System for the in vivo delivery and expression of heterologous genes in the bone marrow

The present invention provides a method of delivering immunogenic or therapeutic proteins to bone marrow cells using alphavirus vectors. The alphavirus vectors disclosed herein target specifically to bone marrow tissue, and viral genomes persist in bone marrow for at least three months post-infection. No or very low levels of virus were detected in quadricep, brain, and sera of treated animals. The sequence of a consensus Sindbis cDNA clone, pTR339, and infectious RNA transcripts, infectious virus particles, and pharmaceutical formulations derived therefrom are also disclosed. The sequence of the genomic RNA of the Girdwood S.A. virus, and cDNA clones, infectious RNA transcripts, infectious virus particles, and pharmaceutical formulations derived therefrom are also disclosed.

FIELD OF THE INVENTION 
The present invention relates to recombinant DNA technology, and in 
particular to introducing and expressing foreign DNA in a eukaryotic cell. 
BACKGROUND OF THE INVENTION 
The Alphavirus genus includes a variety of viruses all of which are members 
of the Togaviridae family. The alphaviruses include Eastern Equine 
Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), 
Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis 
virus (WEE), Sindbis virus, South African Arbovirus No. 86 (S.A.AR 86), 
Girdwood S.A. virus, Ockelbo virus, Semliki Forest virus, Middelburg 
virus, Chikungunya virus, O'Nyong-Nyong virus, Ross River virus, Barmah 
Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una 
virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, 
Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. 
The alphavirus genome is a single-stranded, messenger-sense RNA, modified 
at the 5'-end with a methylated cap, and at the 3'-end with a 
variable-length poly (A) tract. The viral genome is divided into two 
regions: the first encodes the nonstructural or replicase proteins 
(nsP1-nsP4) and the second encodes the viral structural proteins. Strauss 
and Strauss, Microbiological Rev. 58, 491-562, 494 (1994). Structural 
subunits consisting of a single viral protein, C, associate with 
themselves and with the RNA genome in an icosahedral nucleocapsid. In the 
virion, the capsid is surrounded by a lipid envelope covered with a 
regular array of transmembranal protein spikes, each of which consists of 
a heterodimeric complex of two glycoproteins, E1 and E2. See Paredes et 
al., Proc. Natl. Acad. Sci. USA 90, 9095-99 (1993); Paredes et al., 
Virology 187, 324-32 (1993); Pedersen et al., J. Virol. 14:40 (1974). 
Sindbis virus, the prototype member of the alphavirus genus of the family 
Togaviridae, and viruses related to Sindbis are broadly distributed 
throughout Africa, Europe, Asia, the Indian subcontinent, and Australia, 
based on serological surveys of humans, domestic animals and wild birds. 
Kokemot et al., Trans. R. Soc. Trop Med. Hyg. 59, 553-62 (1965); Redaksie, 
S. Afr. Med. J. 42, 197 (1968); Adekolu-John and Fagbami, Trans. R. Soc. 
Trop. Med. Hyg. 77, 149-51 (1983); Darwish et al., Trans. R. Soc. Trop. 
Med. Hyg. 77, 442-45 (1983); Lundstrom et al., Epidemiol. Infect. 106, 
567-74 (1991); Morrill et al., J. Trop. Med. Hyg. 94, 166-68 (1991). The 
first isolate of Sindbis virus (strain AR339) was recovered from a pool of 
Culex sp. mosquitoes collected in Sindbis, Egypt in 1953 (Taylor et al., 
Am. J. Trop. Med. Hyg. 4, 844-62 (1955)), and is the most extensively 
studied representative of this group. Other members of the Sindbis group 
of alphaviruses include South African Arbovirus No. 86, Ockelbo82, and 
Girdwood S.A. These viruses are not strains of the Sindbis virus; they are 
related to Sindbis AR339, but they are more closely related to each other 
based on nucleotide sequence and serological comparisons. Lundstrom et 
al., J. Wildl. Dis. 29, 189-95 (1993); Simpson et al., Virology 222, 
464-69 (1996). Ockelbo82, S.A.AR86 and Girdwood S.A. are all associated 
with human disease, whereas Sindbis is not. The clinical symptoms of human 
infection with Ockelbo82, S.A.AR86, or Girdwood S.A. are a febrile 
illness, general malaise, macropapular rash, and joint pain that 
occasionally progresses to a polyarthralgia sometimes lasting from a few 
months to a few years. 
The study of these viruses has led to the development of beneficial 
techniques for vaccinating against the alphavirus diseases, and other 
diseases through the use of alphavirus vectors for the introduction of 
foreign DNA. See U.S. Pat. No. 5,185,440 to Davis et al., and PCT 
Publication WO 92/10578. It is intended that all United States patent 
references be incorporated in their entirety by reference. 
It is well known that live, attenuated viral vaccines are among the most 
successful means of controlling viral disease. However, for some virus 
pathogens, immunization with a live virus strain may be either impractical 
or unsafe. One alternative strategy is the insertion of sequences encoding 
immunizing antigens of such agents into a vaccine strain of another virus. 
One such system utilizing a live VEE vector is described in U.S. Pat. No. 
5,505,947 to Johnston et al. 
Sindbis virus vaccines have been employed as viral carriers in virus 
constructs which express genes encoding immunizing antigens for other 
viruses. See U.S. Pat. No. 5,217,879 to Huang et al. Huang et al. 
describes Sindbis infectious viral vectors. However, the reference does 
not describe the cDNA sequence of Girdwood S.A. and TR339, nor clones or 
viral vectors produced therefrom. 
Another such system is described by Hahn et al., Proc. Natl. Acad. Sci. USA 
89:2679 (1992), wherein Sindbis virus constructs which express a truncated 
form of the influenza hemagglutinin protein are described. The constructs 
are used to study antigen processing and presentation in vitro and in 
mice. Although no infectious challenge dose is tested, it is also 
suggested that such constructs might be used to produce protective B- and 
T-cell mediated immunity. 
London et al., Proc. Natl. Acad. Sci, USA 89, 207-11 (1992), disclose a 
method of producing an immune response in mice against a lethal Rift 
Valley Fever (RVF) virus by infecting the mice with an infectious Sindbis 
virus containing an RVF epitope. London does not disclose using Girdwood 
S.A. or TR339 to induce an immune response in animals. 
Viral carriers can also be used to introduce and express foreign DNA in 
eukaryotic cells. One goal of such techniques is to employ vectors that 
target expression to particular cells and/or tissues. A current approach 
has been to remove target cells from the body, culture them ex vivo, 
infect them with an expression vector, and then reintroduce them into the 
patient. 
PCT Publication No. WO 92/10578 to Garoff and Liljestrom provide a system 
for introducing and expressing foreign proteins in animal cells using 
alphaviruses. This reference discloses the use of Semliki Forest virus to 
introduce and express foreign proteins in animal cells. The use of 
Girdwood S.A. or TR339 is not discussed. Furthermore, this reference does 
not provide a method of targeting and introducing foreign DNA into 
specific cell or tissue types. 
