein . Typically, a
which are characterized by a sphericity of at least 0.7 , 65 (mature ) mRNA comprises a 5 ' - cap , optionally a 5'UTR , an
preferably at least 0.75 , at least 0.8 , at least 0.85 , at least 0.9 , open reading frame, optionally a 3'UTR and a poly (A)
at least 0.95 or 1. Preferably, the dry powder composition and / or poly ( C ) sequence . Furthermore , the term “ RNA
 US 11,179,337 B2
9 10
molecule ” comprises ribonucleic acids comprising more In a further embodiment, the long -chain RNA molecule
than one open reading frame, such as bicistronic or multi- comprised in the dry powder composition according to the
cistronic RNA molecules . A bicistronic or multicistronic invention is a coding RNA molecule or an immunostimu
RNA molecule is typically an RNA molecule , preferably an latory RNA molecule .
mRNA molecule , that may typically have two (bicistronic ) 5 In a preferred embodiment, the long - chain RNA is a
or more (multicistronic ) open reading frames ( ORF ) . coding RNA , which comprises at least one open reading
Aside from messenger RNA , several non- coding types of frame encoding a peptide or protein .
RNA exist , which may be involved in regulation of tran- In the context of the present invention , the long -chain
scription and / or translation , such as a ribosomal RNA 10 RNA molecule may be aa coding RNA molecule encoding a
( rRNA ) or a transfer RNA ( TRNA ). The terms “ RNA ” or protein or a peptide, which may be selected , without being
“ RNA molecule ” further encompass other coding RNA restricted thereto , e.g. from therapeutically active proteins or
molecules , such as viral RNA , retroviral RNA , self-repli- peptides, selected eng , from adjuvant proteins, from anti
cating RNA (replicon RNA ), small interfering RNA gens , e.g. tumour antigens, pathogenic antigens (e.g.
(siRNA ), microRNA, small nuclear RNA ( snRNA ), small- 15is selected, from animal antigens, from viral antigens, from
hairpin ( sh ) RNA , riboswitches , ribozymes or an aptamers . protozoan antigens, from bacterial antigens ), allergenic anti
The term “ long - chain RNA molecule” ( or “ long -chain gens, autoimmune antigens, or further antigens, preferably
RNA ” ) as used herein typically refers to an RNA molecule , as defined herein , from allergens, from antibodies, from
preferably as described herein , which preferably comprises immunostimulatory proteins or peptides, from antigen - spe
at least 30 nucleotides . Alternatively, the long - chain RNA 20 cific T - cell receptors , or from any other protein or peptide
molecule according to the invention may comprise at least suitable for a specific ( therapeutic ) application, wherein the
35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 100 , 150 , coding RNA molecule may be transported into a cell , a
200 , 250 , 300 , 350 , 400 , 450 or at least 500 nucleotides . In tissue or an organism and the protein may be expressed
a preferred embodiment, the long - chain RNA molecule subsequently in this cell , tissue or organism . In a particularly
comprises at least 100 nucleotides, even more preferably at 25 preferred embodiment, the long - chain RNA molecule is an
least 200 nucleotides. The long -chain RNA molecule further mRNA molecule .
preferably comprises from 30 to 50.000 nucleotides, from 30 The long - chain RNA molecule of the dry powder com
to 20.000 nucleotides, from 100 to 20.000 nucleotides, from position may further be an immunostimulatory RNA mol
200 to 20.000 nucleotides, from 250 to 15.000 nucleotides 30
ecule , such as any RNA molecule known in the art, which is
or from 500 to 20.000 nucleotides . capable of inducing an innate immune response . Particularly
According to a preferred embodiment, the long -chain preferred in this context are immunostimulatory RNA mol
RNA of the dry powder composition as described herein ecules as described in WO 2009/095226 .
comprises more than 200 nucleotides, preferably at least 250 The dry powder composition may further comprise a
nucleotides . Alternatively, the long - chain RNA as described 35 modified RNA molecule . In a preferred embodiment, the
herein may comprise more than 210 , more than 220 , more long - chain RNA molecule comprises at least one modifica
than 230 , more than 240 , more than 250 , more than 260 , tion as described herein . Alternatively or additionally, the
more than 270 , more than 280 , more than 290 , more than dry powder composition may comprise a second or further
300 , more than 350 , more than 400 , more than 450 or more RNA molecule ( distinct from the ( first) long -chain RNA
than 500 nucleotides. More preferably, the long - chain RNA 40 molecule) , which comprises at least one modification as
as described herein may comprise at least about 210 , at least described herein . Preferably, the long -chain RNA molecule
about 220 , at least about 230 , at least about 240 , at least of the dry powder composition according to the invention
about 250 , at least about 260 , at least about 270 , at least comprises an RNA modification , which preferably increases
about 280 , at least about 290 , at least about 300 , at least the stability of the RNA molecule and /or the expression of
about 350, at least about 400, at least about 450 or at least 45 a protein encoded by the RNA molecule . Several RNA
about 500 nucleotides . modifications are known in the art, which can be applied to
The inventive dry powder composition comprises a ( first) an RNA molecule in the context of the present invention .
long -chain RNA molecule and may further comprise a Chemical Modifications:
second or further RNA molecule , which may also be a The term “ RNAmodification ” as used herein may refer to
long-chain RNA molecule, preferably as defined herein . 50 chemical modifications comprising backbone modifications
as well as sugar modifications or base modifications .
Preferably, the second or further RNA molecule comprised In this context, a modified RNA molecule as defined
in the dry powder composition is distinct from the ( first) herein may contain nucleotide analogues /modifications, e.g.
long -chain RNA molecule. backbone modifications, sugar modifications or base modi
In a preferred embodiment, the long -chain RNA molecule
comprised in the dry powder composition according to the 55 fications. A backbone modification in connection with the
present invention is aa modification, in which phosphates of
invention is not an RNA molecule selected from the group the backbone of the nucleotides contained in an RNA
consisting of a small interfering RNA (siRNA ), a molecule as defined herein are chemically modified . A sugar
microRNA, a small nuclear RNA ( snRNA ), a small-hairpin modification in connection with the present invention is a
( sh) RNA or riboswitch, a ribozyme, and an aptamer. More 60 chemical modification of the sugar of the nucleotides of the
preferably, the long - chain RNA as described herein is not an RNA molecule as defined herein . Furthermore, a base modi
siRNA, most preferably not a double - stranded siRNA . fication in connection with the present invention is aa chemi
As used herein , the term “ RNA molecule ” typically refers cal modification of the base moiety of the nucleotides of the
to a single - stranded or a double - stranded RNA molecule . In RNA molecule . In this context, nucleotide analogues or
a preferred embodiment, the long -chain RNA molecule of 65 modifications are preferably selected from nucleotide ana
the inventive dry powder composition is a single - stranded logues , which are applicable for transcription and /or trans
RNA molecule . lation .
 US 11,179,337 B2
11 12
Sugar Modifications: triphosphate , 5 - aminoallylcytidine - 5 '- triphosphate ,
The modified nucleosides and nucleotides, which may be 5 -aminoallyluridine-5'- triphosphate, 5 -bromocytidine -5'- tri
incorporated into a modified RNA molecule as described phosphate, 5 -bromouridine - 5 '- triphosphate, 5 -Bromo-2'-de
herein , can be modified in the sugar moiety . For example, oxycytidine - 5 '-triphosphate, 5 -Bromo- 2'-deoxyuridine -5
the 2 ' hydroxyl group (OH ) can be modified or replaced with 5 triphosphate, 5 - iodocytidine - 5 -triphosphate, 5 - Iodo - 2
a number of different “oxy” or “ deoxy ” substituents . deoxycytidine - 5 '-triphosphate, 5 - iodouridine -5
Examples of “ oxy ” -2' hydroxyl group modifications include, triphosphate , 5 - Iodo - 2 ' -deoxyuridine - 5 '- triphosphate,
but are not limited to , alkoxy or aryloxy ( OR , e.g. , R = H ,
5 -methylcytidine -5 '-triphosphate, 5 -methyluridine -5'-tri
alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar ); poly-phosphate, 5 - Propynyl- 2 '-deoxycytidine - 5 '- triphosphate,
ethyleneglycols ( PEG) , O ( CH2CH2O ), CH2CH2OR ; 10 5 -Propynyl - 2 '- deoxyuridine- 5 '- triphosphate, 6 -azacytidine
“ locked ” nucleic acids ( LNA ) in which the 2 ' hydroxyl is 5 '- triphosphate , 6 - azauridine - 5 ' -triphosphate, 6 -chloropuri
connected, e.g. , by a methylene bridge, to the 4 ' carbon of neriboside - 5 '-triphosphate, 7 - deazaadenosine- 5 '- triphos
the same ribose sugar; and amino groups (0 - amino , phate, 7 - deazaguanosine - 5 - triphosphate , 8 -azaadenosine
wherein the amino group , e.g. , NRR , can be alkylamino , 5 '-triphosphate, 8 - azidoadenosine - 5 '- triphosphate,
dialkylamino , heterocyclyl, arylamino , diarylamino, het- 15 benzimidazole -riboside- 5 '-triphosphate, N1-methyladenos
eroarylamino, or diheteroaryl amino , ethylene diamine, ine - 5 ' -triphosphate, N1-methylguanosine- 5 - triphosphate,
polyamino ) or aminoalkoxy. N6 -methyladenosine - 5 ' -triphosphate, 06 -methylguanosine
“ Deoxy ” modifications include hydrogen , amino (e.g. 5 '- triphosphate, pseudouridine -5 - triphosphate, or puromy
NH ,; alkylamino, dialkylamino, heterocyclyl, arylamino, cin - 5 -triphosphate, Xanthosine - 5 -triphosphate. Particular
diary) amino , heteroaryl amino , diheteroaryl amino , or 20 preference is given to nucleotides for base modifications
amino acid) ; or the amino group can be attached to the sugar selected from the group of base -modified nucleotides con
through a linker, wherein the linker comprises one or more sisting of 5 -methylcytidine - 5 - triphosphate , 7 -deazaguanos
of the atoms C , N , and O. ine - 5 ' -triphosphate, 5 - bromocytidine - 5 '- triphosphate , and
The sugar group can also contain one or more carbons that pseudouridine - 5 -triphosphate.
possess the opposite stereochemical configuration than that 25 In some embodiments, modified nucleosides include pyri
of the corresponding carbon in ribose. Thus, a modified din - 4 - one ribonucleoside, 5 -aza - uridine, 2 -thio -5 -aza -uri
RNA molecule can include nucleotides containing, for dine , 2 - thiouridine, 4 - thio -pseudouridine, 2 - thio -pseudouri
instance, arabinose as the sugar. dine, 5 -hydroxyuridine, 3 -methyluridine, 5 - carboxymethyl
Backbone Modifications: uridine , 1 -carboxymethyl-pseudouridine, 5 - propynyl
The phosphate backbone may further be modified in the 30 uridine, 1 - propynyl -pseudouridine, 5 - taurinomethyluridine,
modified nucleosides and nucleotides, which may be incor- 1 -taurinomethyl -pseudouridine, 5 - taurinomethyl- 2- thio -uri
porated into a modified RNA molecule as described herein . dine , 1 -taurinomethyl - 4 -thio -uridine, 5 -methyl -uridine,
The phosphate groups of the backbone can be modified by 1 -methyl -pseudouridine, 4 - thio - 1 -methyl - pseudouridine,
replacing one or more of the oxygen atoms with aa different 2 -thio - 1 -methyl-pseudouridine, 1-methyl-1-deaza -pseudou
substituent. Further, the modified nucleosides and nucleo- 35 ridine, 2 - thio - 1 -methyl - 1 -deaza -pseudouridine, dihydrouri
tides can include the full replacement of an unmodified dine, dihydropseudouridine, 2 - thio -dihydrouridine , 2 -thio
phosphate moiety with a modified phosphate as described dihydropseudouridine, 2 -methoxyuridine, 2-methoxy -4
herein . Examples of modified phosphate groups include , but thio - uridine, 4 -methoxy -pseudouridine, and 4 -methoxy -2
are not limited to , phosphorothioate, phosphoroselenates, thio - pseudouridine .
borano phosphates, borano phosphate esters, hydrogen 40 In some embodiments, modified nucleosides include
phosphonates , phosphoroamidates, alkyl or aryl phospho- 5 -aza -cytidine, pseudoisocytidine, 3 -methyl- cytidine ,
nates and phosphotriesters. Phosphorodithioates have both N4 -acetylcytidine , 5 - formylcytidine , N4 -methylcytidine,
non - linking oxygens replaced by sulfur. The phosphate 5 -hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyr
linker can also be modified by the replacement of a linking rolo - cytidine, pyrrolo -pseudoisocytidine, 2 -thio -cytidine,
oxygen with nitrogen (bridged phosphoroamidates ), sulfur 45 2 -thio -5 -methyl-cytidine, 4 -thio -pseudoisocytidine, 4 -thio
(bridged phosphorothioates) and carbon ( bridged methyl- 1 -methyl-pseudoisocytidine, 4 - thio -1 -methyl-1- deaza-pseu
ene -phosphonates ). doisocytidine, 1 -methyl - 1 -deaza -pseudoisocytidine, zebu
Base Modifications: larine , 5 - aza -zebularine , 5 -methyl-zebularine, 5 -aza - 2 -thio
The modified nucleosides and nucleotides , which may be zebularine, 2 -thio - zebularine, 2 -methoxy -cytidine,
incorporated into a modified RNA molecule as described 50 2 -methoxy - 5 -methyl-cytidine, 4 -methoxy-pseudoisocyti
herein can further be modified in the nucleobase moiety. dine , and 4 -methoxy - 1 -methyl-pseudoisocytidine.
Examples of nucleobases found in RNA include, but are not In other embodiments, modified nucleosides include
limited to , adenine, guanine, cytosine and uracil. For 2 - aminopurine , 2 , 6 -diaminopurine, 7 - deaza -adenine,
example, the nucleosides and nucleotides described herein 7 -deaza - 8 -aza -adenine, 7 -deaza - 2 -aminopurine, 7 -deaza -8
can be chemically modified on the major groove face. In 55 aza - 2 - aminopurine, 7 -deaza - 2,6 -diaminopurine, 7 -deaza -8
some embodiments, the major groove chemical modifica- aza - 2,6 -diaminopurine, 1 -methyladenosine, N6 -methylad
tions can include an amino group , a thiol group, an alkyl enosine, N6 - isopentenyladenosine, N6-(cis
group , or a halo group . hydroxyisopentenyl )adenosine, 2 -methylthio -N6-(cis
In particularly preferred embodiments of the present hydroxyisopentenyl) adenosine,
invention , the nucleotide analogues/modifications are 60 N6 - glycinylcarbamoyladenosine, N6 - threonylcarbamoylad
selected from base modifications, which are preferably enosine , 2 -methylthio -N6 - threonyl carbamoyladenosine,
selected from 2 -amino -6 -chloropurineriboside -5'-triphos- N6 ,N6 - dimethyladenosine, 7 -methyladenine, 2 -methylthio
phate, 2 - Aminopurine - riboside- 5 - triphosphate; 2- aminoad- adenine, and 2 -methoxy - adenine.
enosine - 5 -triphosphate, 2 - Amino - 2'-deoxycytidine -tri- In other embodiments, modified nucleosides include inos
phosphate , 2 -thiocytidine- 5 '-triphosphate, 2 -thiouridine - 5'- 65 ine , 1 -methyl - inosine, wyosine , wybutosine, 7- deaza
triphosphate , 2 ' -Fluorothymidine- 5 - triphosphate, 2-0- guanosine , 7 -deaza - 8 -aza - guanosine, 6 -thio -guanosine,
Methyl inosine -5 '-triphosphate 4 - thiouridine -5'- 6 - thio - 7 -deaza -guanosine, 6 -thio - 7 -deaza - 8 -aza -guanosine,
 US 11,179,337 B2
13 14
7 -methyl -guanosine, 6 -thio - 7 -methyl- guanosine, 7 -methyl- nucleotide, 1- (beta - D - erythrofuranosyl) nucleotide, 4 ' - thio
inosine , 6 -methoxy - guanosine, 1 -methylguanosine, nucleotide, carbocyclic nucleotide, 1,5 -anhydrohexitol
N2 -methylguanosine, N2 ,N2-dimethylguanosine, 8-oxo- nucleotide, L -nucleotides , alpha-nucleotide, modified base
guanosine, 7 -methyl - 8 -oxo -guanosine, 1 -methyl-6 - thio- nucleotide, threo -pentofuranosyl nucleotide, acyclic 3',4'
guanosine, N2-methyl - 6 - thio - guanosine, and N2N2- dim- 5 seco nucleotide, acyclic 3,4 -dihydroxybutyl nucleotide, acy
ethyl -6 - thio - guanosine. clic 3,5 dihydroxypentyl nucleotide, 3 ' - 3 ' - inverted nucleo
In some embodiments, the nucleotide can be modified on tide moiety, 3 ' - 3 '- inverted abasic moiety , 3 ' - 2 ' - inverted
the major groove face and can include replacing hydrogen nucleotide moiety , 3 ' - 2 '- inverted abasic moiety, 1,4 -butane
on C - 5 of uracil with a methyl group or a halo group . In diol phosphate, 3 ' - phosphoramidate , hexylphosphate , ami
specific embodiments , a modified nucleoside is 5-0-( 1- 10 nohexyl phosphate, 3 '-phosphate, 3'phosphorothioate, phos
Thiophosphate )-Adenosine, 5'-0-( 1- Thiophosphate )-Cyti- phorodithioate, or bridging or non -bridging
dine , 5 '-O-( 1 - Thiophosphate ) -Guanosine, 5-0-( 1 - Thio- methylphosphonate moiety. These modified 5 ' - CAP struc
phosphate)-Uridine or 5 '-0-( 1 - Thiophosphate )- tures are regarded as at least one modification in this context.
Pseudouridine. Particularly preferred modified 5 - CAP structures are
In further specific embodiments, a modified RNA may 15 CAP1 (methylation of the ribose of the adjacent nucleotide
comprise nucleoside modifications selected from 6 -aza- of m7G) , CAP2 (methylation of the ribose of the 2nd
cytidine, 2 - thio -cytidine, a - thio -cytidine, Pseudo - iso - cyti- nucleotide downstream of the m7G) , CAPS (methylation of
dine, 5 -aminoallyl -uridine, 5 - iodo -uridine , N1-methyl- the ribose of the 3rd nucleotide downstream of the m7G) ,
pseudouridine, 5,6 - dihydrouridine, a - thio -uridine, 4 -thio- CAP4 (methylation of the ribose of the 4th nucleotide
uridine, 6 - aza -uridine , 5 -hydroxy -uridine, deoxy -thymidine, 20 downstream of the m7G) , ARCA ( anti- reverse CAP ana
5 -methyl -uridine, Pyrrolo -cytidine, inosine, a -thio -guanos- logue , modified ARCA ( e.g. phosphothioate modified
ine , 6 -methyl - guanosine, 5 -methyl -cytdine, 8 -oxo -guanos- ARCA ), inosine, N1-methyl- guanosine, 2'- fluoro - guanos
ine , 7 -deaza -guanosine, Nl-methyl-adenosine, 2-amino -6- ine , 7 -deaza - guanosine, 8 -oxo - guanosine, 2 -amino -guanos
Chloro -purine, N6 -methyl- 2 -amino -purine, Pseudo -iso- ine , LNA -guanosine, and 2 -azido - guanosine.
cytidine, 6 -Chloro - purine, N6 -methyl-adenosine, a - thio- 25 In a preferred embodiment, the inventive dry powder
adenosine, 8 - azido -adenosine, 7 -deaza - adenosine . composition comprises a modified RNA molecule having at
Lipid Modification: least one open reading frame, which encodes at least one
According to a further embodiment, a modified RNA peptide or protein . Said modified RNA molecule having at
molecule as defined herein can contain a lipid modification . least one open reading frame may be the ( first) long - chain
Such a lipid -modified RNA molecule typically comprises an 30 RNA molecule, preferably a long -chain mRNA molecule , or
RNA as defined herein . Such a lipid -modified RNA mol- a second or further RNA molecule , which may be comprised
ecule as defined herein typically further comprises at least in the dry powder composition in addition to the ( first)
one linker covalently linked with that RNA molecule , and at long -chain RNA molecule . Preferably, the sequence of the
least one lipid covalently linked with the respective linker. open reading frame in such an RNA molecule is modified as
Alternatively, the lipid -modified RNA molecule comprises 35 described herein .
at least one RNA molecule as defined herein and at least one Modification of the G / C Content:
( bifunctional) lipid covalently linked (without a linker) with In a particularly preferred embodiment of the present
that RNA molecule . According to a third alternative , the invention , the G / C content of the coding region of aa modi
lipid -modified RNA molecule comprises an RNA molecule fied RNA comprised in the inventive dry powder composi
as defined herein, at least one linker covalently linked with 40 tion, is modified , particularly increased, compared to the
that RNA molecule , and at least one lipid covalently linked G / C content of its particular wild type coding region , i.e. the
with the respective linker, and also at least one (bifunctional) unmodified coding region . The encoded amino acid
lipid covalently linked (without a linker) with that RNA sequence of the coding region is preferably not modified
molecule . In this context, it is particularly preferred that the compared to the coded amino acid sequence of the particular
lipid modification is present at the terminal ends of a linear 45 wild type coding region . The modification of the G /C
RNA sequence . content of the coding region of the modified RNA as defined
Modification of the 5 ' - End of a Modified RNA Molecule : herein is based on the fact that the sequence of any mRNA
According to another preferred embodiment of the inven- region to be translated is important for efficient translation of
tion, a modified RNA molecule as defined herein , can be that mRNA . Thus, the composition and the sequence of
modified by the addition of aa so - called “ 5 ' CAP ” structure . 50 various nucleotides are important. In particular, mRNA
A 5 ' -cap is an entity , typically a modified nucleotide sequences having an increased G ( guanosine )/ C ( cytosine )
entity, which generally “ caps ” the 5 ' - end of a mature mRNA . content are more stable than mRNA sequences having an
A 5 '-cap may typically be formed by a modified nucleotide, increased A ( adenosine )/ U ( uracil) content . According to the
particularly by a derivative of a guanine nucleotide. Prefer- invention , the codons of the coding region are therefore
ably , the 5 ' -cap is linked to the 5 ' - terminus via a 5 ' -5- 55 varied compared to its wild type coding region, while
triphosphate linkage. A 5 ' -cap may be methylated, e.g. retaining the translated amino acid sequence, such that they
m7GpppN , wherein N is the terminal 5 ' nucleotide of the include an increased amount of G / C nucleotides . In respect
nucleic acid carrying the 5 ' - cap , typically the 5 ' - end of an to the fact that several codons code for one and the same
RNA . m7GpppN is the 5 ' - CAP structure which naturally amino acid ( so - called degeneration of the genetic code ), the
occurs in mRNA transcribed by polymerase II and is there- 60 most favourable codons for the stability can be determined
fore not considered as modification comprised in aa modified ( so - called alternative codon usage ) . Depending on the amino
RNA in this context. Accordingly, a modified RNA of the acid to be encoded by the coding region of the modified
present invention may comprise a m7GpppN as 5 '-CAP, but RNA as defined herein, there are various possibilities for
additionally the modified RNA comprises at least one further modification of the RNA sequence, e.g. the coding region ,
modification as defined herein . 65 compared to its wild type coding region. In the case of amino
Further examples of 5'cap structures include glyceryl, acids , which are encoded by codons, which contain exclu
inverted deoxy abasic residue (moiety ), 4,5' methylene sively G or C nucleotides, no modification of the codon is
 US 11,179,337 B2
15 16
necessary . Thus, the codons for Pro ( CCC or CCG) , Arg “ frequent ” tRNAs are present. In this context, the coding
( CGC or CGG ), Ala (GCC or GCG ) and Gly (GGC or GGG ) region of the modified RNA is preferably modified com
require no modification, since no A or U is present. In pared to the corresponding wild type coding region such that
contrast, codons which contain A and /or U nucleotides can at least one codon of the wild type sequence, which codes for
be modified by substitution of other codons which code for 5 a tRNA which is relatively rare in the cell , is exchanged for
the same amino acids but contain no A and / or U. Examples a codon , which codes for a tRNA which is relatively
of these are : the codons for Pro can be modified from CCU frequent in the cell and carries the same amino acid as the
or CCA to CCC or CCG ; the codons for Arg can be modified relatively rare tRNA . By this modification , the coding region
from CGU or CGA or AGA or AGG to CGC or CGG ; the of the modified RNA as defined herein , is modified such that
codons for Ala can be modified from GCU or GCA to GCC 10 codons , for which frequently occurring tRNAs are available,
or GCG ; the codons for Gly can be modified from GGU or are inserted . In other words , according to the invention , by
GGA to GGC or GGG . In other cases , although A or U this modification all codons of the wild type coding region ,
nucleotides cannot be eliminated from the codons, it is which code for a tRNA which is relatively rare in the cell ,
however possible to decrease the A and U content by using can in each case be exchanged for a codon , which codes for
codons , which contain a lower content of A and / or U 15 a tRNA which is relatively frequent in the cell and which , in
nucleotides. Examples of these are : the codons for Phe can each case , carries the same amino acid as the relatively rare
be modified from UUU to UUC; the codons for Leu can be tRNA . Which tRNAs occur relatively frequently in the cell
modified from UUA , UUG , CUU or CUA LO CUC or CUG ; and which , in contrast, occur relatively rarely is known to a
the codons for Ser can be modified from UCU or UCA or person skilled in the art; cf. e.g. Akashi, Curr. Opin . Genet.
AGU to UCC , UCG or AGC ; the codon for Tyr can be 20 Dev. 2001 , 11 ( 6 ) : 660-666 . The codons which use for the
modified from UAU to UAC ; the codon for Cys can be particular amino acid the tRNA which occurs the most
modified from UGU to UGC ; the codon for His can be frequently, e.g. the Gly codon , which uses the tRNA which
modified from CAU to CAC ; the codon for Gln can be occurs the most frequently in the ( human ) cell , are particu
modified from CAA to CAG ; the codons for Ile can be larly preferred.
modified from AUU or AUAWO AUC ; the codons for Thr can 25 According to the invention, it is particularly preferable to
be modified from ACU or ACA to ACC or ACG ; the codon link the sequential G / C content, which is increased , in
for Asn can be modified from AAU to AAC ; the codon for particular maximized , in the coding region of the modified
Lys can be modified from AAA tO AAG ; the codons for Val RNA as defined herein , with the “ frequent" codons without
can be modified from GUU or GUA to GUC or GUG ; the modifying the amino acid sequence of the peptide or protein
codon for Asp can be modified from GAU to GAC ; the 30 encoded by the coding region of the RNA sequence . This
codon for Glu can be modified from GAA to GAG ; the stop preferred embodiment allows provision of a particularly
codon UAA can be modified to UAG or UGA . In the case efficiently translated and stabilized (modified ) RNA
of the codons for Met ( AUG ) and Trp (UGG) , on the other sequence as defined herein .
hand, there is no possibility of sequence modification . The In the context of the present invention , the long -chain
substitutions listed above can be used either individually or 35 RNA molecule may also comprise a 5'- and / or 3 ' untrans
in any possible combination to increase the G / C content of lated region ( 5 ' - UTR or 3 ' - UTR , respectively ). More pref
the coding region of the modified RNA as defined herein , erably, the long - chain RNA molecule comprises a 5 ' - CAP
compared to its particular wild type coding region ( i.e. the structure .
original sequence ). Thus, for example , all codons for Thr Preferably, the long -chain RNA molecule further com
occurring in the wild type sequence can be modified to ACC 40 prises a poly (A) sequence . The length of the poly (A)
( or ACG) . sequence may vary . For example, the poly (A) sequence may
Preferably, the G / C content of the coding region of the have a length of about 20 adenine nucleotides up to about
modified RNA as defined herein is increased by at least 7 % , 300 adenine nucleotides, preferably of about 40 to about 200
more preferably by at least 15 % , particularly preferably by adenine nucleotides, more preferably from about 50 to about
at least 20% , compared to the G / C content of the wild type 45 100 adenine nucleotides, such as about 60 , 70 , 80 , 90 or 100
coding region . According to a specific embodiment at least adenine nucleotides. Most preferably, the long - chain RNA
5 % , 10% , 20 % , 30 % , 40 % , 50 % , 60 % , more preferably at molecule comprises a poly (A) sequence of about 60 to about
least 70 % , even more preferably at least 80 % and most 70 nucleotides, most preferably 64 adenine nucleotides .
preferably at least 90 % , 95 % or even 100% of the substi- Preferably, the poly ( A ) sequence in the long -chain RNA
tutable codons in the coding region encoding at least one 50 molecule is derived from a DNA template by in vitro
peptide or protein, which comprises a pathogenic antigen or transcription . Alternatively, the poly ( A ) sequence may also
a fragment, variant or derivative thereof, are substituted, be obtained in vitro by common methods of chemical
thereby increasing the G / C content of said coding region. In synthesis without being necessarily transcribed from a
this context, it is particularly preferable to increase the G / C DNA -progenitor.
content of the coding region of the modified RNA as defined 55 Alternatively, the long -chain RNA molecule optionally
herein , to the maximum ( i.e. 100 % of the substitutable comprises a polyadenylation signal , which is defined herein
codons ) , compared to the wild type coding region . as a signal, which conveys polyadenylation to a (tran
Codon Optimization: scribed ) mRNA by specific protein factors ( e.g. cleavage and
According to the invention , a further preferred modifica- polyadenylation specificity factor (CPSF ) , cleavage stimu
tion of the coding region encoding at least one peptide or 60 lation factor ( CstF ) , cleavage factors I and II ( CF I and CF
protein of a modified RNA as defined herein , is based on the II ) , poly ( A ) polymerase ( PAP )). In this context, a consensus
finding that the translation efficiency is also determined by polyadenylation signal is preferred comprising the NN ( U /
a different frequency in the occurrence of tRNAs in cells . T )ANA consensus sequence. In a particularly preferred
Thus, if so - called “ rare codons ” are present in the coding aspect, the polyadenylation signal comprises one of the
region of the wild type RNA sequence , to an increased 65 following sequences: AA ( U / T ) AAA or A ( U / T ) (U / T ) AAA
extent , the mRNA is translated to a significantly poorer (wherein uridine is usually present in RNA and thymidine is
degree than in the case where codons coding for relatively usually present in DNA ).
 US 11,179,337 B2
17 18
In addition or as an alternative to a poly (A) sequence as peptides or proteins , e.g. protamine, cationic or polycationic
described above , the long - chain RNA molecule may also polysaccharides and / or cationic or polycationic lipids .
comprise a poly ( C ) sequence , preferably in the region 3 ' of According to a preferred embodiment, the long -chain
the coding region of the RNA . A poly ( C ) sequence is RNA as described herein may be complexed with lipids to
typically a stretch of multiple cytosine nucleotides, typically 5 form one or more liposomes , lipoplexes , or lipid nanopar
about 10 to about 200 cytidine nucleotides, preferably about ticles . Therefore, in one embodiment, the dry powder com
10 to about 100 cytidine nucleotides, more preferably about position comprises liposomes, lipoplexes, and /or lipid nan
10 to about 70 cytidine nucleotides or even more preferably oparticles comprising the long - chain RNA .
about 20 to about 50 or even about 20 to about 30 cytidine Lipid- based formulations have been increasingly recog
nucleotides . A poly ( C ) sequence may preferably be located 10 nized as one of the most promising delivery systems for
3 ' of the coding region comprised by a nucleic acid . In a RNA due to their biocompatibility and their ease of large
preferred embodiment of the present invention , the long- scale production. Cationic lipids have been widely studied
chain RNA molecule comprises a poly ( A ) sequence and a as synthetic materials for delivery of RNA . After mixing
poly (C ) sequence, wherein the poly (C ) sequence is located 15 together, nucleic acids are condensed by cationic lipids to
3 ' of the poly ( A ) sequence . form lipid /nucleic acid complexes known as lipoplexes .
In a particularly preferred embodiment, the long -chain These lipid complexes are able to protect genetic material
RNA molecule in the context of the present invention from the action of nucleases and to deliver it into cells by
comprises in 5 '- to - 3 ' -direction , a 5 ' - UTR , an open reading interacting with the negatively charged cell membrane.
frame, preferably a modified open reading frame as defined 20 Lipoplexes can be prepared by directly mixing positively
herein , a 3 ' - UTR element and a poly (A) or a poly ( C ) charged lipids at physiological pH with negatively charged
sequence . nucleic acids .
According to a preferred embodiment, the inventive dry Conventional liposomes consist of a lipid bilayer that can
powder composition may comprise the long -chain RNA be composed of cationic, anionic, or neutral (phospho ) lipids
molecule as described herein in free form (“ naked RNA ” ) or 25 and cholesterol, which encloses an aqueous core . Both the
in the form of a complex with another compound, such as a lipid bilayer and the aqueous space can incorporate hydro
transfection or complexation agent. For example, the long- phobic or hydrophilic compounds, respectively. Liposome
chain RNA molecule may be present in the dry powder characteristics and behaviour in vivo can be modified by
composition in a complex with a cationic or polycationic addition of a hydrophilic polymer coating, e.g. polyethylene
carrier or compound, which may serve as transfection or 30 glycol ( PEG) , to the liposome surface to confer steric
complexation agent . In a preferred embodiment, the dry stabilization . Furthermore , liposomes can be used for spe
powder composition comprises both , the long - chain RNA in cific targeting by attaching ligands ( e.g. , antibodies, pep
free form as well in a complex with a cationic or polyca- tides, and carbohydrates) to its surface or the terminal end
tionic carrier or compound . Such a complex of long - chain of the attached PEG chains (Front Pharmacol. 2015 Dec. 1 ;
RNA with a cationic or polycationic carrier or compound 35 6 : 286 ) .
may be present in the inventive dry powder composition or Liposomes are colloidal lipid -based and surfactant -based
in an intermediate product as a nanoparticle, preferably as delivery systems composed of a phospholipid bilayer sur
defined herein . The preparation of RNA complexes with rounding an aqueous compartment. They may present as
polycationic or cationic compounds is known in the art and spherical vesicles and can range in size from 20 nm to a few
is preferably carried out as described in WO2010 / 037539 or 40 microns . Cationic lipid -based liposomes are able to complex
WO2011 /026641 , the entire disclosure of which is herewith with negatively charged nucleic acids via electrostatic inter
incorporated by reference . actions, resulting in complexes that offer biocompatibility,
In this context, the long -chain RNA molecule in the low toxicity, and the possibility of the large -scale production
inventive dry powder composition is preferably complexed required for in vivo clinical applications. Liposomes can
by a compound selected from the group of polymers or 45 fuse with the plasma membrane for uptake; once inside the
complexing agents, typically comprising, without being cell , the liposomes are processed via the endocytic pathway
limited thereto , any polymer suitable for the preparation of and the genetic material is then released from the endosome/
a pharmaceutical composition , such as minor /major groove carrier into the cytoplasm . Liposomes have long been per
binders, nucleic acid binding proteins , lipoplexes , nano- ceived as drug delivery vehicles because of their superior
plexes , non - cationic or non - polycationic compounds, such 50 biocompatibility, given that liposomes are basically analogs
as PLGA, Polyacetate , Polyacrylate, PVA , Dextran , of biological membranes, and can be prepared from both
hydroxymethylcellulose , starch , MMP, PVP, heparin, pectin , natural and synthetic phospholipids ( Int J Nanomedicine.
hyaluronic acid , and derivatives thereof, or cationic or 2014 ; 9 : 1833-1843 ) .
polycationic compound
compo , particularly cationic or polycationic Cationic liposomes have been traditionally the most com
polymers or cationic or polycationic lipids , preferably a 55 monly used non - viral delivery systems for oligonucleotides,
cationic or polycationic polymers. In the context of the including plasmid DNA , antisense oligos , and siRNA / small
present invention, such aa cationic or polycationic compound hairpin RNA -shRNA ). Cationic lipids , such as DOTAP,
is typically selected from any cationic or polycationic com- ( 1,2 - dioleoyl - 3 - trimethylammonium -propane ) and DOTMA
pound , suitable for complexing and thereby stabilizing a (N-[ 1- (2,3 -dioleoyloxy )propyl]-N , N ,N - trimethyl-ammo
long - chain RNA molecule as defined herein , e.g. by asso- 60 nium methyl sulfate ) can form complexes or lipoplexes with
ciating the RNA molecule with the cationic or polycationic negatively charged nucleic acids to form nanoparticles by
compound . electrostatic interaction , providing high in vitro transfection
According to an alternative embodiment, the dry powder efficiency. Furthermore, neutral lipid- based nanoliposomes
composition according to the invention comprises the long- for RNA delivery as e.g. neutral 1,2 -dioleoyl- sn - glycero -3
chain RNA as described herein formulated together with one 65 phosphatidylcholine (DOPC) -based nanoliposomes were
or more cationic or polycationic compounds, preferably with developed . (Adv Drug Deliv Rev. 2014 February; 66 : 110
cationic or polycationic polymers, cationic or polycationic 116. ) .
 US 11,179,337 B2
19 20
Therefore , in one embodiment the long -chain RNA of the one SH moeity, may be , without being restricted thereto ,
dry powder composition as described herein is complexed selected from subformula (la ) :
with aa cationic lipid and / or a neutral lipid and thereby forms { (Arg )i;(Lys )mi (His )ni (Orn ) .; (Xaa '), (Cys ), } subformula (la)
liposomes, lipid nanoparticles, lipoplexes or neutral lipid 5 m