Accordingly, there remains a need in the art for full-length cDNA clones of 
positive-strand RNA viruses, such as Girdwood S.A and TR339. In addition, 
there is an ongoing need in the art for improved vaccination strategies. 
Finally, there remains a need in the art for improved methods and nucleic 
acid sequences for delivering foreign DNA to target cells. 
SUMMARY OF THE INVENTION 
A first aspect of the present invention is a method of introducing and 
expressing heterologous RNA in bone marrow cells, comprising: (a) 
providing a recombinant alphavirus, the alphavirus containing a 
heterologous RNA segment, the heterologous RNA segment comprising a 
promoter operable in bone marrow cells operatively associated with a 
heterologous RNA to be expressed in bone marrow cells; and then (b) 
contacting the recombinant alphavirus to the bone marrow cells so that the 
heterologous RNA segment is introduced and expressed therein. 
As a second aspect, the present invention provides a helper cell for 
expressing an infectious, propagation defective, Girdwood S.A. virus 
particle, comprising, in a Girdwood S.A.-permissive cell: (a) a first 
helper RNA encoding (i) at least one Girdwood S.A. structural protein, and 
(ii) not encoding at least one other Girdwood S.A. structural protein; and 
(b) a second helper RNA separate from the first helper RNA, the second 
helper RNA (i) not encoding the at least one Girdwood S.A. structural 
protein encoded by the first helper RNA, and (ii) encoding the at least 
one other Girdwood S.A. structural protein not encoded by the first helper 
RNA, and with all of the Girdwood S.A. structural proteins encoded by the 
first and second helper RNAs assembling together into Girdwood S.A. 
particles in the cell containing the replicon RNA; and wherein the 
Girdwood S.A. packaging segment is deleted from at least the first helper 
RNA. 
A third aspect of the present invention is a method of making infectious, 
propagation defective, Girdwood S.A. virus particles, comprising: 
transfecting a Girdwood S.A.-permissive cell with a propagation defective 
replicon RNA, the replicon RNA including the Girdwood S.A. packaging 
segment and an inserted heterologous RNA; producing the Girdwood S.A. 
virus particles in the transfected cell; and then collecting the Girdwood 
S.A. virus particles from the cell. Also disclosed are infectious Girdwood 
S.A. RNAs, cDNAs encoding the same, infectious Girdwood S.A. virus 
particles, and pharmaceutical formulations thereof. 
As a fourth aspect, the present invention provides a helper cell for 
expressing an infectious, propagation defective, TR339 virus particle, 
comprising, in a TR339-permissive cell: (a) a first helper RNA encoding 
(i) at least one TR339 structural protein, and (ii) not encoding at least 
one other TR339 structural protein; and (b) a second helper RNA separate 
from the first helper RNA, the second helper RNA (i) not encoding the at 
least one TR339 structural protein encoded by the first helper RNA, and 
(ii) encoding the at least one other TR339 structural protein not encoded 
by the first helper RNA, and with all of the TR339 structural proteins 
encoded by the first and second helper RNAs assembling together into TR339 
particles in the cell containing the replicon RNA; and wherein the TR339 
packaging segment is deleted from at least the first helper RNA. 
A fifth aspect of the present invention is a method of making infectious, 
propagation defective, TR339 virus particles, comprising: transfecting a 
TR339-permissive cell with a propagation defective replicon RNA, the 
replicon RNA including the TR339 packaging segment and an inserted 
heterologous RNA; producing the TR339 virus particles in the transfected 
cell; and then collecting the TR339 virus particles from the cell. Also 
disclosed are infectious TR339 RNAs, cDNAs encoding the same, infectious 
TR339 virus particles, and pharmaceutical formulations thereof. 
As a sixth aspect, the present invention provides a recombinant DNA 
comprising a cDNA coding for an infectious Girdwood S.A. virus RNA 
transcript, and a heterologous promoter positioned upstream from the cDNA 
and operatively associated therewith. The present invention also provides 
infectious RNA transcripts encoded by the above-mentioned cDNA and 
infectious viral particles containing the infectious RNA transcripts. 
As a seventh aspect, the present invention provides a recombinant DNA 
comprising a cDNA coding for a Sindbis strain TR339 RNA transcript, and a 
heterologous promoter positioned upstream from the cDNA and operatively 
associated therewith. The present invention also provides infectious RNA 
transcripts encoded by the above-mentioned cDNA and infectious viral 
particles containing the infectious RNA transcripts. 
The foregoing and other aspects of the present invention are described in 
the detailed description set forth below. 
DETAILED DESCRIPTION OF THE INVENTION 
The production and use of recombinant DNA, vectors, transformed host cells, 
selectable markers, proteins, and protein fragments by genetic engineering 
are well-known to those skilled in the art. See, e.g., U.S. Pat. No. 
4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Pat. No. 
4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Pat. 
No. 4,912,038 to Schilling at Col 3 line 26 to Col 14 line 12; and U.S. 
Pat. No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59. 
The term "alphavirus" has its conventional meaning in the art, and includes 
the various species of alphaviruses such as Eastern Equine Encephalitis 
virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, 
Mucambo virus, Pixuna virus, Western Encephalitis virus (WEE), Sindbis 
virus, South African Arbovirus No. 86, Girdwood S.A. virus, Ockelbo virus, 
Semliki Forest virus, Middelburg virus, Chikungunya virus, O'Nyong-Nyong 
virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, 
Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki 
virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu 
virus, Buggy Creek virus, and any other virus classified by the 
International Committee on Taxonomy of Viruses (ICTV) as an alphavirus. 
The preferred alphaviruses for use in the present invention include 
Sindbis virus strains (e.g., TR339), Girdwood S.A., S.A.AR86, and 
Ockelbo82. 
An "Old World alphavirus" is a virus that is primarily distributed 
throughout the Old World. Alternately stated, an Old World alphavirus is a 
virus that is primarily distributed throughout Africa, Asia, Australia and 
New Zealand, or Europe. Exemplary Old World viruses include SF group 
alphaviruses and SIN group alphaviruses. SF group alphaviruses include 
Semliki Forest virus, Middelburg virus, Chikungunya virus, O'Nyong-Nyong 
virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, 
Bebaru virus, Mayaro virus, and Una virus. SIN group alphaviruses include 
Sindbis virus, South African Arbovirus No. 86, Ockelbo virus, Girdwood 
S.A. virus, Aura virus, Whataroa virus, Babanki virus, and Kyzylagach 
virus. 