based nanoliposomes. wherein (Arg ) ,; (Lys )mi(His ) n ; (Orn ).; and x are as defined
m

Particularly preferred complexation agents in this context herein , Xaa ' is any amino acid selected from native (= natu
are cationic or polycationic compounds, including pro rally, His occurring ) or non - native amino acids except of Arg,
tamine, nucleoline, spermine or spermidine, or other cat Lys , Orn or Cys and y is any number selected from 0 ,
1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ,
ionic peptides or proteins, such as poly - L -lysine ( PLL ) , 10 20 9 2

, 21-30 , 31-40 , 41-50 , 51-60 , 61-70 , 71-80 and 81-90 ,

poly - arginine, oligoarginines as defined above, such as Arg7, provided that the overall content of Arg ( Arginine ), Lys
Args, Argo, Arg , HR ,, R ,H3, H R , Hz , YSSR , SSY, ( Lysine ), His
( RKH )4 , Y ( RKH ) 2R , etc. , basic polypeptides , cell penetrat least 10% of all(Histidine amino
) and Orn ( Ornithine ) represents at
acids of the oligopeptide. Further, the
ing peptides (CPPs ), including HIV - binding peptides, HIV - 1 cationic or polycationic peptide may be selected from sub
Tat ( HIV) , Tat -derived peptides , Penetratin , VP22 derived or 15 formula (Ib ) :
analog peptides, HSV VP22 (Herpes simplex ), MAP, KALA
or protein transduction domains (PTDs ) , PpT620 , proline Cys , { ( Arg ) ;j(Lys )mi (His )ni (Orn ) . ; (Xaa ); } Cys2
m subformula (Ib)
rich peptides, arginine -rich peptides, lysine -rich peptides, wherein empirical formula { (Arg ) :(Lys )m !:(His ), :(Orn ).;
MPG - peptide ( s ) , Pep - 1 , L -oligomers, Calcitonin peptide ( s ) , ( Xaa ); } ( formula (1 ) ) is as defined herein and forms a core
n

Antennapedia -derived peptides (particularly from Droso- 20 of an amino acid sequence according to ( semiempirical)
phila antennapedia ), pAntp , plsl , FGF, Lactoferrin , Trans formula ( IV) and wherein Cys , and Cys , are Cysteines
portan , Buforin - 2 , Bac715-24 , SynB , SynB ( 1 ), pVEC , MCT proximal to , or terminal to ( Arg ) ;; (Lys ) m ;(His ) n ; ( Orn ).; y
derived peptides, SAP, or histones. In a particularly
preferred embodiment, the dry powder composition accord (Xaa ),
Further preferred cationic or polycationic compounds,
ing to the invention comprises protamin , wherein the long 25 which can be used as transfection or complexation agent
chain RNA molecule is preferably complexed by protamine. may include cationic polysaccharides, for example chitosan ,
The dry powder composition according to the invention polybrene, cationic
preferably comprises a cationic or polycationic compound in cationic lipids , e.g.polymers DOTMA
, e.g. polyethyleneimine (PEI ) ,
: [ 1-(2,3- sioleyloxy )propyl)]
solution and / or in complex with the long - chain RNA mol N , N , N -trimethylammonium chloride , DMRIE , di - C14 - ami
ecule . More preferably
tion comprises , theorinventive
a cationic polycationic dry powder composi-
compound , pref 30 dine , DOTIM , SAINT, DC-Chol, BGTC , CTAP, DOPC,
DODAP , DOPE : Dioleyl
erably protamine, and the long -chain RNA molecule at a DOSPA , DODAB , DOIC , DMEPC , DOGS : Dioctadecy phosphatidylethanol - amine,
weight ratio (RNA :protamine, w / w ) in a range from 1:10 to lamidoglicylspermin , DIMRI : Dimyristo -oxypropyl dim
10 : 1 , more preferably from 5 : 1 to 1 : 1 , even more preferably
from 3 :1 to 1 : 1 . Most preferably, the weight ratio of the ethyl hydroxyethyl ammonium bromide, DOTAP : dioleoy
35 loxy - 3 - trimethylammonio ) propane , DC - 6-14 : 0,0
long -chain RNA molecule to cationic or polycationic com
pound, preferably protamine, in the composition is 2 : 1 ditetradecanoyl- N- ( a - trimethylammonioacetyl)
( w / w ). diethanolamine chloride, CLIP1 : rac -[( 2,3
Furthermore, such cationic or polycationic compounds or dioctadecyloxypropyl)(2 -hydroxyethyl) ]
dimethylammonium chloride , CLIP6 : rac-[ 2 ( 2,3
carriers may be cationic or polycationic peptides or proteins
which preferably comprise or are additionally modified to
, 40 dihexadecyloxypropyl -oxymethyloxy)ethyl )
comprise at least one SH moiety . Preferably, a cationic or trimethylammonium , CLIP9 : rac- [2 (2,3
polycationic carrier is selected from cationic peptides having dihexadecyloxypropyl-oxysuccinyloxylethyl]
trimethylammonium , oligofectamine, or cationic or
the following sum formula (1): polycationic polymers, e.g. modified polyaminoacids, such
{ ( Arg )i; (Lys)mi(His ) n ; (Orn ). ; (Xaa )x }; formula ( 1)
45 as B - aminoacid -polymers or reversed polyamides, etc.,
modified polyethylenes, such as PVP (poly (N -ethyl -4 - vi
wherein I +m + n+ o +x =3-100 , and I , m , n or o indepen- nylpyridinium bromide) ) , etc. , modified acrylates , such as
dently of each other is any number selected from 0 , 1 , 2 , 3 , PDMAEMA ( poly (dimethylaminoethyl methylacrylate ) ),
4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ,
2 2 etc. , modified Amidoamines such as PAMAM (poly (amido
)