Acceptable alphaviruses include those containing attenuating mutations. The 
phrases "attenuating mutation" and "attenuating amino acid," as used 
herein, mean a nucleotide sequence containing a mutation, or an amino acid 
encoded by a nucleotide sequence containing a mutation, which mutation 
results in a decreased probability of causing disease in its host (i.e., a 
loss of virulence), in accordance with standard terminology in the art, 
whether the mutation be a substitution mutation or an in-frame deletion 
mutation. See, e.g., B. DAVIS ET AL., MICROBIOLOGY 132 (3d ed. 1980). The 
phrase "attenuating mutation" excludes mutations or combinations of 
mutations which would be lethal to the virus. 
Appropriate attenuating mutations will be dependent upon the alphavirus 
used. Suitable attenuating mutations within the alphavirus genome will be 
known to those skilled in the art. Exemplary attenuating mutations 
include, but are not limited to, those described in U.S. Pat. No. 
5,505,947 to Johnston et al., U.S. Pat. No. 5,792,462 to Johnston et al., 
and U.S. Pat. No. 5,639,650 to Johnston et al. It is intended that all 
United States patent references be incorporated in their entirety by 
reference. 
Attenuating mutations may be introduced into the RNA by performing 
site-directed mutagenesis on the cDNA which encodes the RNA, in accordance 
with known procedures. See, Kunkel, Proc. Natl. Acad. Sci. USA 82, 488 
(1985), the disclosure of which is incorporated herein by reference in its 
entirety. Alternatively, mutations may be introduced into the RNA by 
replacement of homologous restriction fragments in the cDNA which encodes 
for the RNA, in accordance with known procedures. 
I. Methods for Introducing and Expressing Heterologous RNA in Bone Marrow 
Cells 
The present invention provides methods of using a recombinant alphavirus to 
introduce and express a heterologous RNA in bone marrow cells. Such 
methods are useful as vaccination strategies when the heterologous RNA 
encodes an immunogenic protein or peptide. Alternatively, such methods are 
useful in introducing and expressing in bone marrow cells an RNA which 
encodes a desirable protein or peptide, for example, a therapeutic protein 
or peptide. 
The present invention is carried out using a recombinant alphavirus to 
introduce a heterologous RNA into bone marrow cells. Any alphavirus that 
targets and infects bone marrow cells is suitable. Preferred alphaviruses 
include Old World alphaviruses, more preferably SF group alphaviruses and 
SIN group alphaviruses, more preferably Sindbis virus strains (e.g., 
TR339), S.A.AR86 virus, Girdwood S.A. virus, and Ockelbo virus. In a more 
preferred embodiment, the alphavirus contains one or more attenuating 
mutations, as described hereinabove. 
Two types of recombinant virus vector are contemplated in carrying out the 
present invention. In one embodiment employing "double promoter vectors," 
the heterologous RNA is inserted into a replication and propagation 
competent virus. Double promoter vectors are described in U.S. Pat. No. 
5,505,947 to Johnston et al. With this type of viral vector, it is 
preferable that heterologous RNA sequences of less than 3 kilobases are 
inserted into the viral vector, more preferably those less than 2 
kilobases, and more preferably still those less than 1 kilobase. In an 
alternate embodiment, propagation-defective "replicon vectors," as 
described in U.S. Pat. No. 5,792,462, will be used. One advantage of 
replicon viral vectors is that larger RNA inserts, up to approximately 4-5 
kilobases in length can be utilized. Double promoter vectors and replicon 
vectors are described in more detail hereinbelow. 
The recombinant alphaviruses of the claimed method target the heterologous 
RNA to bone marrow cells, where it expresses the encoded protein or 
peptide. Heterologous RNA can be introduced and expressed in any cell type 
found in the bone marrow. Bone marrow cells that may be targeted by the 
recombinant alphaviruses of the present invention include, but are not 
limited to, polymorphonuclear cells, hemopoietic stem cells (including 
megakaryocyte colony forming units (CFU-M), spleen colony forming units 
(CFU-S), erythroid colony forming units (CFU-E), erythroid burst forming 
units (BFU-E), and colony forming units in culture (CFU-C)), erythrocytes, 
macrophages (including reticular cells), monocytes, granulocytes, 
megakaryoctyes, lymphocytes, fibroblasts, osteoprogenitor cells, and 
stromal cells. 
By targeting to the cells of the bone marrow, it is meant that the primary 
site in which the virus will be localized in vivo is the cells of the bone 
marrow. Alternately stated, the alphaviruses of the present invention 
target bone marrow cells, such that titers in bone marrow two days after 
infection are greater than 100 PFU/g crushed bone, preferably greater than 
200 PFU/g crushed bone, more preferably greater than 300 PFU/g crushed 
bone, and more preferably still greater than 500 PFU/g crushed bone. Virus 
may be detected occasionally in other cell or tissue types, but only 
sporadically and usually at low levels. Virus localization in the bone 
marrow can be demonstrated by any suitable technique known in the art, 
such as in situ hybridization. 
Bone marrow cells are long-lived and harbor infectious alphaviruses for a 
prolonged period of time, as demonstrated in the Examples below. These 
characteristics of bone marrow cells render the present invention useful 
not only for the purpose of supplying a desired protein or peptide to 
skeletal tissue, but also for expressing proteins or peptides in vivo that 
are needed by other cell or tissue types. 
The present invention can be carried out in vivo or with cultured bone 
marrow cells in vitro. Bone marrow cell cultures include primary cultures 
of bone marrow cells, serially-passaged cultures of bone marrow cells, and 
cultures of immortalized bone marrow cell lines. Bone marrow cells may be 
cultured by any suitable means known in the art. 
The recombinant alphaviruses of the present invention carry a heterologous 
RNA segment. The heterologous RNA segment encodes a promoter and an 
inserted heterologous RNA. The inserted heterologous RNA may encode any 
protein or a peptide which is desirably expressed by the host bone marrow 
cells. Suitable heterologous RNA may be of prokaryotic (e.g., RNA encoding 
the Botulinus toxin C), or eukaryotic (e.g., RNA encoding malaria 
Plasmodium protein cs1) origin. Illustrative proteins and peptides encoded 
by the heterologous RNAs of the present invention include hormones, growth 
factors, interleukins, cytokines, chemokines, enzymes, and ribozymes. 
Alternately, the heterologous RNAs encode any therapeutic protein or 
peptide. As a further alternative, the heterologous RNAs of the present 
invention encode any immunogenic protein or peptide. 