21-30 , 31-40 , 41-50 , 51-60 , 61-70 , 71-80 , 81-90 and 91-100 50 amine) ) , etc., modified polybetaaminoester ( PBAE) , such as
provided that the overall content of Arg ( Arginine ), Lys diamine end modified 1,4 butanediol diacrylate-co -5 -amino
( Lysine ), His ( Histidine ) and Orn (Ornithine ) represents at 1 - pentanol polymers, etc., dendrimers, such as polypro
least 10 % of all amino acids of the oligopeptide ; and Xaa is pylamine dendrimers or PAMAM based dendrimers , etc.,
any amino acid selected from native ( = naturally occurring ) polyimine ( s ) , such as PEI: poly (ethyleneimine ), poly (pro
or non - native amino acids except of Arg , Lys, His or Orn ; 55 pyleneimine ), etc., polyallylamine , sugar backbone based
and x is any number selected from 0 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ,
2 polymers, such as cyclodextrin based polymers, dextran
9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21-30 , 31-40 ,
9 2 based polymers, chitosan, etc. , silan backbone based poly
41-50 , 51-60 , 61-70 , 71-80 , 81-90 , provided , that the overall mers , such as PMOXA - PDMS copolymers , etc. , blockpoly
content of Xaa does not exceed 90% of all amino acids of the mers consisting of aa combination of one or more cationic
oligopeptide. Any of amino acids Arg, Lys, His , Orn and Xaa 60 blocks ( e.g. selected from aa cationic polymer as mentioned
may be positioned at any place of the peptide . In this above) and of one or more hydrophilic or hydrophobic
context, cationic peptides or proteins in the range of 7-30 blocks ( e.g polyethyleneglycole ); etc.
amino acids are particular preferred. In this context, it is particularly preferred that the long
Further, the cationic or polycationic peptide or protein , chain RNA molecule of the inventive dry powder compo
when defined according to formula { ( Arg ) 7; (Lys )mi (His ) n; 65 sition is complexed at least partially with a cationic or
(Orn ), ; ( Xaa ); } ( formula ( I ) ) as shown above and which polycationic compound, preferably a cationic protein or
comprise or are additionally modified to comprise at least peptide. Partially means that only a part of the long -chain
 US 11,179,337 B2
21 22
RNA molecule is complexed with a cationic or polycationic than 107 nm , equal to or larger than 108 nm , equal to or
compound and that the rest of the long -chain RNA molecule larger than 109 nm , equal to or larger than 110 nm , equal to
is in uncomplexed form ( “ free ” ). Preferably the ratio of or larger than 111 nm , equal to or larger than 112 nm , equal
complexed long -chain RNA to free long - chain RNA is to or larger than 113 nm , equal to or larger than 114 nm ,
selected from a range of about 5 : 1 ( w / w ) to about 1:10 5 equal to or larger than 115 nm , equal to or larger than 116
( w /w ), more preferably from a range of about 4 : 1 ( w / w ) to nm , equal to or larger than 117 nm , equal to or larger than
about 1 : 8 ( w / w ), even more preferably from a range of about 118 nm , equal to or larger than 119 nm , equal to or larger
3 : 1 ( w / w ) to about 1 : 5 ( w / w ) or 1 : 3 ( w / w ), and most than 120 nm , equal to or larger than 125 nm or equal to or
preferably the ratio of complexed long - chain RNA molecule larger than 130 nm . In a particularly preferred embodiment,
to free long - chain RNA molecule is selected from aa ratio of 10 the mean size , preferably the mean size as determined by
about 1 : 1 ( w / w ). NTA , of the nanoparticles comprising or consisting of
In the context of the present invention , a particle of the dry complexed long - chain RNA molecule is equal to or larger
powder composition as defined herein , may thus comprise a than 110 nm , more preferably equal to or larger than 120 nm ,
long -chain RNA molecule in free form or complexed by a most preferably the mean size , preferably the mean size as
cationic or polycationic compound. In a preferred embodi- 15 determined by NTA , of the nanoparticles comprising or
ment, a particle of the inventive dry powder composition as consisting of complexed long -chain RNA molecule is equal
described herein comprises or consists of long - chain RNA to or larger than 130 nm . Alternatively, the mean size ,
complexed by a cationic or polycationic compound, wherein preferably the mean size as determined by NTA , of the
the complex is preferably present as a nanoparticle as nanoparticles comprising or consisting of complexed long
defined herein . As used herein , the term “ nanoparticle” 20 chain RNA molecule is in a range from 100 nm to 200 nm ,
typically refers to a complex of the long - chain RNA mol- preferably from 110 nm to 150 nm .
ecule with a complexation agent as defined herein , prefer- According to a further preferred embodiment, the mode
ably with a cationic or polycationic compound . size , preferably the mode size as determined by NTA , of the
In a preferred embodiment, upon reconstitution of the dry nanoparticles comprising or consisting of complexed long
powder in a suitable solvent, the complexed long - chain 25 chain RNA molecule is equal to or larger than 90 nm , equal
RNA molecule as described herein is present in the solvent to or larger than 91 nm , equal to or larger than 92 nm , equal
in the form of nanoparticles. The size of the nanoparticle to or larger than 93 nm , equal to or larger than 94 nm , equal
comprising or consisting of complexed long -chain RNA to or larger than 95 nm , equal to or larger than 96 nm , equal
molecule after reconstitution is preferably from 50 to 500 to or larger than 97 nm , equal to or larger than 98 nm , equal
nm , more preferably from 50 to 200 nm . In a particularly 30 to or larger than 99 nm , equal to or larger than 100 nm , equal
preferred embodiment, the particle size of the nanoparticle to or larger than 105 nm , equal to or larger than 110 nm ,
comprising or consisting of complexed long - chain RNA equal to or larger than 115 nm , equal to or larger than 120
molecule after reconstitution is from 75 to 180 nm , more nm , equal or larger than 125 nm or equal to or larger than
preferably from 100 to 150 nm . 130 nm . According to a particularly preferred embodiment,
Preferably, the nanoparticle comprising or consisting of 35 the mode size, preferably the mode size as determined by
complexed long -chain RNA molecule is characterized by at NTA , of the nanoparticles comprising or consisting of
least one physico -chemical property. Suitable methods for complexed long -chain RNA molecule is equal to or larger
determining a physico -chemical property of the nanoparticle than 95 nm , more preferably equal to or larger than 100 nm ,
comprising or consisting of complexed long -chain RNA most preferably equal to or larger than 105 nm . Alterna
molecule are known in the art. Preferably, a physico -chemi- 40 tively, the mode size , preferably the mode size as determined
cal property of the nanoparticle comprising or consisting of by NTA , of the nanoparticles comprising or consisting of
complexed long - chain RNA molecule is determined by complexed long -chain RNA molecule is preferably in a
using a method selected from the group consisting of range from 95 nm to 150 nm , more preferably in a range
measurement of turbidity, dynamic light scattering ( DLS ) , from 100 nm to 140 nm .
nanoparticle tracking analysis (NTA ), determining the Zeta 45 In a further preferred embodiment, the D10 size , prefer
potential and micro - flow imaging (MFI ) . More preferably , ably the D10 size as determined by NTA , of the nanopar
such method is used to characterize the nanoparticles com- ticles comprising or consisting of complexed long - chain
prised in a liquid composition obtained after reconstitution RNA molecule is equal to or larger than 70 nm , equal to or
of the inventive dry powder in an appropriate solvent, larger than 71 nm , equal to or larger than 72 nm , equal to or
preferably water, more preferably water for injection. 50 larger than 73 nm , equal to or larger than 74 nm , equal to or
In a preferred embodiment, a physico -chemical property larger than 75 nm , equal to or larger than 76 nm , equal to or
of the nanoparticle comprising or consisting of complexed larger than 77 nm , equal to or larger than 78 nm , equal to or
1

long - chain RNA molecule is determined by nanoparticle larger than 79 nm , equal to or larger than 80 nm , equal to or
tracking analysis (NTA ). As used herein , the term ‘nanopar- larger than 85 nm , equal to or larger than 90 nm , equal to or
ticle tracking analysis ' or ‘NTA’ refers to a method for 55 larger than 95 nm , equal to or larger than 100 nm , equal to
analyzing particles in a liquid that relates the rate of Brown- or larger than 105 nm or equal to or larger than 110 nm . It
ian motion to particle size . Suitable NTA protocols are is particularly preferred that the D10 size , preferably the
known in the art and instruments for NTA are commercially D10 size as determined by NTA , of the nanoparticles
available ( such as the NanoSight instruments, e.g. Nano- comprising or consisting of complexed long - chain RNA
Sight LM20 , NanoSight, Amesbury, UK) . In preferred 60 molecule is equal to or larger than 75 nm , more preferably
embodiments, the mean size , preferably the mean size as equal to or larger than 80 nm , most preferably equal to or
determined by NTA , of the nanoparticles comprising or larger than 85 nm or 90 nm . Alternatively, the D10 size ,
consisting of complexed long -chain RNA molecule is equal preferably the D10 size as determined by NTA , of the
to or larger than 100 nm , equal to or larger than 101 nm , nanoparticles comprising or consisting of complexed long
equal to or larger than 102 nm , equal to or larger than 103 65 chain RNA molecule is in a range from 70 nm to 140 nm ,
nm , equal to or larger than 104 nm , equal to or larger than more preferably in a range from 75 nm to 135 nm , most
105 nm , equal to or larger than 106 nm , equal to or larger preferably in a range from 80 nm to 130 nm .
 US 11,179,337 B2
23 24
According to another embodiment, the D50 size, prefer- composition obtained by reconstitution of the inventive dry
ably the D50 size as determined by NTA , of the nanopar- powder in a suitable solvent, preferably in water, more
ticles comprising or consisting of complexed long - chain preferably in water for injection .
RNA molecule is equal to or larger than 100 nm , equal to or According to a preferred embodiment, the polydispersity
larger than 101 nm , equal to or larger than 102 nm , equal to 5 index (PDI ) , preferably the PDI as determined by DLS , of
or larger than 103 nm , equal to or larger than 104 nm , equal the nanoparticles comprising or consisting of complexed
to or larger than 105 nm , equal to or larger than 106 nm , long - chain RNA molecule is equal to or larger than 0.10 ,
equal to or larger than 107 nm , equal to or larger than 108 equal to or larger than 0.11 , equal to or larger than 0.12 ,
nm , equal to or larger than 109 nm , equal to or larger than equal to or larger than 0.13 , equal to or larger than 0.14 ,
110 nm , equal to or larger than 115 nm , equal to or larger 10 equal to or larger than 0.15 , equal to or larger than 0.16 ,
than 120 nm , equal to or larger than 125 nm , equal to or equal to or larger than 0.17 , equal to or larger than 0.18 ,
larger than 130 nm , equal to or larger than 130 nm or equal equal to or larger than 0.19 , equal to or larger than 0.20 ,
to or larger than 135 nm . It is particularly preferred that the equal to or larger than 0.21 , equal to or larger than 0.22 ,
D50 size , preferably the D50 size as determined by NTA , of equal to or larger than 0.23 , equal to or larger than 0.24 ,
the nanoparticles comprising or consisting of complexed 15 equal to or larger than 0.25 or equal to or larger than 0.26 .
long -chain RNA molecule is equal to or larger than 100 nm , It is particularly preferred that the PDI , preferably the PDI
more preferably equal to or larger than 105 nm , most as determined by DLS , of the nanoparticles comprising or
preferably equal to or larger than 110 nm , equal to or larger consisting of complexed long -chain RNA molecule is equal
than 115 nm , or equal to or larger than 120 nm . Alternatively, to or larger than 0.12 , more preferably equal to or larger than
the D50 size , preferably the D50 size as determined by NTA , 20 0,13 , most preferably equal to or larger than 0.19 or 0.21 .
of the nanoparticles comprising or consisting of complexed Alternatively, the PDI , preferably the PDI as determined by
long -chain RNA molecule is in a range from 100 nm to 150 DLS , of the nanoparticles comprising or consisting of com
nm , more preferably in a range from 105 nm to 145 nm , most plexed long -chain RNA molecule is in a range from 0.10 to
preferably in a range from 110 nm to 140 nm . 0.40 , more preferably in range from 0.13 to 0.30 , most
According to a preferred embodiment, the D90 size , 25 preferably in a range from 0.19 to 0.27 .
preferably the D90 size as determined by NTA , of the It is further preferred that the main peak diameter, pref
nanoparticles comprising or consisting of complexed long- erably the main peak diameter as determined by DLS , of the
chain RNA molecule is equal to or larger than 145 nm , equal nanoparticles comprising or consisting of complexed long
to or larger than 146 nm , equal to or larger than 147 nm , chain RNA molecule is equal to or larger than 320 nm , equal
equal to or larger than 148 nm , equal to or larger than 148 30 to or larger than 325 nm , equal to or larger than 330 nm ,
nm , equal to or larger than 149 nm , equal to or larger than equal to or larger than 335 nm , equal to or larger than 340
150 nm , equal to or larger than 151 nm , equal to or larger nm , equal to or larger than 345 nm , equal to or larger than
than 152 nm , equal to or larger than 153 nm , equal to or 350 nm , equal to or larger than 355 nm , equal to or larger
larger than 154 nm , equal to or larger than 155 nm , equal to than 360 nm , equal to or larger than 365 nm , equal to or
or larger than 160 nm , equal to or larger than 165 nm , equal 35 larger than 370 nm , equal to or larger than 375 nm , equal to
to or larger than 170 nm , equal to or larger than 180 nm or or larger than 380 nm , equal to or larger than 385 nm , equal
equal to or larger than 190 nm . It is particularly preferred to or larger than 390 nm , equal to or larger than 395 nm or
that the D90 size , preferably the D90 size as determined by equal to or larger than 400 nm . It is particularly preferred
NTA , of the nanoparticles comprising or consisting of that the main peak diameter, preferably the main peak
complexed long - chain RNA molecule is equal to or larger 40 diameter as determined by DLS , of the nanoparticles com
than 150 nm , more preferably equal to or larger than 160 nm , prising or consisting of complexed long - chain RNA mol
most preferably equal to or larger than 170 nm or 180 nm . ecule is equal to or larger than 325 nm , more preferably
Alternatively, the D90 size , preferably the D90 size as equal to or larger than 330 nm , most preferably equal to or
determined by NTA , of the nanoparticles comprising or larger than 335 nm or 340 nm . Alternatively, the main peak
consisting of complexed long - chain RNA molecule is in a 45 diameter, preferably the main peak diameter as determined
range from 140 nm to 210 nm , more preferably in a range by DLS , of the nanoparticles comprising or consisting of
from 150 nm to 210 nm , most preferably in a range from 160 complexed long - chain RNA molecule is in a range from 300
nm to 200 nm . nm to 400 nm , more preferably in a range from 330 nm to
In a preferred embodiment, a physico -chemical property 400 nm , most preferably in a range from 327 nm to 390 nm .
of the nanoparticle comprising or consisting of complexed 50 In a preferred embodiment, the Zeta potential of the
long -chain RNA molecule is determined by dynamic light nanoparticles comprising or consisting of complexed long
scattering ( DLS ) . In this context, the term “ dynamic light chain RNA molecule is determined . Methods for determin
scattering' or ' DLS ' refers to a method for analyzing par- ing the Zeta potential are known in the art. For example, the
ticles in a liquid , wherein the liquid is typically illuminated Zeta potential of the nanoparticles comprising or consisting
with a monochromatic light source and wherein the light 55 of complexed long -chain RNA molecule can be determined
scattered by particles in the liquid is detected . Due to by using a Zetasizer Nano Series (Malvern Instruments ,
Brownian motion , smaller particles typically result in time- Worcestershire, UK) . Preferably, the Zeta potential of the
dependent scattering intensity fluctuations that are distinct nanoparticles comprising or consisting of complexed long
from those observed for larger particles. DLS can thus be chain RNA molecule is in a range from –36 mV to -50 mV,
used to measure particle sizes in a liquid. Suitable DLS 60 more preferably from -36 mV to -45 mV.
protocols are known in the art. DLS instruments are com- According to a preferred embodiment, the inventive dry
mercially available ( such as the Zetasizer Nano Series , powder is reconstituted in a suitable solvent, preferably in
Malvern Instruments, Worcestershire, UK ) . Preferably , DLS water for injection, and analyzed with respect to the con
is used in the context of the present invention to determine centration of particles having a certain size , preferably by
the polydispersity index (PDI ) and / or the main peak diam- 65 micro - flow imaging ( MFI ) . For instance, protamine -formu
eter of the nanoparticles comprising or consisting of com- lated RNA , which has preferably been spray -dried and
plexed long -chain RNA molecule , preferably in a liquid reconstituted as described in the Examples 1 to 6 herein ,
 US 11,179,337 B2
25 26
may be analyzed by MFI as described in Example 8.3.6 . In for use in spray drying. Preferably, a solvent is comprised in
that particular embodiment, the concentration of particles the inventive composition , in which the long - chain RNA and
having a diameter of at least 1 um is equal to or less than any other component comprised in the composition, if
175 /ml , equal to or less than 170 /ml, equal to or less than present, are soluble . More preferably, the solvent is volatile
165 /ml , equal to or less than 160 /ml, equal to or less than 5 with a boiling point of preferably below 150 ° C. In addition ,
155 /ml , equal to or less than 150 /ml, equal to or less than the solvent is preferably non- toxic . Preferably, the solvent is
145 /ml or equal to or less than 140 /ml. More preferably , the an aqueous solution . In the case of an organic solvent, the
concentration of particles having a diameter of at least 2 um solvent is preferably miscible with water.
is equal to or less than 35 /ml , equal to or less than 32/ml, In a preferred embodiment, the solvent comprised in the
equal to or less than 31 /ml, equal to or less than 30 /ml, equal 10 dry powder composition according to the invention com
to or less than 29/ml , equal to or less than 28 /ml, equal to or prises an aequeous solution or water, preferably pyrogen
less than 27 /ml, equal to or less than 26 /ml or equal to or less free water or water for injection (WFI ) . In this context, the
than 25 /ml . term “ water for injection ” (WFI ) is a term defined by
According to another embodiment, the dry powder com- standard USP 23. USP 23 monograph states that “ Water for
position as described herein comprises a long - chain RNA , 15 Injection (WFI ) is water purified by distillation or reverse
which is preferably not complexed with poly (lactide - co- osmosis . ” WFI is typically produced by either distillation or
glycolide) PLGA . More preferably, the dry powder compo- 2 - stage reverse osmosis . WFI typically does not contain
sition as described herein does not comprise PLGA . Alter- more than 0.25 USP endotoxin units ( EU ) per ml .
natively, the dry powder composition as described herein Endotoxins are a class of pyrogens that are components of
comprises a long - chain RNA , which is preferably not com- 20 the cell wall of Gram -negative bacteria ( the most common
plexed with a compound selected from the group consisting type of bacteria in water ), preferably in an action limit of 10
of PLGA, poly - lactide (PLA) , polyethylene imine (PEI ) or cfu / 100 ml . The microbial quality may be tested by mem
poly - L - lysine ( PLL) . More preferably , the dry powder com- brane filtration of a 100 ml sample and plate count agar at
position as described herein does not comprise a compound an incubation temperature of 30 to 35 degrees Celsius for a
selected from the group consisting of PLGA , PLA , PEI or 25 48 - hour period. The chemical purity requirements of WFI
PLL . are typically the same as of PW ( purified water) .
According to a further embodiment, the dry powder As a further excipient, the dry powder composition
composition as described herein comprises a long - chain according to the invention may comprise a buffer, preferably
RNA , which is preferably not complexed with DOTAP. selected from a buffer as defined herein , e.g. a buffer
More preferably, the dry powder composition as described 30 containing 2 -hydroxypropanoic acid , preferably including at
herein does not comprise DOTAP. Even more preferably, the least one of its optical isomers L - ( + ) - lactic acid, ( S ) -lactic
dry powder composition as described herein may comprise acid, D - ( - ) - lactic acid or ( R) -lactic acid, more preferably its
a long-chain which is preferably not complexed with biologically active optical isomer L - ( + ) - lactic acid , or a salt
a cationic lipid . More preferably, the dry powder composi- or an anion thereof, preferably selected from sodium - lactate ,
tion as described herein does not comprise a cationic lipid . 35 potassium - lactate , or Alz + -lactate, NH4 + -lactate , Fe - lactate ,
+

In an alternative embodiment, the dry powder composi- Li - lactate, Mg - lactate , Ca - lactate , Mn - lactate or Ag - lactate ,
tion as described herein comprises a long -chain RNA, which or a buffer selected from Ringer's lactate ( RiLa) , lactated
is preferably not complexed with mannitol, trehalose or Ringer's solution (main content sodium lactate , also termed
lactose . More preferably, the dry powder composition as “ Hartmann's Solution ” in the UK) , acetated Ringer's solu
described herein does not comprise mannitol, trehalose or 40 tion , or ortho -lactate -containing solutions (e.g. for injection
lactose . Even more preferably, the dry powder composition purposes ), or lactate containing water. A buffer as defined
as described herein may comprise a long -chain RNA, which herein may also be a mannose containing buffer, an isotonic
is preferably not complexed with a carbohydrate. More buffer or solution, preferably selected from isotonic saline,
preferably, the dry powder composition as described herein a lactate or ortho - lactate -containing isotonic solution , a
does not comprise a carbohydrate. 45 isotonic buffer or solution selected from phosphate -buffered
In some embodiments , the long -chain RNA of the dry saline (PBS ) , TRIS - buffered saline (TBS ) , Hank's balanced
powder composition as described herein is not comprised in salt solution ( HBSS ) , Earle's balanced salt solution ( EBSS ) ,
nanoparticles or in liposomes, preferably as defined herein . standard saline citrate ( SSC ) , HEPES -buffered saline
Furthermore, the dry powder composition may preferably (HBS ) , Grey's balanced salt solution ( GBSS ) , or normal
not comprise a nanoparticle or a liposome , preferably as 50 saline (NaCl ), hypotonic ( saline) solutions with addition of
defined herein . glucose or dextrose, or any solution as defined herein , etc.
In a preferred embodiment, the inventive dry powder These isotonic buffers or solutions are preferably prepared
composition comprising a long - chain RNA molecule com- as defined herein or according to protocols well known in the
prises at least one further component or excipient. art for these specific isotonic buffers or solutions. In this
In a preferred embodiment, the inventive dry powder 55
composition comprises a solvent, preferably in the amounts
context, a buffer or, in particular, a residue thereof, may be
comprised in the dry powder composition according to the
as defined herein with respect to the residual moisture invention , more preferably an aqueous (isotonic solution or
content of the dry powder composition. Typically , the sol- aqueous) buffer, containing a sodium salt , preferably at least
vent is a residue of a solvent, which was used during 50 mM of a sodium salt , a calcium salt , preferably at least
preparation of the dry powder composition , a residue of 60 0.01 mM of a calcium salt , and optionally a potassium salt ,
which may be present in the inventive dry powder compo- preferably at least 3 mM of a potassium salt . According to
sition . Preferably, the solvent contained in the inventive dry a preferred embodiment, the sodium , calcium and, option
powder composition is a residue of a solvent used during ally, potassium salts may occur in the form of their halo
preparation of the dry powder composition by using the genides, e.g. chlorides, iodides , or bromides , in the form of
inventive method as described herein . 65 their hydroxides, carbonates, hydrogen carbonates, or sul
In one embodiment, the solvent comprised in the dry fates, etc. Without being limited thereto , examples of
powder composition according to the invention is suitable sodium salts include e.g. NaCl , Nal , NaBr, Na2CO3 ,
 US 11,179,337 B2
27 28
NaHCO3 , Na2SO4 , examples of the optional potassium salts As a particularly preferred component, the inventive dry
include e.g. KCI , KI , KBr, K2CO3, KHCO3 , K2SO4, and powder composition may additionally contain at least one
examples of calcium salts include e.g. CaCl2 , Calz , CaBrz, suspending agent , preferably mannit.
CaCO3 , CaSO4 , Ca ( OH ) 2 . Typically, the salts are present in As a further component, the inventive dry powder com
such a buffer in a concentration of at least 50 mM sodium 5 position may additionally contain at least one component
selected , e.g. , from proteins , amino acids , alcohols, carbo
chloride (NaCl ) , at least 3 mM potassium chloride ( KCl ) and hydrates
at least 0.01 mM calcium chloride ( CaCl2 ) . Furthermore , tants , polymers , mannose , mannit , metals or metal ions , surfac
organic anions of the aforementioned cations may be con combination thereof or complexing agents, buffers, etc. , or a
tained in the buffer. According to a more preferred embodi 10 In the context of. the present invention , one preferred
ment, the buffer may contain salts selected from sodium component may also be selected from the group of amino
chloride (NaCl ) , calcium chloride ( CaCl2 ) and optionally acids . Such group may comprise , without being limited
potassium chloride ( KCI ) , wherein further anions may be thereto , any naturally occurring amino acid , including ala
present in addition to the chlorides . CaCl2 may also be nine , cysteine , aspartic acid , glutamic acid , phenylalanine,
replaced therein by another salt like KC? . 15 glycine , histidine , isoleucine , lysine , leucine, methionine,
According to a particularly preferred embodiment, the asparagine , pyrrolysine , proline, glutamine, arginine, serine,
inventive dry powder composition, may be reconstituted in threonine, selenocysteine, valine , tryptophan , and tyrosine,
a solvent or a buffer as defined herein , preferably as defined more preferably glycine, arginine, and alanine . Cryopro
above . For example, the inventive dry powder composition tectants and / or lyoprotectants selected from the group of
may be reconstituted in water, Ringer Lactate solution , a 20 amino acids may additionally comprise any modification of
buffer as defined above, or a buffer containing mannose , to a naturally occurring amino acid as defined above .
obtain the desired salt concentration or alternatively the Furthermore , in the context of the present invention, a
desired buffer conditions . The reconstitution of the dry further component may be selected from the group of
powder composition is carried out in WFI (water for injec- alcohols . Such group may comprise, without being limited
tion ), if the dry powder composition was prepared from a 25 thereto , any alcohol suitable for the preparation of a phar
long -chain RNA molecule dissolved in Ringer Lactate solu- maceutical composition , preferably, without being limited
tion (optionally comprising further components ), which thereto, mannitol, polyethyleneglycol, polypropyleneglycol ,
represents an isotonic solution for injection . In a particularly sorbitol, etc.
preferred embodiment, the dry powder composition is Additionally, in the context of the present invention , a
reconstituted in an isotonic solution, preferably as defined 30 further component may be selected from the group of ( free)
herein , more preferably in Ringer Lactate , especially if the carbohydrates. In general, a carbohydrate , such as a sugar ,
dry powder composition was prepared from a long - chain can act , for example, as a bulking agent, enhance cell
RNA molecule dissolved in water, preferably WFI (wherein targeting (e.g. , galactose , lactose) , open cellular junctions
the water optionally comprises further components ). (e.g. , mannitol), and modulate, for instance , the powder's
In a preferred embodiment, the dry powder composition 35 flowability by altering particle density. Such group of ( free )
according to the invention does not comprise a lipid com- carbohydrates may comprise, without being limited thereto ,
pound . any ( free) carbohydrate, suitable for the preparation of a
The inventive dry powder composition may further com- pharmaceutical composition, preferably, without being lim
prise any type of suitable component, which is compatible ited thereto , ( free) monosaccharides, such as e.g. ( free)
with the long -chain RNA molecule . As used herein , the term 40 glucose , ( free ) fructose , ( free ) galactose , ( free ) sorbose,
“ component' preferably comprises any additive or excipient, ( free ) mannose (“ free ” preferably means unbound or uncon
preferably a pharmaceutically acceptable excipient that does jugated, e.g. the mannose is not covalently bound to the
preferably not cause or enhance degradation of the long- long -chain RNA molecule , or in other words, the mannose
chain RNA molecule . Such a component may further be in is unconjugated , preferably with respect to the long - chain
any state , such as liquid , gel - like , solid or semi- solid . A 45 RNA molecule ) , etc. , and mixtures thereof; disaccharides,
component is preferably selected from the group consisting such as e.g. lactose , maltose , sucrose, trehalose, cellobiose ,
of cryoprotectants, lyoprotectants, bulking agents, preserva- etc. , and mixtures thereof; polysaccharides, such as
tives , antioxidants, antimicrobial agents, colorants, carriers, raffinose, melezitose , maltodextrins, dextrans, starches, etc.,
fillers, film formers, redispersants and disintegrants. More- and mixtures thereof; and alditols , such as mannitol, xylitol ,
over, the inventive dry powder composition may also com- 50 maltitol , lactitol , xylitol sorbitol, pyranosyl sorbitol, myo
prise excipients, such as defoamers , surfactants, viscosity inositol , etc. , and mixtures thereof. Examples of sugars that
enhancing agents, force control agents or the like . are preferably used in the composition according to the
Preferably, the inventive dry powder composition com- invention include lactose , sucrose or trehalose . Generally, a
prises at least one component selected from a cryoprotectant, sugar that is preferred in this context, has a high water
a lyoprotectant or a bulking agent. In this context, cryopro- 55 displacement activity and a high glass transition tempera
tectants are understood as excipients, which allow influenc- ture . Furthermore , a sugar suitable for use in the composi
ing the structure of a frozen material and / or the eutectical tion is preferably hydrophilic but not hygroscopic. In addi
temperature of the mixture. Lyoprotectants are typically tion , the sugar preferably has a low tendency to crystallize,
excipients, which partially or totally replace the hydration such as trehalose . Trehalose is particularly preferred .
sphere around a molecule and thus prevent catalytic and 60 In an alternative embodiment, the dry powder composi
hydrolytic processes . A bulking agent ( e.g. a filler) is any tion may comprise a cryoprotectant, which is preferably not
excipient compatible with the long - chain RNA molecule , selected from lactose or trehalose. More preferably, the
which may be comprised in the inventive composition. As cryoprotectant is not a carbohydrate.
used herein , a bulking agent may be used for increasing the The weight ratio of the long -chain RNA molecule in the
volume and /or the mass of the inventive composition. In 65 composition to the carbohydrate component, preferably a
addition , a bulking agent may also protect the long - chain sugar, more preferably trehalose, in the composition is
RNA molecule from degradation . preferably in the range from about 1 : 2.000 to about 1:10 ,
 US 11,179,337 B2
29 30
more preferably from about 1 : 1,000 to about 1 : 100 . Most and Rhenium (Re ), members of subgroup 8 of the periodic
preferably, the weight ratio of the long -chain RNA molecule table : including Iron (Fe ) , Ruthenium (Ru ), and Osmium
in the composition to the carbohydrate excipient, preferably (Os ) , members of subgroup 9 of the periodic table : including
a sugar, more preferably trehalose, in the composition is in Cobalt ( Co ) , Rhodium (Rh ), and Iridium ( Ir) , members of
the range from about 1 : 250 to about 1:10 and more prefer- 5 subgroup 10 of the periodic table : including Nickel (Ni ) ,
ably in the range from about 1 : 100 to about 1:10 and most Palladium (Pd) , and Platin ( Pt) , members of subgroup 11 of
preferably in the range from about 1 : 100 to about 1:50 . the periodic table : including Copper (Cu ) , Silver (Ag ) , and
In preferred embodiment, the dry powder composition Gold ( Au ), members of subgroup 12 of the periodic table :
according to the present invention comprises at least 50 % including Zinc (Zn) , Cadmium (Cd) , and Mercury ( Hg) ;
( w / w ), preferably at least 70 % ( w / w ), at least 80 % ( w /w ), at 10 preferably members of period 4 of any of subgroups 1 to 12
least 90 % ( w / w ), or at least 95 % ( w / w ) of a carbohydrate of the periodic table : including Scandium ( Sc ) , Titanium
component, preferably a sugar, more preferably trehalose. (Ti ) , Vanadium (V) , Chromium (Cr) , Manganese (Mn ), Iron
In a particularly preferred embodiment, the inventive dry (Fe ) , Cobalt (Co ) , Nickel (Ni), Copper (Cu ) and Zinc (Zn)
powder composition comprises trehalose. More preferably, and their metal ions and salts ;
trehalose is present in the inventive dry powder composition 15 earth metals or members of the boron group , including
in a relative amount of about 5 % to about 99.5 % ( w / w ), members of group 3 of the periodic table: including Boron
preferably in aa relative amount of about 20 % to about 98 % (B ) , Aluminium (Al ) , Gallium (Ga ) , Indium ( In) and Thal
( w / w ), more preferably in a relative amount of about 50% to lium ( TI ) and their metal ions and salts ; preferably Boron
about 95 % ( w / w ), even more preferably in aa relative amount (B ) and Aluminium (Al) and their metal ions and salts ;
of about 70 to about 99 % ( w / w ), and most preferably in a 20 metalloids or semi metals: including Boron ( B ) , Silicon
relative amount of about 75 to about 90 % ( w / w ). Preferably, ( Si ) , Germanium ( Ge) , Arsenic ( As ), Antimony ( Sb ) , Tellu
the relative amount of trehalose in the inventive dry powder rium ( Te ), and Polonium ( Po ) , and their semi metal ions and
composition is at least 30% ( w / w ), at least 40% ( w / w ), at salts ;
least 50 % ( w / w ), at least 60 % ( w / w ), at least 70% ( w / w ), at preferably Boron ( B ) and Silicon ( Si ) and their semi metal
least 80 % ( w / w ), at least 90 % ( w / w ) or at least 95 % ( w / w ). 25 ions and salts ;
In the context of the present invention, a further suitable In the context of the present invention , a further compo
component may also be selected from the group of proteins . nent may be selected from the group of surfactants may
Such group may comprise , without being limited thereto , comprise, without being limited thereto , any surfactant,
proteins such as albumin, gelatine , therapeutically active preferably any pharmaceutically acceptable surfactant,
proteins, antibodies, antigens, or any further protein encoded 30 which is preferably suitable for spray drying. More prefer
by the long -chain RNA molecule as defined herein . ably, without being limited thereto , the surfactant is selected
A component, which may be contained in the inventive from the group consisting of Tween , e.g. Tween 80 (0.2 % ) ,
dry powder composition may be selected from the group of Pluronics, e.g. Pluronic L121 ( 1.25 % ) , Triton - X , SDS , PEG ,
metals or metal ions , typically comprising , without being LTAB , Saponin , Cholate , etc.
limited thereto , metals or metal ions or salts selected from 35 As another component, the inventive dry powder compo
alkali metals , including members of group 1 of the
2 sition may additionally contain one or more compatible solid
periodic table : lithium (Li ) , sodium (Na) , potassium (K ), or liquid fillers or diluents or encapsulating compounds,
rubidium (Rb ), caesium ( Cs ) , and francium (Fr), and their which are preferably suitable for administration to a patient
(monovalent) metal alkali metal ions and salts ; preferably to be treated. The term “ compatible” as used here means that
lithium (Li ) , sodium (Na) , potassium (K) , and their (mon- 40 these constituents are capable of being mixed with the
ovalent) metal alkali metal ions and salts ; long -chain RNA molecule ( free or in a complex with a
alkaline earth metals , including members of group 2 of the cationic or polycationic compound ), as defined according to
periodic table : beryllium ( Be) , magnesium (Mg) , calcium the present invention , in such a manner that no interaction
( Ca) , strontium ( Sr ), barium (Ba ) and radium ( Ra ), and their occurs , which would substantially reduce the integrity or
( divalent) alkaline earth metal ions and salts ; preferably 45 biological activity of the long -chain RNA molecule , under
magnesium ( Mg ) , calcium (Ca ) , strontium (Sr ), barium (Ba ) typical use conditions. Pharmaceutically acceptable carriers ,
and their ( divalent) alkaline earth metal ions and salts ; fillers and diluents must, of course , have sufficiently high
transition metals , including members of groups 3 to 13 of purity and sufficiently low toxicity to make them suitable for
the periodic table and their metal ions and salts . The administration to a person to be treated . Some examples of
transition metals typically comprise the 40 chemical ele- 50 compounds, which can be used as pharmaceutically accept
ments 21 to 30 , 39 to 48 , 71 to 80 , and 103 to 112. The name able carriers , fillers or constituents thereof are sugars , such
transition originates from their position in the periodic table as , for example, lactose , glucose and sucrose ; starches , such
of elements . In each of the four periods in which they occur, as , for example, corn starch or potato starch ; cellulose and
these elements represent the successive addition of electrons its derivatives, such as , for example , sodium carboxymeth
to the d atomic orbitals of the atoms. In this way, the 55 ylcellulose, ethylcellulose , cellulose acetate; powdered tra
transition metals represent the transition between subgroup gacanth ; malt ; gelatin ; tallow ; solid glidants, such as , for
2 elements and subgroup 12 (or 13 ) elements. Transition example, stearic acid, magnesium stearate ; calcium sulfate ;
metals in the context of the present invention particularly vegetable oils , such as , for example , groundnut oil , cotton
comprise members of subgroup 3 of the periodic table: seed oil , sesame oil , olive oil , corn oil and oil from theo
including Scandium ( Sc ) , Yttrium (Y) , and Lutetium (Lu ) , 60 broma; polyols , such as , for example, polypropylene glycol ,
members of subgroup 4 of the periodic table : including Titan glycerol, sorbitol, mannitol and polyethylene glycol ; alginic
( Ti ), Zirconium (Zr) , and Hafnium (Hf), members of sub- acid .
group 5 of the periodic table : including Vanadium ( V ), In addition , the dry powder composition according to the
Niobium ( Nb ), and Tantalum ( Ta ), members of subgroup 6 invention may optionally contain further excipients or
of the periodic table : including Chrome ( Cr), Molybdenum 65 agents, such as stabilizers , for example EDTA , Tween,
( Mo ) , and Tungsten (W ) , members of subgroup 7 of the benzoic acid derivatives or RNAse inhibitors. Preferably, the
periodic table : including Manganese (Mn ), Technetium ( TC ), dry powder composition may further comprise any type of
 US 11,179,337 B2
31 32
component or additive , which is compatible with the long- molecule , preferably a long - chain RNA in particulate form
chain RNA molecule . Such an excipient is preferably as described herein , wherein a liquid comprising the RNA
selected from the group consisting of preservatives, antioxi- molecule is provided and wherein the liquid is dried by
dants, antimicrobial agents, colorants, carriers , fillers , film spray -drying. In one embodiment, the invention concerns a
formers, redispersants and disintegrants. Moreover, the dry 5 method for drying a liquid comprising a long -chain RNA
powder composition may also comprise a component or molecule .
additive, preferably in very small amounts, that were added With respect to the following description of the inventive
during the manufacturing process , such as defoamers, sur- method , it is noted that the definitions and specifications
factants, viscosity enhancing agents, force control agents or provided above with respect to the inventive dry powder
the like . 10 composition may likewise apply to the inventive method. In
In a preferred embodiment, the dry powder composition particular, the description of the long - chain RNA molecule
of the invention is obtained by the method as described and further components of the dry powder composition
herein . apply to the inventive method as well . In addition , further
As explained herein , the dry powder composition accord- definitions may apply to the inventive method as specifically
ing to the invention is particularly suitable as storage - stable 15 indicated in the following.
form of a long -chain RNA molecule . The inventors have In a preferred embodiment, the invention concerns a
surprisingly found that the storage stability of the long - chain method for preparing a dry powder comprising a long - chain
RNA molecule in the dry powder composition is excellent RNA molecule , wherein the method comprises the following
and the long - chain RNA molecule remains functional after steps :
extended storage periods. The storage stability of the long- 20 a ) providing a liquid comprising
? the long -chain RNA
chain RNA molecule is typically determined through deter- molecule,
mination of the relative ( structural) integrity and the bio- b ) drying the liquid provided in step a) by spray -drying.
logical activity after a given storage period, e.g. via time- The inventors found that a storage form of a long -chain
course in vitro expression studies. RNA molecule may be obtained by the method as described
The relative integrity is preferably determined as the 25 herein . In particular, the inventors found that — by using the
percentage of full - length RNA ( i.e. non -degraded long -chain method according to the invention a dry powder compris
RNA ) with respect to the total amount of RNA (i.e. long- ing a long - chain RNA molecule can be obtained . Advanta
chain RNA and degraded RNA fragments (which appear as geously, the method according to the invention is suitable for
smears in gel electrophoresis )), preferably after deduction of application at an industrial scale . In addition, the invention
the LOD ( 3x background noise ), for example, by using the 30 provides a method that can be carried out by the skilled
software QuantityOne from BioRad . person using standard equipment, thus providing a cost- and
The dry powder composition according to the invention time - effective solution . Moreover, the method can be carried
thus provides the advantageous characteristics of a powder out in bulk as well as continuously. The storage form ,
and the potential of such a composition for, e.g. packaging preferably the long -chain RNA in particulate form , obtained
and dosage , while it also allows significantly longer storage 35 by using the method according to the invention therefore
at temperatures from -80 ° C. to 60 ° C. than the correspond-
-
represents an effective means for extending the stability of
ing RNAs in WFI or other injectable solutions . Particularly, long -chain RNA as an API ( active pharmaceutical ingredi
it can be stored at room temperature, which simplifies ent ), especially during storage at a variety of different
shipping and storage. Preferably, the dry powder composi- temperatures and in different packaging formats.
tion is stored with or without shielding gas . In one embodi- 40 In step a ) of the method according to the invention , a
ment, single doses of the dry powder composition are liquid is provided that comprises a long - chain RNA mol
packaged and sealed. Alternatively, multiple doses can be ecule . The long - chain RNA molecule comprised in the liquid
packaged in one packaging unit. Single dose packaging in provided in step a) of the inventive method is characterized
blisters or capsules is preferably used in order to prevent by any feature or any combination of features described
cross -contamination . 45 herein with respect to the long -chain RNA molecule that is
Preferably, the relative integrity is at least 70 % , more comprised in the inventive dry powder composition .
preferably at least 75 % , at least 80% , at least 85 % , at least Typically, the liquid in step a ) of the method according to
90 % or at least 95 % after storage at room temperature for the invention is provided by diluting or dissolving the
preferably at least one week, more preferably for at least one long - chain RNA molecule in a suitable solvent. The solvent
month , even more preferably for at least 6 months and most 50 is preferably a solvent suitable for use in spray drying.
preferably for at least one year. Preferably, a solvent is used , in which the long - chain RNA
Further preferably, the biological activity of the long- and any other component, if present, are soluble . Suitable
chain RNA molecule of the dry powder composition after solvents are described above with respect to the inventive
storage at room temperature , preferably as defined above dry powder composition. The liquid in step a ) of the
with respect to the relative integrity of the long -chain RNA 55 inventive method preferably comprises the long - chain RNA
molecule , is preferably at least 70 % , more preferably at least molecule and a solvent or buffer as described above with
75 % , at least 80% , at least 85 % , at least 90 % or at least 95 % respect to the inventive dry powder composition. Preferably,
of the biological activity of the freshly prepared long -chain step a ) of the inventive method comprises dissolving or
RNA molecule . The biological activity is preferably deter- diluting the long -chain RNA molecule as defined herein in
mined by analysis of the amounts of protein expressed from 60 a solvent or buffer as defined herein , preferably in an
reconstituted RNA and from freshly prepared RNA , respec- aqueous solution , such as Ringer Lactate , or water, more
tively, e.g. after transfection into a mammalian cell line. preferably pyrogen -free water or WFI .
Alternatively, the biological activity may be determined by In a further preferred embodiment, the liquid comprising
measuring the induction of an ( adaptive or innate ) immune the long -chain RNA molecule comprises at least one further
response in a subject. 65 component, preferably as described herein with respect to
In aa further aspect of the invention, a method is provided the dry powder composition disclosed herein . In particular,
that allows preparing a storage form of a long - chain RNA the liquid provided in step a ) of the inventive method
 US 11,179,337 B2
33 34
preferably comprises a further component selected from the In another embodiment, the liquid provided in step a ) of
group consisting of buffers, cryoprotectants, lyoprotectants, the inventive method does not comprise a lipid compound.
bulking agents, suspending agents, proteins, amino acids , As a particularly preferred component, the liquid pro
alcohols , carbohydrates, metals , metal ions , salts , surfac- vided in step a) of the method may additionally contain at
tants, fillers, diluents, carriers, glidants, vegetable oils, poly- 5 least one suspending agent, preferably mannit, preferably in
ols , encapsulating compounds, stabilizers, preservatives, a concentration of about 1 to 15 % ( w / w ), more preferably in
antioxidants, antimicrobial agents, colorants, film formers, a concentration of about 3 to 10 % ( w / w ), and even more
redispersants, disintegrants, defoamers , viscosity enhancing preferably in a concentration of about 4 to 6 % ( w / w ).
agents and force control agents, wherein the respective In aa further embodiment, the liquid provided in step a) of
component is preferably as defined above with respect to the 10 the method comprises a carbohydrate component, preferably
a sugar, more preferably trehalose . In a preferred embodi
inventive dry powder composition . ment, a carbohydrate component, preferably a sugar, more
The long -chain RNA molecule as defined herein may be preferably trehalose is present in the liquid provided in step
present in the liquid provided in step a ) of the inventive a ) of the method at a concentration of about 0.01 to about
method in free form (asor “naked
with a polycationic cationicRNA ” ) and/,or preferably
compound as a complexas 15 20 % (w /w ),preferably in a concentration of about 0.01 to
about 15 % ( w / w ), more preferably in a concentration of
described herein . The long - chain RNA molecule as defined about 0.1 to about 10% ( w / w ), even more preferably in a
herein and a cationic or polycationic compound may be concentration of about 0.5 to about 10 % ( w / w ), and most
comprised in the liquid provided in step a ) , either as a preferably in a concentration of about 2.5 to about 7.5 %
complex, preferably in the form of a nanoparticle as defined 20 (w /w ),e.g. at a concentration of about 4 to about 7 % ( w /w ),
herein , or both in free form , i.e. in solution without being in such as about 5 % ( w / w ).
a complex with each other. The preparation of RNA com- The pH of the liquid provided in step a ) of the method
plexes with complexation agents, preferably with polyca- may be in the range of about 4 to 8 , preferably in the range
tionic or cationic compounds, is known in the art and is of about 6 to about 8 , more preferably from about 7 to about
preferably carried out as described in WO2010 / 037539 or 25 8 .
WO2011 /026641 , the entire disclosure of which is herewith Preferably, the liquid provided in step a ) of the method
incorporated by reference . contains the herein defined contents, optional components ,
In a particularly preferred embodiment, the liquid pro- additives, etc. in such a concentration so as to lead to an
vided in step a ) of the inventive method comprises a osmolarity comparable to that of blood plasma . In this
complexation agent, preferably as defined herein, more 30 context, the term " osmolarity ” is typically to be understood
preferably a cationic or polycationic compound as defined as a measure of all contents, optional components, additives ,
herein, such as protamine, nucleoline, spermin, spermidine, etc. of the liquid as defined herein . More precisely, osmo
oligoarginines as defined such as Arg7, Args, Argo, larity is typically the measure of solute concentration,
Arg7, H3R9 , R.H3 , H3R, Hz , YSSR , SSY, (RKH )44,, Y (RKH ) 2 defined as the number of osmoles (Osm) of all solubilized
R , etc. The complexation agent, preferably a cationic or 35 contents, optional components , additives , etc. per liter (I ) of
polycationic compound as defined herein , is preferably solution ( osmol/ l or osm / 1). In the present context, the liquid
present in the liquid provided in step a ) in free form (in provided in step a ) of the method may comprise an osmo
solution ) or in a complex with the long - chain RNA mol- larity preferably in the range of about 200 mosmol/l to about
ecule . Protamine is particularly preferred and is preferably 400 mosmol/ l , more preferably in the range of about 250
comprised in the liquid provided in step a ) of the method at 40 mosmol /l to about 350 mosmol/l , even more preferably in
a concentration in a range from 0.01 g /I to 10 g/I , from 0.05 the range of about 270 mosmol/l to about 330 mosmol/ l or
g/I to 5 g / I, or from 0.05 g/I to 2 g/I . More preferably, the in the range of about 280 mosmol/ l to about 320 mosmo1/1 ,
concentration of protamine in the liquid provided in step a ) or in the range of about e.g. about 290 mosmol/l to about 310
of the method is in a range from 0.05 g/ I to 3 g/I or from 0.1 mosmol /l , e.g. about 295 mosmol/l , about mosmol/ l , about
2