An immunogenic protein or peptide, or "immunogen," may be any protein or 
peptide suitable for protecting the subject against a disease, including 
but not limited to microbial, bacterial, protozoal, parasitic, and viral 
diseases. For example, the immunogen may be an orthomyxovirus immunogen 
(e.g., an influenza virus immunogen, such as the influenza virus 
hemagglutinin (HA) surface protein or the influenza virus nucleoprotein 
gene, or an equine influenza virus immunogen), or a lentivirus immunogen 
(e.g., an equine infectious anemia virus immunogen, a Simian 
Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus 
(HIV) immunogen, such as the HIV envelope GP160 protein and the HIV 
matrix/capsid proteins). The immunogen may also be an arenavirus immunogen 
(e.g., Lassa fever virus immunogen, such as the Lassa fever virus 
nucleocapsid protein gene and the Lassa fever envelope glycoprotein gene), 
a poxvirus immunogen (e.g., vaccinia), a flavivirus immunogen (e.g., a 
yellow fever virus immunogen or a Japanese encephalitis virus immunogen), 
a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus 
immunogen), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or 
a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, 
such as the human coronavirus envelope glycoprotein gene, or a 
transmissible gastroenteritis virus immunogen for pigs, or an infectious 
bronchitis virus immunogen for chickens). 
Alternatively, the present invention can be used to express heterologous 
RNAs encoding antisense oligonucleotides. In general, "antisense" refers 
to the use of small, synthetic oligonucleotides to inhibit gene expression 
by inhibiting the function of the target mRNA containing the complementary 
sequence. Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993). 
Gene expression is inhibited through hybridization to coding (sense) 
sequences in a specific mRNA target by hydrogen bonding according to 
Watson-Crick base pairing rules. The mechanism of antisense inhibition is 
that the exogenously applied oligonucleotides decrease the mRNA and 
protein levels of the target gene. Milligan, J. F. et al., J. Med. Chem. 
36(14), 1923-1937 (1993). See also Helene, C. and Toulme, J., Biochim. 
Biophys. Acta 1049, 99-125 (1990); Cohen, J. S., Ed., 
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC 
Press:Boca Raton, Fla. (1987). 
Antisense oligonucleotides may be of any suitable length, depending on the 
particular target being bound. The only limits on the length of the 
antisense oligonucleotide is the capacity of the virus for inserted 
heterologous RNA. Antisense oligonucleotides may be complementary to the 
entire mRNA transcript of the target gene or only a portion thereof. 
Preferably the antisense oligonucleotide is directed to an mRNA region 
containing a junction between intron and exon. Where the antisense 
oligonucleotide is directed to an intron/exon junction, it may either 
entirely overlie the junction or may be sufficiently close to the junction 
to inhibit splicing out of the intervening exon during processing of 
precursor mRNA to mature mRNA (e.g., with the 3' or 5' terminus of the 
antisense oligonucleotide being positioned within about, for example, 10, 
5, 3 or 2 nucleotides of the intron/exon junction). Also preferred are 
antisense oligonucleotides which overlap the initiation codon. 
When practicing the present invention, the antisense oligonucleotides 
administered may be related in origin to the species to which it is 
administered. When treating humans, human antisense may be used if 
desired. 
Promoters for use in carrying out the present invention are operable in 
bone marrow cells. An operable promoter in bone marrow cells is a promoter 
that is recognized by and functions in bone marrow cells. Promoters for 
use with the present invention must also be operatively associated with 
the heterologous RNA to be expressed in the bone marrow. A promoter is 
operably linked to a heterologous RNA if it controls the transcription of 
the heterologous RNA, where the heterologous RNA comprises a coding 
sequence. Suitable promoters are well known in the art. The Sindbis 26S 
promoter is preferred when the alphavirus is a strain of Sindbis virus. 
Additional preferred promoters beyond the Sindbis 26S promoter include the 
Girdwood S.A. 26S promoter when the alphavirus is Girdwood S.A., the 
S.A.AR86 26S promoter when the alphavirus is S.A.AR86, and any other 
promoter sequence recognized by alphavirus polymerases. Alphavirus 
promoter sequences containing mutations which alter the activity level of 
the promoter (in relation to the activity level of the wild-type) are also 
suitable in the practice of the present invention. Such mutant promoter 
sequences are described in Raju and Huang, J. Virol. 65, 2501-2510 (1991), 
the disclosure of which is incorporated in its entirety by reference. 
The heterologous RNA is introduced into the bone marrow cells by contacting 
the recombinant alphavirus carrying the heterologous RNA segment to the 
bone marrow cells. By contacting, it is meant bringing the recombinant 
alphavirus and the bone marrow cells in physical proximity. The contacting 
step can be performed in vitro or in vivo. In vitro contacting can be 
carried out with cultures of immortalized or non-immortalized bone marrow 
cells. In one particular embodiment, bone marrow cells can be removed from 
a subject, cultured in vitro, infected with the vector, and then 
introduced back into the subject. Contacting is performed in vivo when the 
recombinant alphavirus is administered to a subject. Pharmaceutical 
formulations of recombinant alphavirus can be administered to a subject 
parenterally (e.g., subcutaneous, intracerebral, intradermal, 
intramuscular, intravenous and intraarticular) administration. 
Alternatively, pharmaceutical formulations of the present invention may be 
suitable for administration to the mucus membranes of a subject (e.g., 
intranasal administration, by use of a dropper, swab, or inhaler). Methods 
of preparing infectious virus particles and pharmaceutical formulations 
thereof are discussed in more detail hereinbelow. 
By "introducing" the heterologous RNA segment into the bone marrow cells it 
is meant infecting the bone marrow cells with recombinant alphavirus 
containing the heterologous RNA, such that the viral vector carrying the 
heterologous RNA enters the bone marrow cells and can be expressed 
therein. As used with respect to the present invention, when the 
heterologous RNA is "expressed," it is meant that the heterologous RNA is 
transcribed. In particular embodiments of the invention in which it is 
desired to produce a protein or peptide, expression further includes the 
steps of post-transcriptional processing and translation of the mRNA 
transcribed from the heterologous RNA. In contrast, where the heterologous 
RNA encodes an antisense oligonucleotide, expression need not include 
post-transcriptional processing and translation. With respect to 
embodiments in which the heterologous RNA encodes an immunogenic protein 
or a protein being administered for therapeutic purposes, expression may 
also include the further step of post-translational processing to produce 
an immunogenic or therapeutically-active protein. 
The present invention also provides infectious RNAs, as described 
hereinabove, and cDNAs encoding the same. Preferably the infectious RNAs 
and cDNAs are derived from the S.A.AR86, Girdwood S.A., TR339, or Ockelbo 
viruses. The cDNA clones can be generated by any of a variety of suitable 
methods known to those skilled in the art. A preferred method is the 
method set forth in U.S. Pat. No. 5,185,440 to Davis et al., the 
disclosure of which is incorporated in its entirety by reference, and 
Gubler et al., Gene 25:263 (1983). 
RNA is preferably synthesized from the DNA sequence in vitro using purified 
RNA polymerase in the presence of ribonucleotide triphosphates and cap 
analogs in accordance with conventional techniques. However, the RNA may 
also be synthesized intracellularly after introduction of the cDNA. 
A. Double Promoter Vectors 
In one embodiment of the invention, double promoter vectors are used to 
introduce the heterologous RNA into the target bone marrow cells. A double 
promoter virus vector is a replication and propagation competent virus. 