to 1 g /l. 45 296 mosmol /l , about 297 mosmol/ l , about 298 mosmo1/1 ,

In a preferred embodiment, the liquid provided in step a ) about 299 mosmol/ l , about, 300 mosmol/ l , about 301 mos
further comprises lactate , wherein the lactate concentration mol/ l , about 302 mosmol/l , about 303 mosmol/l , about 304
is preferably in the range of about 3 mM to about 300 mM , mosmol /l , about 305 mosmol /l , about 306 mosmol /l , about
preferably in the range of about 5 mM to about 200 mm , 307 mosmol/ l , about 308 mosmol/ 1 .
more preferably in the range of about 10 mM to about 150 50 The method according to the present invention further
mM , even more preferably about 15 mM to about 35 mM , comprises a step b ) , wherein the liquid provided in step a )
and most preferably 20 mM to about 31 mM . Alternatively, of the method is dried by spray - drying.
the liquid provided in step a ) of the method typically As used herein , the term 'spray -drying' typically relates to
comprises a Ringer's lactate concentration (or a concentra- a process that involves breaking up a liquid into small
tion of any of the afore mentioned lactate containing solu- 55 droplets (atomization) and rapidly removing solvent from
tions ) e.g. in the range of about 10 % ( w / w ) to about 100 % the droplets in a spray -drying apparatus, where there is a
( w / w ), e.g. in the range of about 20 % ( w / w ) to about 100 % strong driving force for evaporation of solvent from the
( w / w ), in the range of about 30% ( w / w ) to about 100 % droplets, which provide a favourable surface to mass ratio .
( w / w ), in the range of about 40% ( w / w ) to about 100 % The strong driving force for solvent evaporation is generally
( w / w ), in the range of about 50% ( w / w ) to about 90% ( w /w ), 60 provided by a high surface to mass ratio of the droplets and
preferably in the range of about 60 % ( w / w ) to about 90 % by maintaining the partial pressure of solvent in the spray
( w / w ), more preferably in the range of about 70 % ( w / w ) to drying apparatus well below the vapor pressure of the
about 90 % ( w / w ), e.g. about 80 % ( w / w ), of Ringer's lactate solvent at the temperature of the drying droplets. This may
( or the afore mentioned lactate containing solution ). In this be achieved , for example, by maintaining the pressure in the
context, Ringer's lactate ( 100 % ( w / w )) is typically defined 65 spray -drying apparatus at a partial vacuum or by mixing the
as a solution comprising 131 mM Na + , 5.36 mM K+ , 1.84 droplets with a warm drying gas or a combination of both .
mM Ca2 + , and 28.3 mm Lactate ).
9 As aa result of the spray - drying process , particles, preferably
 US 11,179,337 B2
35 36
dry particles, more preferably in the form of a dry powder 1 to 200 um . In a particularly preferred embodiment, the
composition , are typically obtained . MMAD of the droplets is at least 3 um , at least 5 um or at
The method according to the invention may be carried out least 20 yum .
in bulk or as a continuous process. In a preferred embodi- Preferably, the droplet size distribution is narrow , i.e. the
ment, the method is carried out as a continuous process. In 5 size of the individual droplets that are formed by the
particular, the spray - drying process may be carried out in atomizer is relatively uniform . More preferably , the droplets
bulk or as a continuous process in the context of the formed by the atomizer are characterized by using the span
inventive method . Most preferably, the spray -drying process of the droplet size distribution as a parameter. Therein , the
is carried out in a continuous process . span ( for a volume weighted distribution ) is defined as
Preferably, the liquid provided in step a) of the method is 10 outlined above with respect to the particle size of the
inventive dry powder composition. In a preferred embodi
used as liquid feed in a spray -drying process .
Typically, the liquid comprising the long -chain RNA ment, the droplet size distribution is characterized by a low
molecule , which is provided in step a ) , is first brokenup into span value , which preferably results in a narrow particle size
distribution in the dry powder composition . Typically, a
aa gasplurality of small
or a gas droplets
mixture , suchthat
as airare. The
preferably
obtainedsuspended in 15 narrow droplet size distribution after atomization results in
mixture of
droplets and gas is typically referred to as 'spray ' or ' fog '. increased flowability of the resulting dry powder. Preferably,
The process of breaking up the liquid feed into droplets is the span of the droplets formed by the atomizer is equal to
known as “ atomization and may be carried out using any or less than 5 , more preferably equal to or less than 4 , and
even more preferably equal to or less than 3. In a particularly
suitable device
atomizers knownininthetheartart, which
are known (atomizer
are ).suitable
Variousfortypes of 20 preferred embodiment, the particle size distribution of the
being dry powder composition according to the invention is char
used in the inventive method, such as rotary atomizers , acterized by a span of less than about 2 or less than about
pressure nozzles, two - fluid nozzles , fountain nozzles, ultra 1.5 .
sonic nebulizers and vibrating orifice aerosol generators. In a preferred embodiment, atomization of the liquid feed
In one embodiment, a rotary atomizer is used as atomizer 25 results in spherical droplets. As used herein , the term
in the spray -drying. Rotary atomizers exploit the energy of " spherical " comprises not only geometrically perfect
high -speed rotation to produce fine droplets. The liquid feed spheres, but also more irregular shapes, such as spheroidal,
is introduced into a reservoir, typically in the center of the elipsoid, oval or rounded droplet. Waddell's sphericity w
rotary wheel . ( herein also referred to as “ sphericity” or “ circularity ” ) may
According to a preferred embodiment, a two - fluid nozzle 30 be calculated, e.g. by using the following equation
is used in the spray -drying process . Two - fluid nozzles com
bine two fluids, where one fluid is typically the liquid feed
to be dried and the second fluid is typically a compressed gas surface area of sphere of equal
( e.g. air, nitrogen or CO2 at , for example, 0.1 to 7 bar ). The 35 y= volume to the droplet
energy of the compressed gas is used to atomize the liquid surface area of the droplet
feed . Once the liquid feed is atomized , the produced spray
droplets are usually mixed with a drying gas stream , allow In a preferred embodiment, a droplet formed by atomi
ing the liquid to quickly evaporate . The rapid evaporation zation of the liquid feed is characterized by a sphericity of
typically results in a cooling effect, so that the dried particles 40 at least 0.7 , preferably at least 0.75 , at least 0.8 , at least 0.85 ,
do not reach the drying air temperature , which is particularly at least 0.9 , at least 0.95 or 1. Preferably, the atomizer
advantageous if heat sensitive material is dried . generates a plurality of droplets comprising at least one
In a preferred embodiment, a pressure nozzle is used as droplet with a sphericity in the range from 0.7 to 1 , more
atomizer. Preferably, a pressure nozzle is used , which com- preferably in the range from 0.8 to 1 , from 0.85 to 1 , or from
prises a swirl chamber, causing the liquid passing through 45 0.9 to 1 .
them to rotate . Preferably, a pressure nozzle is used as It is further preferred that the average sphericity of the
atomizer, wherein the nozzle pressure is preferably not droplets, which are formed by the atomizer, is at least 0.7 ,
higher than about 1 bar, more preferably not higher than preferably at least 0.75 , at least 0.8 , at least 0.85 , at least 0.9 ,
about 0.7 bar, not higher than about 0.5 bar or not higher at least 0.95 or 1. Preferably, the average sphericity of the
than about 0.3 bar. Preferably, the nozzle pressure is in a 50 droplets, which are formed by the atomizer, is in the range
range from about 1 to about 0.1 bar, more preferably in a from 0.7 to 1 , more preferably in the range from 0.8 to 1 ,
range from about 0.7 to about 0.3 bar. In a particularly from 0.85 to 1 , or from 0.9 to 1 .
preferred embodiment, the nozzle pressure is not higher than Alternatively, the sphericity of those droplets that have a
about about 0.3 bar. particle size ( i.e. droplet size) equal to Dv50 in the droplet
In the context of the present invention , an atomizer is 55 size distribution as defined herein is at least 0.7 , preferably
preferably used that produces droplets , which are preferably at least 0.75 , at least 0.8 , at least 0.85 , at least 0.9 , at least
characterized by a mass median aerodynamic diameter 0.95 or 1. Preferably , the sphericity of those droplets that
(MMAD ), preferably as defined herein , of at least 0.3 um . have a particle size equal to Dv50 as defined herein is in the
Alternatively, the MMAD of the droplets according to the range from 0.7 to 1 , more preferably in the range from 0.8
invention is at least 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 60 to 1 , from 0.85 to 1 , or from 0.9 to 1. Even more preferably,
.

30 , 35 , 40 , 45 or 50 um . Preferably, the MMAD of the those droplets that have a particle size equal to Dv90 as
droplets is equal to or less than 1,500 , 1,250 , 1,000 , 750 , defined herein have a sphericity of at least 0.7 , preferably of
600 , 500 , 400 , 300 , 200 or 100 um . Further preferably, the at least 0.75 , at least 0.8 , at least 0.85 , at least 0.9 , at least
MMAD of the droplets may be in a range from 0.5 um to 0.95 or 1. Preferably, the sphericity of those droplets that
2,000 um , from 1 um to 1,000 um , from 2 um to 500 um or 65 have a particle size equal to Dv90 as defined herein is in the
from 2 um to 200 um . In a preferred embodiment, the range from 0.7 to 1 , more preferably in the range from 0.8
MMAD of the droplets is at least 1 um or in the range from to 1 , from 0.85 to 1 , or from 0.9 to 1 .
 US 11,179,337 B2
37 38
Further preferably, the average sphericity of those drop- be used in the method according to the invention . In a
lets that have a particle size equal to or lower than Dv50 as preferred embodiment the spray - drying device is set up as a
defined herein is at least 0.7 , preferably at least 0.75 , at least co - current flow device ( spray and drying gas move into the
0.8 , at least 0.85 , at least 0.9 , at least 0.95 or 1. Preferably, same directions ), as a counter -current flow device ( spray and
the average sphericity of those droplets that have a particle 5 drying gas move into opposite directions ) or as a mixed flow
size equal to or lower than Dv50 as defined herein is in the device ( co -current and counter - current flow combined ). In a
range from 0.7 to 1 , more preferably in the range from 0.8 particularly preferred embodiment, the spray -drying device
to 1 , from 0.85 to 1 , or from 0.9 to 1. Even more preferably, is a co - current flow device .
the average sphericity of those droplets that have a particle Moreover, common spray -drying devices may be catego
size equal to or lower than Dv90 as defined herein is at least 10 rized depending on the type of air cycle that is used . A
0.7 , preferably of at least 0.75 , at least 0.8 , at least 0.85 , at spray -drying device as used herein is preferably an open
least 0.9,at least 0.95 or 1. Preferably, the average sphericity cycle device (drying air that enters the system through the
of those droplets that have a particle size equal to or lower inlet is exhausted through the outlet into the atmosphere ) or
than Dv90 as defined herein is in the range from 0.7 to 1 , a closed cycle device ( drying air that enters the system
more preferably in the range from 0.8 to 1 , from 0.85 to 1 , 15 through the inlet , exits the system via the outlet and is
or from 0.9 to 1 . reused ). A closed cycle device is preferably employed, if a
In a preferred embodiment, the droplets formed by the drying gas is used that should not accumulate in the facility,
atomizer are dried by evaporation of the solvent from the where the device is located ( such as nitrogen or CO2 ) , and / or
droplets . Typically, this takes place in a drying chamber, if toxic substances are released from the drying material.
which may be of any shape and which may consist of one or 20 The spray -drying device preferably reduces the residual
more chambers. Preferably, the droplets are contacted with moisture content of the composition to the desired level ,
a gas stream that is preferably capable of absorbing, at least preferably as defined herein , in one pass through the system .
partially, the solvent that evaporates from the droplets. The If the moisture content of the product after one cycle is
gas stream is preferably introduced into the drying chamber higher than desired , the moisture content of the powder may
via an inlet , such as a disperser, which is preferably located 25 be further reduced by a second drying stage ( or several of
in the upper half of the drying chamber, more preferably in those ) until the desired residual moisture content of the
the vicinity of the atomizer, thus allowing rapid mixing of product is achieved .
the drying gas and the droplets. The gas stream leaves the An example of a spray - drying apparatus is shown in FIG .
drying chamber through an outlet, which is preferably 1 , which further illustrates the principle of spray -drying .
located at the bottom of the drying chamber. In a preferred 30 Liquid input stream (A) is sprayed through a nozzle (3 ) into
embodiment, the drying chamber comprises a cone - shaped a hot vapor stream ( 1 , 2 ) and is vaporized ( 4 ) . Upon
part, wherein the tip of the cone comprises the outlet, introduction into the hot air stream the droplets are cooled
preferably for the drying gas as well as for dried particles. down due to the evaporation of water or a chemical solvent
Preferably, the characteristics of the drying chamber are from the concentrate . Solid particles form , while moisture
matched with , amongst others, the atomizer that is used . In 35 quickly leaves the droplets. In the example illustrated in
order to ensure uniform product quality, the droplets pref- FIG . 3 , a nozzle is used in order to achieve a sufficiently
erably contact a surface only when they are sufficiently dry . small droplet size ( atomizer ) and in order to maximize heat
Spray- drying devices that use centrifugal atomizer typically transfer and the rate of water /solvent evaporation. A co
require relatively larger diameter vessels , but less cylinder current flow of hot drying gas is used in the exemplary
height for optimal drying. Spray -drying devices that use 40 device shown in FIG . 3. Particles are further dried and
pressure nozzles usually require relatively small diameters separated in a cyclone device ( 6 ) . The dry particles are
with larger cylinder height for sufficient drying. The dry cooled and collected , ready for packaging in different for
powder composition is preferably collected at the bottom of mats ( 8) .
the drying chamber that is preferably designed as a cone . In The final product is collected as described above and is
the center of the cone area , the outlet of the gas - stream is 45 preferably in the form of a dry powder comprising the
preferably positioned , where cool and moist air is removed particles as defined herein with respect to the inventive dry
from the drying chamber. Such a design of the cone and powder composition .
outlet is acting as a cyclone separator and leads to an The spray -drying process , in the context of the present
accumulation of the dry powder composition at the bottom invention , may be carried out using any suitable spray
of the drying chamber. Cyclonic separation is preferably 50 drying device known in the art. Examples of commercially
used to separate dry particles or fine droplets from the drying available devices that may be employed include , but are not
gas , preferably without the use of filters , through vortex limited to the following examples: Mini Spray Dryer B - 290
separation . To this end, a high speed rotating flow is pref- ( Buchi); Nano Spray Dryer B - 90 ( Buchi ) ; Anhydro
erably established within a cylindrical or conical container, MicraSpray Dryer GMP ( SPX.com) ; Anhydro MicraSpray
the cyclone . Typically, drying gas flows in aa helical pattern , 55 Dryer Aseptic series ( SPX.com) ; MDL - 50 series; B , C , S , M
from the top (wide end) of the cyclone to the bottom sub - types. ( fujisaki electric ); MDL - 015 ( C ) MGC lab - scale .
(narrow ) end before exiting the cyclone in a straight stream ( fujisaki electric ); MDL - 050 (C ) MGC lab - scale . ( fujisaki
through the center of the cyclone and out the top . Larger or electric ); LSD - 1500 Mini spray dryer ( cndryer.com );
denser particles in the rotating stream do not follow the tight MSD -8 Multi - functional laboratory spray dryer ( cndryer.
curve of the stream , but strike the outside wall and fall to the 60 com) ; PSD - 12 Precision pharmacy spray dryer ( cndryer.
bottom of the cyclone, where they can be collected . Alter- com) ; PSD - 12 Precision pharmacy spray dryer (cndryer .
natively, a filter, e.g. a bag filter or a combination of a com ) ; TALL FORM DRYERTM TFD ( GEA Process Engi
cyclone separator and a filter may be used for separation . neering ); COMPACT DRYERTM_CD (GEA Process Engi
Depending on the type of flow , i.e. the relative positions neering); Multi - Stage Dryer - MSDTM (GEA Process Engi
of atomizer and drying gas inlet or, respectively, the relative 65 neering ); FILTERMATTM Spray Dryer - FMD (GEA
movement of the spray and the drying gas , several types of Process Engineering ); SDMICROTM (GEA Process Engi
spray -drying devices may be distinguished , all of which may neering ), MOBILE MINORTM (GEA Process Engineering );
 US 11,179,337 B2
39 40
PRODUCTION MINORTM (GEA Process Engineering ); spray -drying process ( for example, Toutle > 60 ° C. ) would
VERSATILE - SDTM (GEA Process Engineering ); FSDTM result in degradation of long - chain RNA . This allows the
Fluidized Spray Dryer (GEA Process Engineering ); Stan- preparation of a particulate, storage - stable form of long
dard GEA Niro PHARMASDTM spray dryers (GEA Process chain RNA , which advantageously has an outstandingly low
Engineering ); R& D Spray Dryer - SDMICROTM ( GEA Pro- 5 residual moisture content that allows for long -term storage
cess Engineering ). without degradation .
In the spray -drying process according to the invention , the One particular advantage of the inventive dry powder
drying gas may be any suitable gas or mixture of gases , such composition
as air. Preferably, an inert gas is used as drying gas , for composition and the inventive method is that a dry powder
example nitrogen , nitrogen - enriched air, helium or argon . 10 ages useful for shipping,, which
is provided can be divided into pack
storage and use as medicament.
The spray -drying process is influenced to a considerable Furthermore dry powder formation
degree by the temperature of the drying gas . That tempera represents a cost- and time effective ofprocess long - chain RNA
, which can
ture is typically characterized by two parameters, i.e. the readily be scaled -up for commercial production . In this
inlet temperature ( Tiniet) and the outlet temperature ( Toutlet).
As used herein, the term ‘ inlet temperature' refers to the 15 context , it is particularly advantageous that spray drying can
drying gas temperature as measured at the drying gas inlet be carried out as a continuous process . One advantage of a
of the drying chamber as described herein . Analogously, the continuous process is that the product produced in one run
term “outlet temperature ' refers to the drying gas tempera has the same properties, therefore reducing the amount of
ture as measured at the drying gas outlet of the drying required quality controls .
chamber as described herein . 20 Preferably, the residual moisture content of the dry pow
For heat sensitive material, the inlet temperature is pref- der composition obtained by the method according to the
erably chosen sufficiently high in order to allow rapid and invention is as defined above with regard to the inventive dry
efficient drying , while at the same time avoiding degradation powder composition .
of the material. If heat sensitive material is dried , the More preferably, the relative integrity and the biological
residence time of the material in the drying chamber is 25 activity of the long - chain RNA molecule in the dry powder
preferably increased, e.g. by using larger drying chambers , composition obtained by using the inventive method is
which allows operating at lower temperatures. preferably as defined above for the inventive dry powder
In a preferred embodiment, the outlet temperature is at composition comprising a long - chain RNA molecule .
least about 65 ° C. , at least about 66 ° C. , at least about 67 ° The inventive method thus provides long - chain RNA as
C. , at least about 68 ° C. , at least about 69 ° C. , at least about 30 defined herein in a particulate formulation . In a particularly
)

70 ° C. , at least about 71 ° C. , at least about 72 ° C. , at least preferred embodiment, the particles comprised in the dry
about 73 ° C. , at least about 74 ° C. , at least about 75 ° C. , at powder composition obtained by the inventive method are
least about 76 ° C. , at least about 77 ° C. , at least about 78 ° characterized by a size distr utic which is preferably as
C. , at least about 79 ° C. , at least about 80 ° C. , at least about defined herein for the particles of the inventive dry powder
85 ° C. , at least about 86 ° C. , at least about 87 ° C. , at least 35 composition. In a particularly preferred embodiment, the
about 88 ° C. , at least about 89 ° C. , at least about 90 ° C. , at product, which is obtained from the method according to the
least about 91 ° C. , at least about 92 ° C. , at least about 93 ° invention , is the inventive dry powder composition as
C. , at least about 94 ° C. , at least about 95 ° C. , at least about described herein .
96 ° C. , at least about 97 ° C. , at least about 98 ° C. , at least In one aspect , the invention concerns a particle, or a
about 99 ° C. , at least about 100 ° C. , at least about 101 ° C. , 40 plurality of particles, comprising a long - chain RNA mol
at least about 102 ° C. , at least about 103 ° C. , at least about ecule , which is preferably obtainable by the inventive
104 ° C. , at least about 105 ° C. , at least about 106 ° C. , at least method . Furthermore , the invention is directed to a dry
about 107 ° C. , at least about 108 ° C. , at least about 109 ° C. , powder composition comprising a long - chain RNA mol
or at least about 110 ° C. ecule , which is obtainable by the inventive method as
According to a particularly preferred embodiment, the 45 defined herein .
outlet temperature is at least about 65 ° C. Alternatively, the In a preferred embodiment, the inventive dry powder
outlet temperature is at least about 69 ° C. More preferably, composition , the particle obtainable by the inventive method
the outlet temperature is at least about 70 ° C. , most prefer- or the dry powder composition obtainable by the inventive
ably at least about 71 ° C. or at least about 72 ° C. method are packaged in single dosages after the drying
Typically , higher temperatures increase the efficiency of 50 process is completed. Alternatively, the method may further
the spray -drying process. The disadvantage of higher tem- comprise purification or selection steps, for example in order
peratures may be , however, an increased risk of degradation to separate particles of a certain size or shape .
depending on the active ingredient or the excipients. Advantageously, the inventive dry powder composition ,
Another caveat with spray -drying in general are shear forces the particle obtainable by the inventive method or the dry
that are acting on the liquid to be dried , in particular during 55 powder composition obtainable by the inventive method
atomization . The shear stress is typically expected to be may also be extended at any stage after the production
particularly detrimental to larger molecules . Before the process per se is finished . For instance, an excipient, pref
present invention, it was therefore altogether unexpected erably as described herein , may be added to the inventive
that a long - chain RNA molecule may be dried by spray- dry powder composition or to the product of the inventive
drying, while the long -chain RNA molecule’s integrity and 60 method , respectively. In that manner , the product of the
biological activity is retained notwithstanding the mechani- inventive method provides considerable flexibility and
cal stress involved in the process . Even more surprisingly, it allows extension of weight/volume (e.g. for better handling )
has been found by the inventors of the present invention that as well as combination with other active ingredients and
the long - chain RNA molecule is not degraded, even when excipients. For example, a suitable excipient, preferably as
relatively high temperatures, in particular outlet tempera- 65 defined herein , such as a carbohydrate, may be added , for
tures, are used . In contrast, the skilled person would have example insulin, starch or trehalose . Preferably, the excipi
expected that high outlet temperatures (Toutlet) used in the ent, which is added to inventive dry powder composition or
 US 11,179,337 B2
41 42
to the particles or dry powder composition obtained by the sition , water or preferably a buffer, more preferably an
inventive method , is characterized by a low osmolarity. aqueous buffer, may be used , containing a sodium salt ,
If the inventive dry powder composition , the particles or preferably at least 50 mM of a sodium salt , a calcium salt ,
the dry powder composition obtainable by the inventive preferably at least 0.01 mM of a calcium salt , and optionally
method is used in the manufacture of a pharmaceutical 5 a potassium salt , preferably at least 3 mM of a potassium
composition , the powder or the particles can easily be salt . According to a preferred aspect, the sodium , calcium
further processed to other dosage forms, such as tablets, and, optionally, potassium salts may occur in the form of
capsules, granules or the like . their halogenides , e.g. chlorides, iodides, or bromides , in the
In a further aspect , the present invention provides a form of their hydroxides, carbonates, hydrogen carbonates,
pharmaceutical composition, comprising or consisting of the 10 or sulfates, etc. Without being limited thereto , examples of
inventive dry powder composition, the particles as obtain- sodium salts include e.g. NaCl , Nal , NaBr, Na2CO3 ,
able by the inventive method or the dry powder composition NaHCO3 , Na2SO4, examples of the optional potassium salts
obtainable by the inventive method . In a preferred embodi- include e.g. KCI , KI , KBr, K2CO3, KHCO3 , K2SO4, and
ment, the inventive dry powder composition, the particles as examples of calcium salts include e.g. CaCl2 , Calz , CaBrz,
obtainable by the inventive method or the dry powder 15 CaCO3 , CaSO4 , Ca (OH ) 2 . Furthermore, organic anions of
composition obtainable by the inventive method are phar- the aforementioned cations may be contained in the buffer .
maceutical compositions. Alternatively, the inventive phar- According to a more preferred aspect , the buffer suitable for
maceutical composition comprises the inventive dry powder injection purposes as defined above, may contain salts
composition , the particles as obtainable by the inventive selected from sodium chloride (NaCl), calcium chloride
method or the dry powder composition obtainable by the 20 (CaCl2 ) and optionally potassium chloride (KCI ) , wherein
inventive method and optionally a pharmaceutically accept- further anions may be present additional to the chlorides .
able carrier and / or vehicle . The inventive pharmaceutical CaCl2 can also be replaced by another salt like KCl . Typi
composition may optionally be supplemented with further cally, the salts in the injection buffer are present in a
components as defined above for the inventive dry powder concentration of at least 50 mM sodium chloride (NaCl ) , at
composition or for the inventive method . The inventive 25 least 3 mM potassium chloride (KCl ) and at least 0.01 mM
pharmaceutical composition may be prepared as a whole by calcium chloride ( CaCl2 ). The injection buffer may be
the inventive method . hypertonic, isotonic or hypotonic with reference to the
As a first ingredient, the inventive pharmaceutical com- specific reference medium , i.e. the buffer may have a higher,
position comprises the long - chain RNA in particulate form identical or lower salt content with reference to the specific
as defined herein . In particular, the first ingredient of the 30 reference medium , wherein preferably such concentrations
inventive pharmaceutical composition is the inventive dry of the afore mentioned salts may be used , which do not lead
powder composition , the particles as obtainable by the to damage of cells due to osmosis or other concentration
inventive method or the dry powder composition obtainable effects. Reference media are e.g. liquids occurring in “ in
by the inventive method , as defined above . Preferably, the vivo " methods, such as blood, lymph , cytosolic liquids, or
long -chain RNA molecule as defined herein represents a 35 other body liquids, or e.g. liquids, which may be used as
pharmaceutically active ingredient of the pharmaceutical reference media in “ in vitro ” methods, such as common
composition. buffers or liquids. Such common buffers or liquids are
As a second ingredient the inventive pharmaceutical known to a skilled person and may be as defined above .
composition may comprise another class of compounds, However, one or more compatible solid or liquid fillers or
which may be added to the inventive pharmaceutical com- 40 diluents or encapsulating compounds may be used as well
position in this context, may be selected from at least one for the inventive pharmaceutical composition , which are
pharmaceutically active component. A pharmaceutically suitable for administration to a patient to be treated . The
active component in this context is a compound that has a term “ compatible” as used here means that these constitu
therapeutic effect against a particular medical indication , ents of the inventive pharmaceutical composition are
preferably cancer diseases , autoimmune disease , allergies, 45 capable of being mixed with the dry powder composition or
infectious diseases or a further disease as defined herein . the particles as defined herein in such a manner that no
Such compounds include, without implying any limitation , interaction occurs , which would substantially reduce the
preferably compounds including, without implying any limi- pharmaceutical effectiveness of the inventive pharmaceuti
tation, peptides or proteins ( e.g. as defined herein ), nucleic cal composition under typical use conditions . Pharmaceuti
acid molecules , ( therapeutically active) low molecular 50 cally acceptable carriers, fillers and diluents must, of course ,
weight organic or inorganic compounds (molecular weight have sufficiently high purity and sufficiently low toxicity to
less than 5,000 , preferably less than 1,000) , sugars , antigens make them suitable for administration to a person to be
or antibodies (e.g. as defined herein ), therapeutic agents treated . Some examples of compounds, which can be used as
already known in the prior art, antigenic cells , antigenic pharmaceutically acceptable carriers, fillers or constituents
cellular fragments, cellular fractions; modified , attenuated or 55 thereof are sugars , such as , for example , lactose , glucose and
de - activated ( e.g. chemically or by irridation ) pathogens sucrose ; starches, such as , for example, corn starch or potato
( virus, bacteria etc. ) , etc. starch; cellulose and its derivatives, such as , for example ,
Furthermore , the inventive pharmaceutical composition sodium carboxymethylcellulose, ethylcellulose, cellulose
may comprise a pharmaceutically acceptable carrier and / or acetate ; powdered tragacanth ; malt ; gelatin ; tallow ; solid
vehicle. In the context of the present invention , a pharma- 60 glidants, such as , for example, stearic acid, magnesium
ceutically acceptable carrier typically includes the liquid or stearate; calcium sulfate; vegetable oils , such as , for
non - liquid basis of the inventive pharmaceutical composi- example , groundnut oil ,cottonseed oil , sesame oil , olive oil ,
tion . If the inventive pharmaceutical composition is pro- corn oil and oil from theobroma; polyols , such as , for
vided in liquid form , the carrier will typically be pyrogen- example , polypropylene glycol, glycerol, sorbitol , mannitol
free water; isotonic saline or buffered ( aqueous) solutions , 65 and polyethylene glycol ; alginic acid .
e.g phosphate, citrate etc. buffered solutions . Particularly for The inventive pharmaceutical composition may be
injection of the inventive inventive pharmaceutical compo- administered orally, parenterally, by inhalation , topically,
 US 11,179,337 B2
43 44
rectally, nasally, buccally, vaginally or via an implanted oxypropylene compound , emulsifying wax and water . Alter
reservoir. The term parenteral as used herein includes sub- natively, the inventive pharmaceutical composition can be
cutaneous, intravenous, intramuscular, intra - articular, intra- formulated in a suitable lotion or cream . In the context of the
synovial, intrasternal, intrathecal, intrahepatic, intralesional, present invention , suitable carriers include , but are not
intracranial, transdermal, intradermal, intrapulmonal, intra- 5 limited to , mineral oil , sorbitan monostearate , polysorbate
peritoneal, intracardial, intraarterial, and sublingual injec- 60 , cetyl esters wax , cetearyl alcohol, 2 -octyldodecanol,
tion or infusion techniques. benzyl alcohol and water.
Preferably, the inventive pharmaceutical composition In a particularly preferred embodiment, the inventive
may be administered by parenteral injection , more prefer- pharmaceutical composition, preferably as an aerosolizable
ably by subcutaneous , intravenous, intramuscular, intra- 10 formulation , is for mucosal, intranasal, inhalation or pulmo
articular, intra - synovial, intrasternal, intrathecal, intrahe- nary delivery . In a preferred embodiment, the pharmaceuti
patic , intralesional, intracranial, transdermal, intradermal, cal composition comprises the powder or the particles,
intrapulmonal, intraperitoneal, intracardial, intraarterial, and preferably as defined herein, which are respirable, i.e. which
sublingual injection or via infusion techniques. Sterile can readily be dispersed in air or in a gas ( e.g. by using an
injectable forms of the inventive pharmaceutical composi- 15 inhalation device) and inhaled by a subject. Preferably, the
tions may be aqueous or oleaginous suspension . These particles of the pharmaceutical composition are as defined
suspensions may be formulated according to techniques herein with respect to the inventive dry powder composition
known in the art using suitable dispersing or wetting agents so that at least a portion of the aerosolized particles reaches
and suspending agents. The sterile injectable preparation the lungs . In a preferred embodiment, the pharmaceutical
may also be a sterile injectable solution or suspension in a 20 composition according to the invention comprises the inven
non- toxic parenterally -acceptable diluent or solvent, for tive dry powder composition, wherein the dry powder com
example as a solution in 1,3 - butanediol. Among the accept- position comprises particles that have aa MMAD of 10 um or
able vehicles and solvents that may be employed are water, less .
Ringer's solution and isotonic sodium chloride solution . In The inventive pharmaceutical composition typically com
addition, sterile, fixed oils are conventionally employed as a 25 prises a “ safe and effective amount ” of the components of
solvent or suspending medium . For this purpose , any bland the inventive pharmaceutical composition as defined above,
fixed oil may be employed including synthetic mono- or particularly of long - chain RNA molecule as comprised in
di -glycerides. Fatty acids , such as oleic acid and its glycer- the inventive dry powder composition or in the particles
ide derivatives are useful in the preparation of injectables, as obtainable by the inventive method . As used herein , a “ safe
are natural pharmaceutically -acceptable oils , such as olive 30 and effective amount ” means an amount of the long - chain
oil or castor oil, especially in their polyoxyethylated ver- RNA molecule that is sufficient to significantly induce a
sions . These oil solutions or suspensions may also contain a positive modification of a disease or disorder as defined
long - chain alcohol diluent or dispersant, such as carboxym- herein . At the same time , however, a " safe and effective
ethyl cellulose or similar dispersing agents that are com- amount” is small enough to avoid serious side -effects, that
monly used in the formulation of pharmaceutically accept- 35 is to say to permit a sensible relationship between advantage
able dosage forms including emulsions and suspensions. and risk . The determination of these limits typically lies
Other commonly used surfactants, such as Tweens, Spans within the scope of sensible medical judgment. A " safe and
and other emulsifying agents or bioavailability enhancers effective amount " of the components of the inventive phar
which are commonly used in the manufacture of pharma- maceutical composition , particularly of the long - chain RNA
ceutically acceptable solid, liquid , or other dosage forms 40 molecule will furthermore vary in connection with the
may also be used for the purposes of formulation of the particular condition to be treated and also with the age and
inventive pharmaceutical composition . physical condition of the patient to be treated, the body
The inventive pharmaceutical composition as defined weight, general health , sex , diet , time of administration , rate
above may also be administered orally in any orally accept- of excretion , drug combination, the activity of the specific
able dosage form including, but not limited to , capsules, 45 (lyophilized) nucleic acid ( sequence ) employed, the severity
tablets , aqueous suspensions or solutions . In the case of of the condition, the duration of the treatment, the nature of
tablets for oral use , carriers commonly used include lactose the accompanying therapy, of the particular pharmaceuti
and corn starch. Lubricating agents, such as magnesium cally acceptable carrier used, and similar factors, within the
stearate , are also typically added . For oral administration in knowledge and experience of the accompanying doctor. The
a capsule form , useful diluents include lactose and dried 50 inventive pharmaceutical composition may be used for
cornstarch . When aqueous suspensions are required for oral human and also for veterinary medical purposes, preferably
use , the active ingredient, i.e. the dry powder composition or for human medical purposes , as a pharmaceutical composi
the particles, as defined above , is combined with emulsify- tion in general or as a vaccine .
ing and suspending agents. If desired , certain sweetening, According to a specific aspect , the inventive dry powder
flavoring or coloring agents may also be added . 55 composition , the particles or the dry powder composition
The inventive pharmaceutical composition may also be obtainable by the inventive method or the inventive phar
administered topically, especially when the target of treat- maceutical composition may be provided as a vaccine. Such
ment includes areas or organs readily accessible by topical an inventive vaccine is typically composed like the inventive
application , e.g. including diseases of the skin or of any pharmaceutical composition, i.e. it contains a long -chain
other accessible epithelial tissue . Suitable topical formula- 60 RNA molecule formulated as defined above and optionally
tions are readily prepared for each of these areas or organs. a pharmaceutically acceptable carrier and /or vehicle. Further
For topical applications, the inventive pharmaceutical com- components may be as defined above for the inventive
position may be formulated in aa suitable ointment, contain- pharmaceutical composition . The inventive vaccine prefer
ing the components as defined above suspended or dissolved ably supports at least an innate immune response of the
in one or more carriers . Carriers for topical administration 65 immune system of a patient to be treated . Additionally , the
include , but are not limited to , mineral oil , liquid petrolatum , inventive vaccine furthermore may also elicit an adaptive
white petrolatum , propylene glycol , polyoxyethylene, poly- immune response , preferably, if the long -chain RNA mol
 US 11,179,337 B2
45 46
ecule of the inventive vaccine encodes any of the antigens TLR8 , TLR9 , TLR10 , or due to its binding affinity (as
( or antibodies ) mentioned herein , which elicit an adaptive ligands ) to murine Toll - like receptors TLR1 , TLR2 , TLR3 ,
immune response or any antigen as defined herein is added TLR4 , TLR5 , TLR6 , TLR7 , TLR8 , TLR9 , TLR10 , TLR11 ,
to the inventive vaccine , which can effectively induce an TLR12 or TLR13 .
adaptive immune response . 5 The present invention furthermore provides several appli
The inventive vaccine may also comprise a pharmaceu- cations and uses of the inventive dry powder composition ,
tically acceptable carrier, adjuvant, and / or vehicle as defined the particles obtainable by the inventive method or the dry
above for the inventive pharmaceutical composition. In the powder composition obtainable by the inventive method.
specific context of the inventive vaccine , the choice of a According to one aspect , the invention concerns the use of
pharmaceutically acceptable carrier is determined in prin- 10 the inventive dry powder composition , the particles obtain
ciple by the manner in which the inventive vaccine is able by the inventive method or the dry powder composition
administered . The inventive vaccine can be administered, obtainable by the inventive method for the preparation of a
for example, systemically or locally. Routes for systemic medicament for the prophylaxis, treatment and / or amelio
administration in general include, for example, transdermal, ration of a disorder or a disease , preferably as defined herein .
oral, parenteral routes, including subcutaneous , intravenous, 15 According to a further aspect , the present invention is
intramuscular, intraarterial, intradermal and intraperitoneal directed to the use of the long - chain RNA molecule in
injections and / or intranasal /intrapulmonal administration particulate form as defined herein in the treatment or pre
routes . Routes for local administration in general include, vention of a disease . Further, the invention concerns the use
for example , topical administration routes but also intrad- of the inventive dry powder composition, the particles
ermal, transdermal, subcutaneous , or intramuscular injec- 20 obtainable by the inventive method or the dry powder
tions or intralesional, intracranial, intrapulmonal, intracar- composition obtainable by the inventive method, in the
dial , and sublingual injections. More preferably, vaccines treatment or the prevention of a disease , preferably as
herein may be administered by an intradermal, subcutane- defined herein . In particular, the present invention concerns
ous , or intramuscular route . Inventive vaccines are therefore the first medical use of the inventive dry powder composi
preferably formulated in liquid (or sometimes in solid , e.g. 25 tion , the particles obtainable by the inventive method or the
as an aerosol) form . The suitable amount of the inventive dry powder composition obtainable by the inventive method
vaccine to be administered can be determined by routine as a medicament. The medicament may be in the form of a
experiments with animal models . Such models include , pharmaceutical composition or in the form of a vaccine as a
without implying any limitation , rabbit , sheep , mouse , rat , specific form of pharmaceutical compositions. A pharma
dog and non -human primate models . Preferred unit dose 30 ceutical composition in the context of the present invention
forms for injection include sterile solutions of water, physi- typically comprises or consists of the inventive dry powder
ological saline or mixtures thereof. The pH of such solutions composition, the particles obtainable by the inventive
should be adjusted to about 7.4 . Suitable carriers for injec- method or the dry powder composition obtainable by the
tion include hydrogels, devices for controlled or delayed inventive method as defined above , optionally further ingre
release , polylactic acid and collagen matrices. Suitable phar- 35 dients, preferably as defined above , and optionally a phar
maceutically acceptable carriers for topical application maceutically acceptable carrier and /or vehicle, preferably as
include those , which are suitable for use in lotions , creams, defined above .
gels and the like . If the inventive vaccine is to be adminis- According to a further aspect , the present invention con
tered orally, tablets , capsules and the like are the preferred cerns a method of treating or preventing a disorder or a
unit dose form . The pharmaceutically acceptable carriers for 40 disease by administering to a subject in need thereof a
the preparation of unit dose forms, which can be used for pharmaceutically effective amount, preferably as defined
oral administration , are well known in the prior art. The herein, of the inventive dry powder composition , the inven
choice thereof will depend on secondary considerations such tive pharmaceutical composition , or the inventive vaccine .
as taste , costs and storability, which are not critical for the Preferably , the method is for treating or preventing a disor
purposes of the present invention , and can be made without 45 der or a disease selected from cancer or tumor diseases ,
difficulty by a person skilled in the art. infectious diseases, preferably ( viral, bacterial or protozoo
Further additives which may be included in the inventive logical ) infectious diseases, autoimmune diseases , allergies
vaccine are emulsifiers, such as , for example, Tween® ; or allergic diseases , monogenetic diseases, i.e. ( hereditary )
wetting agents , such as , for example , sodium lauryl sulfate ; diseases , or genetic diseases in general, diseases which have
colouring agents; taste -imparting agents, pharmaceutical 50 a genetic inherited background and which are typically
carriers; tablet- forming agents; stabilizers; antioxidants; pre- caused by a single gene defect and are inherited according
servatives. to Mendel's laws , cardiovascular diseases, neuronal dis
According to a specific embodiment, the inventive vac- eases , or any further disease mentioned herein .
cine may comprise an adjuvant. In this context, an adjuvant According to one further aspect , the present invention is
may be understood as any compound, which is suitable to 55 directed to the use of the long - chain RNA molecule in
initiate or increase an immune response of the innate particulate form , preferably in the form of the inventive dry
immune system , i.e. a non -specific immune response . In powder composition , the particles obtainable by the inven
other terms, when administered , the inventive vaccine pref- tive method or the dry powder composition obtainable by
erably elicits an innate immune response due to the adjuvant, the inventive method , for the prophylaxis, treatment and / or
optionally contained therein. Preferably, such an adjuvant 60 amelioration of a disease or disorder as defined herein,
may be selected from an adjuvant known to a skilled person wherein the disease or disorder is preferably selected from
and suitable for the present case , i.e. supporting the induc- cancer or tumor diseases, infectious diseases , preferably
tion of an innate immune response in a mammal. ( viral, bacterial or protozoological) infectious diseases ,
In this context, the adjuvant is preferably selected from autoimmune diseases , allergies or allergic diseases , mono
compounds, which are known to be immune - stimulating due 65 genetic diseases, i.e. ( hereditary ) diseases , or genetic dis
to their binding affinity (as ligands ) to human Toll- like eases in general, diseases which have a genetic inherited
receptors TLR1 , TLR2, TLR3 , TLR4, TLR5 , TLR6 , TLR7 , background and which are typically caused by a single gene
 US 11,179,337 B2
47 48
defect and are inherited according to Mendel's laws , car- inventive pharmaceutical composition or the inventive vac
diovascular diseases , neuronal diseases , or any further dis- cine or any further liquid and / or buffer as described herein
ease mentioned herein . for lyophilization , transfection and / or injection. Without
According to another aspect , the present invention is being limited thereto , further ingredients of the kit may
directed to the second medical use of the long - chain RNA in 5 include components as defined above , e.g. ( solutions com
particulate form as defined herein , preferably in the form of prising) proteins , amino acids , alcohols , carbohydrates, met
the inventive dry powder composition , the particles obtain als or metal ions , surfactants , polymers or complexing
able by the inventive method or the dry powder composition agents, and / or buffers, preferably all as defined above . These
obtainable by the inventive method for the treatment of further ingredients may be contained in different parts of the
diseases
the as defined
preparation of aaherein , preferably
medicament to the
for the use thereof
prophylaxis for 10
, treat kit (or kitofparts ). The kit or kit of parts as describedabove
ment and / or amelioration of various diseases as defined may contain optionally technical instructions with informa
herein , preferably selected from cancer or tumor diseases , tion on the administration and dosage of the inventive
infectious diseases , preferably ( viral, bacterial or protozoo composition. Such a kit , preferably kit of parts, may be
logical ) infectious diseases, autoimmune diseases , allergies 15 applied , e.g. , for any of the above mentioned applications or
or allergic diseases, monogenetic diseases, i.e. (hereditary ) uses .