Double promoter vectors are described in U.S. Pat. No. 5,505,947 to 
Johnston et al., the disclosure of which is incorporated in its entirety 
by reference. Preferred alphaviruses for constructing the double promoter 
vectors are S.A.AR86, Girdwood S.A., TR339 and Ockelbo viruses. More 
preferably, the double promoter vector contains one or more attenuating 
mutations. Attenuating mutations are described in more detail hereinabove. 
The double promoter vector is constructed so as to contain a second 
subgenomic promoter (i.e., 26S promoter) inserted 3' to the virus RNA 
encoding the structural proteins. The heterologous RNA is inserted between 
the second subgenomic promoter, so as to be operatively associated 
therewith, and the 3' UTR of the virus genome. Heterologous RNA sequences 
of less than 3 kilobases, more preferably those less than 2 kilobases, and 
more preferably still those less than 1 kilobase, can be inserted into the 
double promoter vector. In a preferred embodiment of the invention, the 
double promoter vector is derived from Girdwood S.A., and the second 
subgenomic promoter is a duplicate of the Girdwood S.A. subgenomic 
promoter. In an alternate preferred embodiment, the double promoter vector 
is derived from TR339, and the second subgenomic promoter is a duplicate 
of the TR339 subgenomic promoter. 
B. Replicon Vectors 
Replicon vectors, which are propagation-defective virus vectors can also be 
used to carry out the present invention. Replicon vectors are described in 
more detail in U.S. Pat. No. 5,792,462, the disclosure of which is 
incorporated in its entirety by reference. Preferred alphaviruses for 
constructing the replicon vectors are S.A.AR86, Girdwood S.A., TR339, and 
Ockelbo. 
In general, in the replicon system, a foreign gene to be expressed is 
inserted in place of at least one of the viral structural protein genes in 
a transcription plasmid containing an otherwise full-length cDNA copy of 
the alphavirus genome RNA. RNA transcribed from this plasmid contains an 
intact copy of the viral nonstructural genes which are responsible for RNA 
replication and transcription. Thus, if the transcribed RNA is transfected 
into susceptible cells, it will be replicated and translated to give the 
nonstructural proteins. These proteins will transcribe the transfected RNA 
to give high levels of subgenomic mRNA, which will then be translated to 
produce high levels of the foreign protein. The autonomously replicating 
RNA (i.e., replicon) can only be packaged into virus particles if the 
alphavirus structural protein genes are provided on one or more "helper" 
RNAs, which are cotransfected into cells along with the replicon RNA. The 
helper RNAs do not contain the viral nonstructural genes for replication, 
but these functions are provided in trans by the replicon RNA. Similarly, 
the transcriptase functions translated from the replicon RNA transcribe 
the structural protein genes on the helper RNA, resulting in the synthesis 
of viral structural proteins and packaging of the replicon into virus-like 
particles. As the packaging or encapsidation signal for alphavirus RNAs is 
located within the nonstructural genes, the absence of these sequences in 
the helper RNAs precludes their incorporation into virus particles. 
Alphavirus-permissive cells employed in the methods of the present 
invention are cells which, upon transfection with the viral RNA 
transcript, are capable of producing viral particles. Preferred 
alphavirus-permissive cells are TR339-permissive cells, Girdwood 
S.A.-permissive cells, S.A.AR86-permissive cells, and Ockelbo-permissive 
cells. Alphaviruses have a broad host range. Examples of suitable host 
cells include, but are not limited to Vero cells, baby hamster kidney 
(BHK) cells, and chicken embryo fibroblast cells. 
The phrase "structural protein" as used herein refers to the encoded 
proteins which are required for encapsidation (e.g., packaging) of the RNA 
replicon, and include the capsid protein, E1 glycoprotein, and E2 
glycoprotein. As described hereinabove, the structural proteins of the 
alphavirus are distributed among one or more helper RNAs (i.e., a first 
helper RNA and a second helper RNA). In addition, one or more structural 
proteins may be located on the same RNA molecule as the replicon RNA, 
provided that at least one structural protein is deleted from the replicon 
RNA such that the resulting alphavirus particle is propagation defective. 
As used herein, the terms "deleted" or "deletion" mean either total 
deletion of the specified segment or the deletion of a sufficient portion 
of the specified segment to render the segment inoperative or 
nonfunctional, in accordance with standard usage. See, e.g., U.S. Pat. No. 
4,650,764 to Temin et al. The term "propagation defective" as used herein, 
means that the replicon RNA cannot be encapsidated in the host cell in the 
absence of the helper RNA. The resulting alphavirus particles are 
propagation defective inasmuch as the replicon RNA does not include all of 
the alphavirus structural proteins required for encapsidation, at least 
one of the required structural proteins being deleted therefrom, such that 
the packaged replicon RNA is not capable of replicating the entire viral 
genome. 
The helper cell for expressing the infectious, propagation defective 
alphavirus particle comprises a set of RNAs, as described above. The set 
of RNAs principally include a first helper RNA and a second helper RNA. 
The first helper RNA includes RNA encoding at least one alphavirus 
structural protein but does not encode all alphavirus structural proteins. 
In other words, the first helper RNA does not encode at least one 
alphavirus structural protein; the at least one non-coded alphavirus 
structural protein being deleted from the first helper RNA. In one 
embodiment, the first helper RNA includes RNA encoding the alphavirus E1 
glycoprotein, with the alphavirus capsid protein and the alphavirus E2 
glycoprotein being deleted from the first helper RNA. In another 
embodiment, the first helper RNA includes RNA encoding the alphavirus E2 
glycoprotein, with the alphavirus capsid protein and the alphavirus E1 
glycoprotein being deleted from the first helper RNA. In a third, 
preferred embodiment, the first helper RNA includes RNA encoding the 
alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, with the 
alphavirus capsid protein being deleted from the first helper RNA. 
The second helper RNA includes RNA encoding at least one alphavirus 
structural protein which is different from the at least one structural 
protein encoded by the first helper RNA. Thus, the second helper RNA 
encodes at least one alphavirus structural protein which is not encoded by 
the first helper RNA. The second helper RNA does not encode the at least 
one alphavirus structural protein which is encoded by the first helper 
RNA, thus the first and second helper RNAs do not encode duplicate 
structural proteins. In the embodiment wherein the first helper RNA 
includes RNA encoding only the alphavirus E1 glycoprotein, the second 
helper RNA may include RNA encoding one or both of the alphavirus capsid 
protein and the alphavirus E2 glycoprotein which are deleted from the 
first helper RNA. In the embodiment wherein, the first helper RNA includes 
RNA encoding only the alphavirus E2 glycoprotein, the second helper RNA 
may include RNA encoding one or both of the alphavirus capsid protein and 
the alphavirus E1 glycoprotein which are deleted from the first helper 
RNA. In the embodiment wherein the first helper RNA includes RNA encoding 
both the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, 
the second helper RNA may include RNA encoding the alphavirus capsid 
protein which is deleted from the first helper RNA. 