diseases , or genetic diseases in general, diseases which have BRIEF DESCRIPTION OF THE FIGURES
a genetic inherited background and which are typically
caused by a single gene defect and are inherited according
to Mendel's laws , cardiovascular diseases, neuronal dis- 20 The figures shown in the following are merely illustrative
eases , or any further disease mentioned herein . and shall describe the present invention in a further way.
The present invention also allows treatment of diseases , These figures shall not be construed to limit the present
which have not been inherited , or which may not be sum invention thereto .
marized under the above categories . Such diseases may FIG . 1 : Scheme of a co - current spray - drying apparatus A :
include e.g. the treatment of patients, which are in need of 25 solution or suspension to be dried inlet, B : atomization gas
a specific protein factor, e.g. a specific therapeutically active ( e.g. nitrogen ) inlet, 1 : drying gas ( e.g. nitrogen ) inlet, 2 :
protein as mentioned above . This may e.g. include dialysis heating of drying gas , 3 : spraying of solution or suspension ,
patients, e.g. patients, which undergo a (regular ) a kidney or 4 : drying chamber, 5 : part between drying chamber and
renal dialysis , and which may be in need of specific thera- cyclone , 6 : cyclone , 7 : drying gas outlet, 8 : product collec
peutically active proteins as defined above , e.g. erythropoi- 30 tion vessel .
etin (EPO ) , etc. FIG . 2 : Sequence of the mRNA used in this study ( R2564 ;
Likewise, diseases in the context of the present invention SEQ ID NO : 1 ) .
may include cardiovascular diseases chosen from , without FIG . 3 : Photograph of powder of protamine - formulated
being limited thereto , coronary heart disease, arteriosclero RNA ( T - SD1, T - SD2, T -SD3) spray -dried with outlet tem
sis , apoplexy and hypertension , etc. 35
peratures of 47 , 69 and 87 ° C. respectively and of placebo
Finally, diseases in the context of the present invention sample ( T -SD - P ) spray -dried at 84 ° C.
may be chosen from neuronal diseases including e.g. FIG . 4 : Residual water content of dry powder formula
Alzheimer's disease , amyotrophic lateral sclerosis, dystonia , tions obtained by spray -drying using different outlet tem
epilepsy, multiple sclerosis and Parkinson's disease etc.
According to aa further embodiment, the present invention 40 peratures .
FIG . 5 : X - ray powder diffraction analysis of protamine
also provides a kit, particularly as a kit of parts. Such a kit
of parts may contain e.g. the inventive dry powder compo formulated RNA ( T - SD1, T - SD2, T -SD3) spray -dried with
sition, the inventive pharmaceutical composition or the outlet temperatures of 47 , 69 and 87 ° C. respectively and
inventive vaccine as defined above , preferably divided into placebo sample ( T - SD - P ) spray -dried at 84 ° C.
different parts of the kit . As an example, the inventive 45 FIG . 6 : Particle size distributions of protamine -formu
pharmaceutical composition or the inventive vaccine may be lated RNA ( T -SD1, T -SD2, T -SD3) spray -dried with outlet
prepared as a kit of parts, e.g. by incorporating into one or temperatures of 47 , 69 and 87 ° C. respectively and placebo
more parts of the kit ( all or at least some components of) the sample ( T -SD - P ) spray -dried at 84 ° C. as determined by
inventive pharmaceutical composition or the inventive vac- laser diffraction analysis.
cine as described herein ( whereby at least the long - chain 50 FIGS . 7A - D : Scanning electron microscope ( SEM )
RNA in particulate form is included ), or the inventive dry images of protamine - formulated RNA powder particles
powder composition as such , as a dry formulation , i.e. ( T - SD - 1 ( FIG . 7A , 5100x) , T -SD - 2 ( FIG . 7B , 4950x) ,
devoid of any liquid component, and in at least one further T - SD - 3 ( FIG . 7C , 2080x ) and placebo powder particles
separate part of the kit a solvent and /or a buffer as described ( T -SD - P, FIG . 7D , 2660x ).
herein with respect to the liquid provided in step a) of the 55 FIG . 8 : Particle size distribution of protamine- formulated
inventive method, the inventive pharmaceutical composition RNA before ( TO) and after ( T -SD 1 , T -SD 2 , T - SD 3 )
or the inventive vaccine or any further solvent and /or buffer spray -drying with outlet temperatures of 47 , 69 and 87 ° C.
as described herein for lyophilization, transfection and / or respectively and particle size of spray -dried placebo sample
injection. Alternatively, the inventive pharmaceutical com- ( T -SD - P ) with an outlet temperature of 84 ° C. The particle
position or the inventive vaccine may be prepared as a kit of 60 size was determined by dynamic light scattering ( DLS ) .
parts, e.g. by incorporating into one or more parts of the kit FIG . 9 : Particle size distribution of protamine - formulated
only the inventive dry powder composition, the particles RNA before ( TO) and after ( T -SD 1 , T -SD 2 , T -SD 3 )
obtainable by the inventive method, or the dry powder spray -drying with outlet temperatures of 47 , 69 and 87 ° C.
composition obtainable by the inventive method, as respectively and particle size of spray -dried placebo sample
described herein , and in at least one further separate part of 65 ( T -SD - P ) with an outlet temperature of 84 ° C. The particle
the kit a solvent and /or a buffer as described herein for the size was determined by nanoparticle tracking analysis
liquid provided in step a) of the inventive method , for the (NTA ).
 US 11,179,337 B2
49 50
EXAMPLES mary, three spray - drying experiments were performed at
different outlet temperatures using the protamine -formulated
The Examples shown in the following are merely illus- RNA prepared according to Example 3. As a control, the
trative and shall describe the present invention in a further placebo formulation as described in Example 3 was pro
way. These Examples shall not be construed to limit the 5 cessed in parallel .
present invention thereto . Protamine - formulated RNA ( Example 3 ) or placebo
sample was thawed and each aliquot was homogenized by
Example 1 gentle mixing using a magnetic stirrer before spray -drying.
10
Spray -drying of protamine - formulated RNA and placebo
Preparation of DNA and RNA Constructs formulation was carried out using a Büchi Mini Spray - Dryer
B - 290 equipped with a two - fluid nozzle and a high perfor
A vector for in vitro transcription was constructed con mance cyclone . The spray -dryer was operated using nitro
taining a 17 promoter followed by a GC - enriched sequence gen as drying gas in aa closed cycle mode . The spray -drying
encoding the hemagglutinin ( HA ) protein of influenza A
virus (A /Netherlands/602/2009(H1N1) and used for subse- 15 experiments were1 . carried out under the process parameters
listed in Table
quent in vitro transcription reactions . According to a first
preparation, the DNA sequence coding for the above men TABLE 1
tioned mRNA was prepared. The constructs R2564 ( SEQ ID
NO : 1 ) was prepared by introducing a 5 ' - TOP -UTR derived 20
Process parameters spray -drying
from the ribosomal protein 32L4 , modifying the wild type Process T - SD1 T - SD2 T - SD3
coding sequence by introducing a GC - optimized sequence parameter ( verum ) ( verum ) ( verum ) T -SP - P
for stabilization , followed by a stabilizing sequence derived
from the albumin - 3 ' -UTR , a stretch of 64 adenosines (poly Nozzle type two - fluid
nozzle
two - fluid
nozzle
two - fluid
nozzle
two - fluid
nozzle
( A ) -sequence ), a stretch of 30 cytosines ( poly ( C ) -sequence ), 25 Atomization 30 mm 30 mm I 30 mm + 30 mm =
and aa histone stem loop . In SEQ ID NO : 1 ( see FIG . 2 ) and gas flow 5 mm 5 mm 5 mm 5 mm
the sequence of the corresponding mRNA is shown. setting * (~ 357 l/ h ) (~ 357 l/h ) (~ 357 1/h ) (~ 357 l / h )
(theoretical
Example 2 Inlet
volume flow )
65 ° C. 100 ° C. 128 ° C. 128 ° C.
temperature
In Vitro Transcription and Purification of RNA 30
Outlet 47 ° C. 69 ° C. 87 ° C. 84 ° C.
temperature
The respective DNA plasmids prepared according to Drying gas
Drying gas
nitrogen
100 %
nitrogen
100 %
nitrogen
100 %
nitrogen
100 %
section 1 above were transcribed in vitro using 17 poly rate
merase . The in vitro transcription of influenza HA encoding Pump/aspirator
speed
( ~ 35 m3/h ) (~35 m3/h) ( ~ 35 m3/h ) (~ 35 m /h )
3% 3% 3% 3%
R2564 was performed in the presence of a CAP analog 35 Yield [g] (~ 1 ml/min ) ( ~ 1 ml/min ) (~ 1 ml/min ) (~ 1 ml/min )
( m7GpppG ) . Subsequently the RNA was purified using Calculated 44.2
0.788 0.915
51.4
1.017
56.5
0.909 *
48.3 *
PureMessenger® ( CureVac, Tubingen , Germany; WO2008 / relative
077592A1). yield [ % ]
Example 3 40 0.23
* Atomization
bar
gas flow setting of 30 mm correlates with a pressure drop at the nozzle of

Preparation of Protamine - Formulated RNA Following spray -drying, the produced powders were filled
into vials ( see section 5 ) and characterized ( see section 6 ) .
RNA was diluted ( 0.87 g/L RNA final concentration ) and 5. Powder Filling
a protamine /trehalose mixture was prepared ( 43000 anti- 45 The powder obtained after spray -drying was collected in
heparin IU / L protamine; 10.87 % trehalose in water for a glass container at the product outlet of the cyclone . For
injection ). One volume unit of each solution was mixed to storage , shipment and further analysis the powder was
yield a ratio of protamine to RNA of 50 anti -heparin IU per divided into aliquots and transferred into 10 R vials under
mg RNA. controlled humidity conditions ( < 15 % RH ) in a glove box .
The solution of RNA /protamine complexes were supple- 50 Vials were stoppered inside the glove box. The exact weight
mented with R2564 to yield final concentrations of 0.4 g/L of the dry powder was documented during filling. FIG . 3
RNA complexed with 20000 anti- heparin IU / L of protamine shows a photograph of glass vials containing the spray -dried
( corresponding to a protamine concentration of about 1.5 powder.
g/L ) , 0.4 g / L free RNA and 5 % trehalose ( w / w ). 6. Analytical Characterization
Such formulated RNA was used for spray -drying experi- 55 A sampling scheme, including the sampling time points
ments . and analytical methods is shown in Table 2 .
As a placebo, 5 % trehalose was prepared in water for
injection . TABLE 2
Example 4 60 sampling time points for samples from spray -drying
Time
Spray -Drying of Protamine -Formulated RNA and point Description analytical methods
Placebo Formulation TO Liquid verum sample before DLS , NTA , MFI , turbidity,
TO(verum
- P ) spray - drying. ZP
The objective of the experiments presented in this section 65 Liquid placebo formulation before DLS , NTA , MFI , turbidity
was to test the feasibility of large scale production of the spray - drying
inventive dry powder composition by spray - drying. In sum
 US 11,179,337 B2
51 52
TABLE 2 - continued 6.1.2 Differential Scanning Calorimetry ( DSC)
sampling time points for samples from spray- drying Differential scanning calorimetry (DSC ) in a Mettler
Toledo 821e (Mettler Toledo, Giessen , Germany ) was used
Time
point Description 5 to determine thermal events of the spray -dried samples (e.g.
analytical methods glass transition temperature ( Tg) , crystallization or endo
T - SDX Spray - dried verum sample obtained DLS , ZP, NTA , MFI , thermal melting ) . Approximately 10 mg of the spray -dried
from experiment T - SD1, T -SD2, turbidity Karl -Fischer
T -SD3, T -SD4 titration, DSC , XRD ,
samples were analyzed in crimped Al - crucibles (Mettler
laser diffraction Toledo, Giessen , Germany ). The samples were cooled to 0 °
analysis, SEM 10 C. at a cooling rate of 10 K /min and reheated to 120 ° C. with
T -SD -P Spray - dried placebo sample DLS , NTA , MFI , turbidity a rate of 10 K /min . The measurement of the temperature
obtained from experiment T - SD - P profile was repeated in a second cycle in order to evaluate
reversibility of thermal events . The Tg was determined as
6.1 Methods for Physico -Chemical Characterization of the midpoint of the endothermic shift of the baseline during
Spray -Dried Powder 15 the heating scan ( see Table 5 ) . The maximum of exothermic /
Spray -dried powders were characterized with respect to endothermic peaks were reported as Tcryst/ Tm .
physico - chemical properties of the spray -dried formulation
using various methods ( Table 3 ) . TABLE 5
20 Tg values of spray-dried formulation
TABLE 3
Sample Tg (1st scan ) [ ° C . ]
Analytical methods for physico - chemical characterization of spray -dried
powder . T - SD1 59.0
T - SD2 72.1
Sample Abbreviation Full term T -SD3 87.9
25 T - SD - P 87.8
Dry powder DSC Differential scanning calorimetry
KF Karl Fischer titration
XRD X -ray powder diffraction
Laser diffraction Laser diffraction analysis Tg values correlate with the water contents of the
SEM Scanning electron microscopy samples. No relaxation phenomenon ( exothermic event) was
30 detectable in spray - dried samples, in contrast to thermo
6.1.1 Karl Fischer Titration grams obtained for lyophilized samples.
The residual moisture content of the dried powders were 6.1.3 X - Ray Powder Diffraction ( XRD )
determined using the coulometric Karl Fischer titrator Aqua Wide angle X - ray powder diffraction (XRD ) was used to
40.00 (Analytik Jena GmbH , Jena, Germany ), which is 35 study the morphology of lyophilized products. The X - ray
equipped with a headspace module . diffractometer Empyrean (Panalytical, Almelo , The Nether
As a system suitability check , the residual moisture con lands ) equipped with a copper anode (45 kV, 40 mA , Kal
tent of a Pure Water Standard ( Apura 1 water standard oven emission at a wavelength of 0.154 nm ) and a PIXcel3D
1.0 , Merck KGaA) was analyzed prior to sample measure- detector was used . Approximately 100 mg of the spray -dried
ment. The residual moisture content of the standard had to 40 samples were analyzed in reflection mode in the angular
be within 1.00 + 0.03 % in order to comply with the manu- range from 5-45 ° 20 , with a step size of 0.04 ° 20 and a
facturer specifications. counting time of 100 seconds per step . The respective XRD
For the measurement, about 20 mg of sample were diagrams are shown in FIG . 5 .
weighed into 2 R glass vials and heated to a measurement All samples showed an amorphous pattern and no indi
temperature of 120 ° C. in the oven connected to the reaction 45 cation of crystalline phases .
vessel via a tubing system . The evaporated water was 6.1.4 Laser Diffraction Analysis
transferred into the titration solution and the amount of
water was determined . The measurement was performed Size distribution of spray -dried powders were measured
until no more water evaporation was detectable ( actual drift by laser diffraction . Laser diffraction measurements were
comparable to drift at the beginning of the measurement). 50 performed using a Partica LA - 950 Laser Diffraction Particle
Ambient moisture was determined by measurement of three Size Distribution Analyzer (Horiba, Kyoto , Japan ) equipped
blanks ( empty vials prepared in the preparation environ- with a 605 nm laser diode for detecting particles > 500 nm
ment ). Results obtained for samples were corrected for the and 405 nm blue light emitting diode (LED ) for detecting
determined ambient moisture by blank subtraction . Samples particles < 500 nm . The powder samples were dispersed in
were measured in duplicates. The results are shown in FIG . 55 Miglyol 812 by ultra sonication for up to 5 min . Prior to
4 and Table 4 . measurement, the system was blanked with Miglyol 812 .
Each sample dispersion was measured 3 times . Measure
TABLE 4 ment results were analyzed using Horiba LA - 950 Software .
Water content of spray- dried formulation The results were reported as
Sample Water content [% ]
60 D10 : particle diameter corresponding to 10 % of the
cumulative undersize distribution ;
T - SD1
T - SD2
4.37
2.81
D50 : particle diameter corresponding to 50 % of the
T - SD3 1.78 cumulative undersize distribution ;
T - SD - P 1.70 65 D90 : particle diameter corresponding to 90 % of the
cumulative undersize distribution .
The results are summarized in Table 6 and FIG . 6 .
 US 11,179,337 B2
53 54
TABLE 6
Particle size distribution of spray - dried powders as measured by laser diffraction
Median Modal Mean
Sample Absorbance Diameter Diameter Value St. Dev. D10 D50 D90
T - SDP 0.126 4.21 6.75 4.032 0.394 1.204 4.21 12.516
T - SD3 0.217 4.132 6.75 3.615 0.408 0.938 4.132 11.151
T - SD2 0.187 4.31 6.75 3.728 0.418 0.933 4.31 11.588
T - SD1 0.051 4.608 6.75 3.927 0.375 1.075 4.608 10.854
(sizes are indicated in um )