In one embodiment, the packaging segment (RNA comprising the encapsidation 
or packaging signal) is deleted from at least the first helper RNA. In a 
preferred embodiment, the packaging segment is deleted from both the first 
helper RNA and the second helper RNA. 
In the preferred embodiment wherein the packaging segment is deleted from 
both the first helper RNA and the second helper RNA, the helper cell is 
co-transfected with a replicon RNA in addition to the first helper RNA and 
the second helper RNA. The replicon RNA encodes the packaging segment and 
an inserted heterologous RNA. The inserted heterologous RNA may be RNA 
encoding a protein or a peptide. In a preferred embodiment, the replicon 
RNA, the first helper RNA and the second helper RNA are provided on 
separate molecules such that a first molecule, i.e., the replicon RNA, 
includes RNA encoding the packaging segment and the inserted heterologous 
RNA, a second molecule, i.e., the first helper RNA, includes RNA encoding 
at least one but not all of the required alphavirus structural proteins, 
and a third molecule, i.e., the second helper RNA, includes RNA encoding 
at least one but not all of the required alphavirus structural proteins. 
For example, in one preferred embodiment of the present invention, the 
helper cell includes a set of RNAs which include (a) a replicon RNA 
including RNA encoding an alphavirus packaging sequence and an inserted 
heterologous RNA, (b) a first helper RNA including RNA encoding the 
alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, and (c) a 
second helper RNA including RNA encoding the alphavirus capsid protein so 
that the alphavirus E1 glycoprotein, the alphavirus E2 glycoprotein and 
the capsid protein assemble together into alphavirus particles in the host 
cell. 
In an alternate embodiment, the replicon RNA and the first helper RNA are 
on separate molecules, and the replicon RNA and RNA encoding a structural 
gene not encoded by the first helper RNA are on another single molecule 
together, such that a first molecule, i.e., the first helper RNA, 
including RNA encoding at least one but not all of the required alphavirus 
structural proteins, and a second molecule, i.e., the replicon RNA, 
including RNA encoding the packaging segment, the inserted heterologous 
RNA, and the remaining structural proteins not encoded by the first helper 
RNA. For example, in one preferred embodiment of the present invention, 
the helper cell includes a set of RNAs including (a) a replicon RNA 
including RNA encoding an alphavirus packaging sequence, an inserted 
heterologous RNA, and an alphavirus capsid protein, and (b) a first helper 
RNA including RNA encoding the alphavirus E1 glycoprotein and the 
alphavirus E2 glycoprotein so that the alphavirus E1 glycoprotein, the 
alphavirus E2 glycoprotein and the capsid protein assemble together into 
alphavirus particles in the host cell, with the replicon RNA packaged 
therein. 
In one preferred embodiment of the present invention, the RNA encoding the 
alphavirus structural proteins, i.e., the capsid, E1 glycoprotein and E2 
glycoprotein, contains at least one attenuating mutation, as described 
hereinabove. Thus, according to this embodiment, at least one of the first 
helper RNA and the second helper RNA includes at least one attenuating 
mutation. In a more preferred embodiment, at least one of the first helper 
RNA and the second helper RNA includes at least two, or multiple, 
attenuating mutations. The multiple attenuating mutations may be 
positioned in either the first helper RNA or in the second helper RNA, or 
they may be distributed randomly with one or more attenuating mutations 
being positioned in the first helper RNA and one or more attenuating 
mutations positioned in the second helper RNA. Alternatively, when the 
replicon RNA and the RNA encoding the structural proteins not encoded by 
the first helper RNA are located on the same molecule, an attenuating 
mutation may be positioned in the RNA which codes for the structural 
protein not encoded by the first helper RNA. The attenuating mutations may 
also be located within the RNA encoding non-structural proteins (e.g., the 
replicon RNA). 
Preferably, the first helper RNA and the second helper RNA also include a 
promoter. It is also preferred that the replicon RNA also includes a 
promoter. Suitable promoters for inclusion in the first helper RNA, second 
helper RNA and replicon RNA are well known in the art. One preferred 
promoter is the Girdwood S.A. 26S promoter for use when the alphavirus is 
Girdwood S.A. Another preferred promoter is the TR339 26S promoter for use 
when the alphavirus is TR339. Additional promoters beyond the Girdwood 
S.A. and TR339 promoters include the VEE 26S promoter, the Sindbis 26S 
promoter, the Semliki Forest 26S promoter, and any other promoter sequence 
recognized by alphavirus polymerases. Alphavirus promoter sequences 
containing mutations which alter the activity level of the promoter (in 
relation to the activity level of the wild-type) are also suitable in the 
practice of the present invention. Such mutant promoter sequences are 
described in Raju and Huang, J. Virol. 65, 2501-2510 (1991), the 
disclosure of which is incorporated herein in its entirety. In the system 
wherein the first helper RNA, the second helper RNA, and the replicon RNA 
are all on separate molecules, the promoters, if the same promoter is used 
for all three RNAs, provide a homologous sequence between the three 
molecules. It is preferred that the selected promoter is operative with 
the non-structural proteins encoded by the replicon RNA molecule. 
In cases where vaccination with two immunogens provides improved protection 
against disease as compared to vaccination with only a single immunogen, a 
double-promoter replicon would ensure that both immunogens. are produced 
in the same cell. Such a replicon would be the same as the one described 
above, except that it would contain two copies of the 26S RNA promoter, 
each followed by a different multiple cloning site, to allow for the 
insertion and expression of two different heterologous proteins. Another 
useful strategy is to insert the IRES sequence from the picornavirus, EMC 
virus, between the two heterologous genes downstream from the single 26S 
promoter of the replicon described above, thus leading to expression of 
two immunogens from the single replicon transcript in the same cell. 
C. Uses of the Present Invention 
The alphavirus vectors, RNAs, cDNAs, helper cells, infectious virus 
particles, and methods of the present invention find use in in vitro 
expression systems, wherein the inserted heterologous RNA encodes a 
protein or peptide which is desirably produced in vitro. The RNAs, cDNAs, 
helper cells, infectious virus particles, methods, and pharmaceutical 
formulations of the present invention are additionally useful in a method 
of administering a protein or peptide to a subject in need of the protein 
or peptide, as a method of treatment or otherwise. In this embodiment of 
the invention, the heterologous RNA encodes the desired protein or 
peptide, and pharmaceutical formulations of the present invention are 
administered to a subject in need of the desired protein or peptide. In 
this manner, the protein or peptide may thus be produced in vivo in the 
subject. The subject may be in need of the protein or peptide because the 
subject has a deficiency thereof, or because the production of the protein 
or peptide in the subject may impart some therapeutic effect, as a method 
of treatment or otherwise. 
Alternately, the claimed methods provide a vaccination strategy, wherein 
the heterologous RNA encodes an immunogenic protein or peptide. 
The methods and products of the invention are also useful as antigens and 
for evoking the production of antibodies in animals such as horses and 
rabbits, from which the antibodies may be collected and then used in 
diagnostic assays in accordance with known techniques. 
A further aspect of the present invention is a method of introducing and 
expressing antisense oligonucleotides in bone marrow cell cultures to 
regulate gene expression. Alternately, the claimed method finds use in 
introducing and expressing a protein or peptide in bone marrow cell 
cultures. 
II. Girdwood S.A. and TR339 Clones 
Disclosed hereinbelow are genomic RNA sequences encoding live Girdwood S.A. 
virus, live S.A.AR86 virus, and live Sindbis strain TR339 virus, cDNAs 
derived therefrom, infectious RNA transcripts encoded by the cDNAs, 
infectious viral particles containing the infectious RNA transcripts, and 
pharmaceutical formulations derived therefrom. 
The cDNA sequence of Girdwood S.A. is given herein as SEQ ID NO:4. 
Alternatively, the cDNA may have a sequence which differs from the cDNA of 
SEQ ID NO:4, but which has the same protein sequence as the cDNA given 
herein as SEQ ID NO:4. Thus, the cDNA may include one or more silent 
mutations. 
The phrase "silent mutation" as used herein refers to mutations in the CDNA 
coding sequence which do not produce mutations in the corresponding 
protein sequence translated therefrom. 
Likewise, the cDNA sequence of TR339 is given herein as SEQ ID NO:8. 
Alternatively, the cDNA may have a sequence which differs from the cDNA of 
SEQ ID NO:8, but which has the same protein sequence as the cDNA given 
herein as SEQ ID NO:8. Thus, the cDNA may include one or more silent 
mutations. 
The cDNAs encoding infectious Girdwood S.A. and TR339 virus RNA transcripts 
of the present invention include those homologous to, and having 
essentially the same biological properties as, the cDNA sequences 
disclosed herein as SEQ ID NO:4 and SEQ ID NO:8, respectively. Thus, cDNAs 
that hybridize to cDNAs encoding infectious Girdwood S.A. or TR339 virus 
RNA transcripts disclosed herein are also an aspect of this invention. 
Conditions which will permit other cDNAs encoding infectious Girdwood S.A. 
or TR339 virus transcripts to hybridize to the cDNAs disclosed herein can 
be determined in accordance with known techniques. For example, 
hybridization of such sequences may be carried out under conditions of 
reduced stringency, medium stringency, or even high stringency conditions 
(e.g., conditions represented by a wash stringency of 35-40% formamide 
with 5.times. Denhardt's solution, 0.5% SDS and 1.times. SSPE at 
37.degree. C.; conditions represented by a wash stringency of 40-45% 
formamide with 5.times. Denhardt's solution, 0.5% SDS, and 1.times. SSPE 
at 42.degree. C.; and conditions represented by a wash stringency of 50% 
formamide with 5.times. Denhardt's solution, 0.5% SDS and 1.times. SSPE at 
42.degree. C., respectively, to cDNA encoding infectious Girdwood S.A. or 
TR339 virus RNA transcripts disclosed herein in a standard hybridization 
assay. See J. SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL (2d 
ed. 1989)). In general, cDNA sequences encoding infectious Girdwood S.A. 
or TR339 virus RNA transcripts that hybridize to the cDNAs disclosed 
herein will be at least 30% homologous, 50% homologous, 75% homologous, 
and even 95% homologous or more with the cDNA sequences encoding 
infectious Girdwood S.A. or TR339 virus RNA transcripts disclosed herein. 
Promoter sequences and Girdwood S.A. virus or Sindbis virus strain TR339 
cDNA clones are operatively associated in the present invention such that 
the promoter causes the cDNA clone to be transcribed in the presence of an 
RNA polymerase which binds to the promoter. The promoter is positioned on 
the 5' end (with respect to the virion RNA sequence), of the cDNA clone. 
An excessive number of nucleotides between the promoter sequence and the 
cDNA clone will result in the inoperability of the construct. Hence, the 
number of nucleotides between the promoter sequence and the cDNA clone is 
preferably not more than eight, more preferably not more than five, still 
more preferably not more than three, and most preferably not more than 
one. 
Examples of promoters which are useful in the cDNA sequences of the present 
invention include, but are not limited to T3 promoters, T7 promoters, 
cytomegalovirus (CMV) promoters, and SP6 promoters. The DNA sequence of 
the present invention may reside in any suitable transcription vector. The 
DNA sequence preferably has a complementary DNA sequence bound thereto so 
that the double-stranded sequence will serve as an active template for RNA 
polymerase. The transcription vector preferably comprises a plasmid. When 
the DNA sequence comprises a plasmid, it is preferred that a unique 
restriction site be provided 3' (with respect to the virion RNA sequence) 
to the cDNA clone. This provides a means for linearizing the DNA sequence 
to allow the transcription of genome-length RNA in vitro. 
The cDNA clones can be generated by any of a variety of suitable methods 
known to those skilled in the art. A preferred method is the method set 
forth in U.S. Pat. No. 5,185,440 to Davis et al., the disclosure of which 
is incorporated in its entirety by reference, and Gubler et al., Gene 
25:263 (1983). 
RNA is preferably synthesized from the DNA sequence in vitro using purified 
RNA polymerase in the presence of ribonucleotide triphosphates and cap 
analogs in accordance with conventional techniques. However, the RNA may 
also be synthesized intracellularly after introduction of the cDNA. 
The Girdwood S.A. and TR339 cDNA clones and the infectious RNAs and 
infectious virus particles produced therefrom of the present invention are 
useful for the preparation of pharmaceutical formulations, such as 
vaccines. In addition, the cDNA clones, infectious RNAs, and infectious 
viral particles of the present invention are useful for administration to 
animals for the purpose of producing antibodies to the Girdwood S.A. virus 
or the Sindbis virus strain TR339, which antibodies may be collected and 
used in known diagnostic techniques for the detection of Girdwood S.A. 
virus or Sindbis virus strain TR339. Antibodies can also be generated to 
the viral proteins expressed from the cDNAs disclosed herein. As another 
aspect of the present invention, the claimed cDNA clones are useful as 
nucleotide probes to detect the presence of Girdwood S.A. or TR339 genomic 
RNA or transcripts. 
III. Infectious Virus Particles and Pharmaceutical Formulations 
The infectious virus particles of the present invention include those 
containing double promoter vectors and those containing replicon vectors 
as described hereinabove. Alternately, the infectious virus particles 
contain infectious RNAs encoding the Girdwood S.A. or TR339 genome. When 
the infectious RNA comprises the Girdwood S.A. genome, preferably the RNA 
has the sequence encoded by the cDNA given as SEQ ID NO:4. When the 
infectious RNA comprises the TR339 genome, preferably the RNA has the 
sequence encoded by the cDNA given as SEQ ID NO:8. 