6.1.5 Scanning Electron Microscopy ( SEM) TABLE 7 - continued

Images of spray - dried powder particles were generated by
using the bench top scanning electron microscope Phenom 15 Reconstitution behaviour
( Phenom - World B.V., Eindhoven , Netherlands ). The instru
2

ment is equipped with a CCD camera and a diaphragm Sample Reconstitution time [mm :ss] Foam formation
vacuum pump. Each sample was prepared in a glove box
under controlled humidity conditions (<20 % relative humid T -SD3 01:37 0
ity ) by using the following method: a small amount of the 20 T - SD - P 01:04 0
powder was carefully put on a self -adhesive carbon foil
placed on a sample holder. The sample was analyzed under
vacuum with a light optical magnification of 24x and 5 kV In conclusion, reconstitution times for the spray -dried
acceleration voltage . The electron optical magnification was formulations were below 2 minutes . A slightly shorter reso
adjusted between 1160x and 1700x and images
from representative sections of each sample.
were made 25 lution time was observed for the placebo formulation ( T-SD
P) .
The obtained images ( see FIG . 7) demonstrate that the
obtained powder particles have spherical shape and that the 6.3 Particle Characterization
size of the powder particles is in the range from less than 1 The particles comprised in liquid samples of the pro
um to approximately 20 um . 30
tamine - formulated RNA as described above were character
6.2 Reconstitution of Spray -Dried Samples ized before spray -drying, and after reconstitution of the
For reconstitution of the spray -dried samples, the recon- spray -dried samples ( Table 8 ).
stitution volume was calculated for each sample individually
based on the amount of powder weighed into the vial . The TABLE 8
calculation was based on the method for reconstitution of 35
lyophilized samples ( addition of 600 ul water for injection to Analytical methods for characterization of liquid samples (before and after
30.6 mg powder per vial ) . spray- drying and reconstitution ).
The reconstitution volume for varying amounts of spray Sample Abbreviation Full term
dried powder was calculated according to the following Liquid sample Visual inspection
equation : 40 Visual
DLS Dynamic light scattering
Vreconst.. =mpowder* 1000 ul/51 mg MFI Micro - Flow Imaging
Turbidity Turbidity
Vreconst..:: reconstitution volume in ml NTA Nanoparticle tracking analysis
mmpowder: mass of powder to be reconstituted in mg ZP Zeta potential
(based on a theoretical solid content of 51 mg per ml ( 50 45
mg /ml trehalose , 0.8 mg /ml RNA ( free + complexed ), 20 6.3.1 Visual Inspection
anti-heparin IU /mL protamine ))
The spray - dried samples were reconstituted under laminar For visual inspection, the reconstituted vials were
flow conditions using a procedure comparable to the proce- inspected for the presence or absence of visible particles
dure for the lyophilized product: cap and stopper were 50 under gentle, manual, radial agitation for 5 sec in front of a
removed from the vial and the calculated volume of water white background and for 5 sec in front of a black back
for injection was added to the dry powder ( into the center of ground according to the European Pharmacopoeia. The
the vial ) by using a multipette with 10 ml combitip . The vial inspection was performed by two independent examiners . To
was carefully slewed ( shaking was avoided ), until all pow further classify the particle content, the method described in
der particles were dissolved . The reconstitution time was 55
measured as the time required in order to achieve full the “ Deutscher Arzneimittel- Codex ” ( DAC ) was used .
reconstitution of the dry powder after the liquid has been The classification can be described as follows :
added . The reconstitution behavior was judged , mainly with No particles visible within 5 sec : O point
respect to foaming, and recorded ( see Table 7 ) .
60 Few particles visible within 5 sec : 1 point
TABLE 7 Medium number of particles visible within 5 sec : 2 points
Reconstitution behaviour Large number of particles directly visible : 10 points
Sample Reconstitution time [mm :ss ] Foam formation (Particles that were on the limit of being visible as distinct
T - SD1 01:27 0 65 particles ( cloudiness , schlieren ) were rated with 2 points .)
T - SD2 01:20 The results of the visual inspection of the liquid samples
are recorded in Table 9 .
 US 11,179,337 B2
55 56
TABLE 9 TABLE 10
Results of visual inspection Turbidity of the liquid samples
Sample Score [Exp 1/2 ] Sample Turbidity [FNU ]
5
T - SD1 2 * /2 * TO 20.1
T - SD2 1 * /2 T - SD1 26.1
T - SD3 1/1 T - SD2 17.7
T - SD - P 2 * /2 * T - SD3 18.0
T -SD - P 3.5
* fiber - like particle (s) 10

As a result , visible particles were observed in all of the In summary, the turbidity of the spray -dried protamine
analyzed samples. The majority of the observed particles formulated RNA after reconstitution varied from 18 to 26
FNU .
were fiber -like particles that were likely due to a contami
nation . 6.3.3 Dynamic Light Scattering ( DLS )
15
6.3.2 Turbidity DLS measurements were carried out by using a Zetasizer
The NEPHLA turbidimeter (Dr. Lange, Dusseldorf, Ger Nano Series ( Malvern Instruments, Worcestershire, UK)
instrument . 150 ul of the sample were analyzed in small
many ), operating at 860 nm and detecting at 90 ° angle , was volume disposable cuvettes (UVette ) by using an automated
used for turbidity measurements . The system was calibrated mode for each sample
with formazin as a standard and the results were given in 20 formulated RNA before. As a control ( TO) , the protamine
spray -drying was used .
formazin nephelometric units ( FNU ).
For the measurement, 2.0 ml solution were analyzed in a Z -The Malvern Zetasizer Software was used to calculate
clean glass cuvette . The turbidity of the individual samples intensity sizediameter
average , polydispersity index (PDI ) and an
distribution (refractive index and viscosity of
is indicated in Table 10. After analysis, the sample material water was selected in the software ). The results are shown in
was used for further analysis ( e.g. DLS , MFI , etc. ). Table 11 and FIG . 8 .
TABLE 11
Z -average diameter, polydispersity index , main peak diameter and main peak
intensity as determined by Dynamic light scattering
Z -average Main peak Main peak
diameter diameter intensity Derived Count
Sample [nm ] PdI [ nm ] [%] Rate
TO 218.7 + 0.5 0.183 + 0.012 263.1 + 3.6 100 +0 55383 + 219
T - SD1 282.9 + 8.7 0.128 + 0.103 325.6 + 19.4 100 =0 54725 + 363
T - SD2 267.3 + 4.9 0.208 + 0.021 328.3 + 4.6 100 +0 48428 + 394
T - SD3 292.7 + 4.9 0.252 + 0.012 380.4 + 3.6 100 =0 39503 + 42
T - SD - P 425.2 + 36.5 0.418 + 0.050 252.3 + 15.3 98.3 + 3.0 1989 – 197

40
As aa result, slightly increased Z - average and main peak
diameters were determined for spray dried samples.
6.3.4 Nanoparticle Tracking Analysis (NTA )
NTA experiments were carried out with a NanoSight
LM20 (NanoSight, Amesbury, UK) . The instrument is
45 equipped with a 405 nm blue laser, a sample chamber and a
Viton fluoroelastomer O - Ring . The samples were diluted
with ultra -pure water in order to achieve suitable concen
trations for NTA measurement. After the measurement, all
results were normalized to the original concentration .
50 Samples were loaded into the measurement cell using a 1
ml syringe. The results of the NTA analysis are shown in
Table 12 and FIG . 11. Movements of the particles in the
samples were recorded as videos for 60 seconds at room
temperature using the NTA 2.0 Software . The recorded
videos were analyzed with the NTA 2.0 Software .
TABLE 12
Results from NTA analysis
Mean size Mode size D10 size D50 size D90 size Total Concentration
Sample [nm ] [nm ] [nm ] [nm] [nm ] [# /ml]
TO 108 + 2 96 + 7 71 + 1 102 +3 150 + 5 4.74 ( +0.16 ) E + 11
T - SD1 109 + 6 92 + 9 73 + 5 102 +9 149 + 5 3.96 ( +0.89 ) E + 11
T - SD2 120 + 3 107 + 6 83 + 3 114 5 161 = 2 5.71 ( +0.42 ) E + 11
T - SD3 136 + 5 116 10 86 + 4 126 +3 197 + 13 6.04 ( +0.59 ) E + 11
T - SD - P 139 + 30 131 + 40 117 + 41 136 + 33 161 + 19 8.31 ( 19.65 ) E + 09
 US 11,179,337 B2
57 58
The particle size values determined for the spray -dried
and 210 um : 250,000 particles /ml), samples were diluted
samples were comparable or slightly increased with respect
before analysis by adding ultrapure water ( e.g. Milli - Q
to the control ( TO) . water ).
6.3.5 Zeta Potential Measurements For the analysis of the liquid samples, a pre -run volume
Zeta potential measurements were carried out with a 5 of 0.17 ml was followed by a sample run of 0.26 ml .
Zetasizer Nano Series instrument (Malvern Instruments , Approximately 1100 images were taken per sample.
Worcestershire, UK) . 750 ul of each formulation were Between measurements, the flow cell was cleaned with
analyzed in disposable folded capillary cells. For each water and the background illumination was optimized by
sample , 3 zeta potential measurements consisting of 100 using ultrapure water. The MFI View System Software
sub - runs were performed and the mean value for zeta 10 (MVSS ) version 2 - R2-6.1.20.1915 was used to perform the
potential was calculated . For all samples, a negative zeta measurements and the MFI View Analysis Suite (MVAS)
potential was determined ( see Table 13 ) . software version 1.3.0.1007 was used to analyze the data .
The particle counts of the diluted samples were normalized
TABLE 13 to the original concentration . Significantly increased particle
Results of Zeta potential measurements
15 concentrations were determined for the spray -dried samples
including placebo (see Table 14 ) , pointing towards particle
Sample Zeta potential [mV ] contamination .
TO -32.4 TABLE 14
T - SD1 -35.8 20
T - SD2 -36.7
T - SD3 -41.2 Particle concentration as determined by MFI
T - SD - P -22.5
Particle concentration (# /ml]
Sample 21 um 22 um 210 um 225 um
6.3.6 Micro - Flow Imaging ( MFI ) 25
Micro -Flow Imaging measurements were conducted by TO 12,509 4,115 203 11
using a DPA - 5200 particle analyzer system (ProteinSimple, T - SD1
T - SD2
174,199
128,351
32,426
27,151
218
289
4
18
Santa Clara , Calif ., USA) equipped with aa silane coated high T - SD3 139,397 28,440 225 7
resolution 100 um flow cell . Samples were analyzed undi T - SD - P 183,275 35,053 86 0
luted . In case of excess of the MFI concentrations limits
( 22.5 um : 900,000 particles /ml, 25 um : 400,000 particles /ml
SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 1

< 210 > SEQ ID NO 1

< 211 > LENGTH : 2083
< 212 > TYPE : RNA
< 213 > ORGANISM : Artificial Sequence
< 220 > FEATURE :
< 223 > OTHER INFORMATION : R2564

< 400 > SEQUENCE : 1

ggggcgcugc cuacggaggu ggcagccauc uccuucucgg caucaagcuu accaugaagg 60

ccauccuggu gguccuccug uacaccuucg ccaccgcgaa cgccgacacg cugugcaucg 120

gcuaccacgc caacaacagc accgacaccg uggacaccgu gcucgagaag aacgucacgg 180

ugacccacuc cgugaaccug cuggaggaca agcacaacgg gaagcucugc aagcugcggg 240

gcgucgcccc gcugcaccuc gggaagugca acaucgccgg cuggauccug gggaacccgg 300

agugcgagag ccuguccacc gcgagcuccu ggagcuacau cguggagacc uccagcuccg 360

acaacggcac gugcuacccc ggcgacuuca ucgacuacga ggagcuccgc gagcagcuga 420

gcuccgugag cuccuucgag cgguucgaga ucuuccccaa gaccagcucc uggcccaacc 480

acgacagcaa caaggggguc accgccgccu goccgcacgc cggcgcgaag uccuucuaca 540

agaaccugau cuggcucgug aagaagggga acagcuaccc caagcugucc aagagcuaca 600

ucaacgacaa gggcaaggag gugcuggucc ucugggggau ccaccacccc agcaccuccg 660

ccgaccagca gagccuguac cagaacgccg acgccuacgu guucgugggc uccagccgcu 720

acuccaagaa guucaagccc gagaucgcca uc?ggccgaa gguccgcgac caggagggcc 780

ggaugaacua cuacuggacg cugguggagc ccggggacaa gaucaccuuc gaggcgaccg 840

 US 11,179,337 B2
59 60
- continued
gcaaccucgu ggucccccgc uacgccuucg ccauggagcg gaacgccggg agcggcauca 900

ucaucuccga cacccccgug cacgacugca acacgaccug ccagaccccg aagggcgcca 960

ucaacaccag ccugcccuuc cagaacaucc accccaucac gaucgggaag ugccccaagu 1020

acgugaaguc caccaagcug cgccucgcga ccggccugcg gaacgucccg agcauccagu 1080

cccgcgggcu guucggcgcc aucgccgggu ucaucgaggg cggcuggacc gggauggugg 1140

acggcuggua cggguaccac caccagaacg agcagggcag cggguacgcc gccgaccuca 1200

aguccacgca gaacgcgauc gacgagauca ccaacaaggu gaacagcguc aucgagaaga 1260

ugaacaccca guucaccgcc gugggcaagg aguucaacca ccuggagaag cggaucgaga 1320

accugaacaa gaaggucgac gacggcuucc ucgacaucug gacguacaac gccgagcugc 1380

uggugcuccu ggagaacgag cgcacccugg acuaccacga cuccaacgug aagaaccucu 1440

acgagaaggu ccggagccag cugaagaaca acgccaagga gaucgggaac ggcugcuucg 1500

aguucuacca caagugcgac aacaccugca uggaguccgu gaagaacggg accuacgacu 1560

accccaagua cagcgaggag gccaagcuga accgcgagga gaucgacggc gugaagcucg 1620

aguccacgcg gaucuaccag auc?uggega ucuacagcac cgucgccagc ucccuggugc 1680

ucguggucag ccugggggcc aucuccuucu ggaugugcag caacggcucc cugcagugcc 1740

gcaucugcau cugaccacua gugcaucaca uuuaaaagca ucucagccua ccaugagaau 1800

aagagaaaga aaaugaagau caauagcuua uucaucucuu uuucuuuuuc guugguguaa 1860

agccaacacc cugucuaaaa aacauaaauu ucuuuaauca uuuugccucu uuucucugug 1920

cuucaauuaa uaaaaaaugg aaagaaccua gaucuaaaaa aaaaaaaaaa aaaaaaaaaa 1980

?????????? aaaaaaaaaa ?????????? ?????????u gcaucccccccccccccccc 2040

cccccccccccccccaaagg cucuuuucag agccaccaga auu 2083

The invention claimed is : 6. The method according to claim 2 , wherein the liquid of
1. A method for expression of a polypeptide in a subject step a ) is atomized and the droplets resulting from the
comprising administering to the subject an effective amount 40 atomization of the liquid are characterized by a mass median
of a pharmaceutical composition comprising a dry powder aerodynamic diameter of 300 nm to 200 um .
composition comprising a long -chain RNA molecule com has7.aThe method according to claim 1 , wherein the subject
disorder or disease selected from the group consisting
prising at least 200 nucleotides, wherein the dry powder has of cancer or tumor diseases , infectious diseases, autoim
a residual moisture content of 7 % ( w / w ) or less , wherein the 45 mune diseases , allergies or allergic diseases , monogenetic
long - chain RNA molecule is in a complex with a cationic or
polycationic carrier, wherein the pharmaceutical composi diseases , cardiovascular diseases and neuronal diseases .
tion is formulated for mucosal, intranasal, inhalation, or maceuticalmethod
8. The according to claim 1 , wherein the phar
pulmonary delivery, and wherein the long - chain RNA maceutically acceptable further
composition comprises at least one phar
excipient.
encodes the polypeptide. 50 9. The method according to claim 1 , wherein the phar
2. The method of claim 1 , wherein the dry powder maceutical composition comprises a plurality of particles.
composition is prepared by a method comprising the fol 10. The method according to claim 8 , wherein the median
lowing steps: particle size in a volume weighted distribution of the result
a ) providing a liquid comprising the long - chain RNA ing dried powder is at least 1 um .
molecule, 55 11. The method according to claim 8 , wherein the average
b ) spray -drying the liquid of step a ) with a spray - drying sphericity of the particles in the resulting dried powder is in
device having a drying gas inlet and a drying gas outlet , a range from 0.7 to 1.0 .
wherein the drying gas has a temperature at the inlet of 12. The method according to claim 1 , wherein the long
at least 85 ° C. , wherein the drying gas has a tempera- chain RNA molecule comprises more than 200 nucleotides.
ture at the outlet of at least 50 ° C. 60 13. The method according to claim 1 , wherein the long
3. The method according to claim 2 , wherein the liquid of chain RNA molecule comprises at least one modification .
step a) further comprises at least one excipient selected from 14. The method according to claim 2 , wherein the liquid
a cryoprotectant, a lyoprotectant and a bulking agent. of step a) further comprises a suspending agent and / or an
4. The method according to claim 2 , wherein the liquid of osmolality of about 200 mosmol/ 1 to about 400 mosmol / 1 .
step a) does not contain a lipid compound. 65 15. The method according to claim 6 , wherein at least a
5. The method according to claim 2 , wherein the liquid of first pressure nozzle is used as an atomizer, optionally
step a ) comprises a spray -drying compatible solvent . wherein the pressure is less than about 1 bar.
 US 11,179,337 B2
61 62
16. The method of claim 1 , wherein the dry powder 25. A method for expression of a polypeptide in a subject
comprises an average particle size of 1 um to 20 um . comprising :
17. The method of claim 1 , wherein the pharmaceutical a ) providing a dry powder composition comprising a
composition is administered by inhalation . long - chain RNA molecule comprising at least 200
18. The method of claim 1 , wherein the pharmaceutical 5 nucleotides, wherein the dry powder has a residual
composition is administered to the lungs of the patient. moisture content of 7 % ( w / w ) or less , wherein the
19. The method of claim 1 , wherein the pharmaceutical long - chain RNA molecule is in a complex with a
composition is administered as an aerosolized formulation . cationic or polycationic carrier and wherein the long
chain RNA encodes the polypeptide;
20. The method of claim 1 , wherein the dry powder has 10 b ) reconstituting the dry powder composition in a phar
a residual moisture content of 2 % to 5 % ( w / w ). maceutically acceptable aqueous solvent to provide a
21. The method of claim 1 , wherein the wherein the liquid pharmaceutical composition; and
cationic or polycationic carrier comprises a cationic or c ) administering an effective amount of a pharmaceutical
polycationic polypeptide. composition to the subject .
22. The method of claim 1 , wherein the wherein the 15 26. The method of claim 24 , wherein the pharmaceutical
cationic or polycationic carrier comprises a cationic or composition is administered by injection.
polycationic lipid . 27. The method of claim 24 , wherein the pharmaceutical
23. The method of claim 1 , wherein the wherein the composition is administered as an aerosolized formulation
cationic or polycationic carrier comprises a cationic or for mucosal, intranasal, inhalation, or pulmonary delivery.
polycationic polymer. 20 28. The method of claim 26 , wherein the RNA encodes an
24. The method of claim 1 , wherein the pharmaceutical antigen, said method further defined as a method for stimu
composition is administered by mucosal, intranasal, inhala lating an immune response .
tion , or pulmonary delivery . * *