The infectious, alphavirus particles of the present invention may be 
prepared according to the methods disclosed herein in combination with 
techniques known to those skilled in the art. These methods include 
transfecting an alphavirus-permissive cell with a replicon RNA including 
the alphavirus packaging segment and an inserted heterologous RNA, a first 
helper RNA including RNA encoding at least one alphavirus structural 
protein, and a second helper RNA including RNA encoding at least one 
alphavirus structural protein which is different from that encoded by the 
first helper RNA. Alternately, and preferably, at least one of the helper 
RNAs is produced from a cDNA encoding the helper RNA and operably 
associated with an appropriate promoter, the cDNA being stably transfected 
and integrated into the cells. More preferably, all of the helper RNAs 
will be "launched" from stably transfected cDNAs. The step of transfecting 
the alphavirus-permissive cell can be carried out according to any 
suitable means known to those skilled in the art, as described above with 
respect to propagation-competent viruses. 
Uptake of propagation-competent RNA into the cells in vitro can be carried 
out according to any suitable means known to those skilled in the art. 
Uptake of RNA into the cells can be achieved, for example, by treating the 
cells with DEAE-dextran, treating the RNA with LIPOFECTIN.RTM. before 
addition to the cells, or by electroporation, with electroporation being 
the currently preferred means. These techniques are well known in the art. 
See e.g., U.S. Pat. No. 5,185,440 to Davis et al., and PCT Publication No. 
WO 92/10578 to Bioption AB, the disclosures of which are incorporated 
herein by reference in their entirety. Uptake of propagation-competent RNA 
into the cell in vivo can be carried out by administering the infectious 
RNA to a subject as described in Section I above. 
The infectious RNAs may also contain a heterologous RNA segment, where the 
heterologous RNA segment contains a heterologous RNA and a promoter 
operably associated therewith. It is preferred that the infectious RNA 
introduces and expresses the heterologous RNA in bone marrow cells as 
described in Section I above. According to this embodiment, it is 
preferable that the promoter operatively associated with the heterologous 
RNA is operable in bone marrow cells. The heterologous RNA may encode any 
protein or peptide, preferably an immunogenic protein or peptide, a 
therapeutic protein or peptide, a hormone, a growth factor, an 
interleukin, a cytokine, a chemokine, an enzyme, a ribozyme, or an 
antisense oligonucleotide as described in more detail in Section I above. 
The step of facilitating the production of the infectious viral particles 
in the cells may be carried out using conventional techniques. See e.g., 
U.S. Pat. No. 5,185,440 to Davis et al., PCT Publication No. WO 92/10578 
to Bioption AB, and U.S. Pat. No. 4,650,764 to Temin et al. (although 
Temin et al., relates to retroviruses rather than alphaviruses). The 
infectious viral particles may be produced by standard cell culture growth 
techniques. 
The step of collecting the infectious virus particles may also be carried 
out using conventional techniques. For example, the infectious particles 
may be collected by cell lysis, or collection of the supernatant of the 
cell culture, as is known in the art. See e.g., U.S. Pat. No. 5,185,440 to 
Davis et al., PCT Publication No. WO 92/10578 to Bioption AB, and U.S. 
Pat. No. 4,650,764 to Temin et al. Other suitable techniques will be known 
to those skilled in the art. Optionally, the collected infectious virus 
particles may be purified if desired. Suitable purification techniques are 
well known to those skilled in the art. 
Pharmaceutical formulations, such as vaccines, of the present invention 
comprise an immunogenic amount of the infectious, virus particles in 
combination with a pharmaceutically acceptable carrier. An "immunogenic 
amount" is an amount of the infectious virus particles which is sufficient 
to evoke an immune response in the subject to which the pharmaceutical 
formulation is administered. An amount of from about 10.sup.3 to about 
10.sup.7 particles, and preferably about 10.sup.4 to 10.sup.6 particles 
per dose is believed suitable, depending upon the age and species of the 
subject being treated, and the immunogen against which the immune response 
is desired. 
Pharmaceutical formulations of the present invention for therapeutic use 
comprise a therapeutic amount of the infectious virus particles in 
combination with a pharmaceutically acceptable carrier. A "therapeutic 
amount" is an amount of the infectious virus particles which is sufficient 
to produce a therapeutic effect (e.g., triggering an immune response or 
supplying a protein to a subject in need thereof) in the subject to which 
the pharmaceutical formulation is administered. The therapeutic amount 
will depend upon the age and species of the subject being treated, and the 
therapeutic protein or peptide being administered. Typical dosages are an 
amount from about 10.sup.1 to about 10.sup.5 infectious units. 
Exemplary pharmaceutically acceptable carriers include, but are not limited 
to, sterile pyrogen-free water and sterile pyrogen-free physiological 
saline solution. Subjects which may be administered immunogenic amounts of 
the infectious virus particles of the present invention include but are 
not limited to human and animal (e.g., pig, cattle, dog, horse, donkey, 
mouse, hamster, monkeys) subjects. 
Pharmaceutical formulations of the present invention include those suitable 
for parenteral (e.g., subcutaneous, intracerebral, intradermal, 
intramuscular, intravenous and intraarticular) administration. 
Alternatively, pharmaceutical formulations of the present invention may be 
suitable for administration to the mucus membranes of a subject (e.g., 
intranasal administration by use of a dropper, swab, or inhaler). The 
formulations may be conveniently prepared in unit dosage form and may be 
prepared by any of the methods well known in the art. 
The following examples are provided to illustrate the present invention, 
and should not be construed as limiting thereof. In these examples, PBS 
means phosphate buffered saline, EDTA means ethylene diamine tetraacetate, 
ml means milliliter, .mu.l means microliter, mM means millimolar, .mu.M 
means micromolar, u means unit, PFU means plaque forming units, g means 
gram, mg means milligram, .mu.g means microgram, cpm means counts per 
minute, ic means intracerebral or intracerebrally, ip means 
intraperitoneal or intraperitoneally, iv means intravenous or 
intravenously, and sc means subcutaneous or subcutaneously. 
Amino acid sequences disclosed herein are presented in the amino to 
carboxyl direction, from left to right. The amino and carboxyl groups are 
not presented in the sequence. Nucleotide sequences are presented herein 
by single strand only in the 5' to 3' direction, from left to right. 
Nucleotides and amino acids are represented herein in the manner 
recommended by the IU-IUB Biochemical Nomenclature Commission, or (for 
amino acids) by either one letter or three letter code, in accordance with 
37 CFR .sctn. 1.822 and established usage. Where one letter amino acid 
code is used, the same sequence is also presented elsewhere in three 
letter code.