U.S. patent application number 10/357095 was filed with the patent office on 2003-09-11 for invasive bacterial vectors for expressing alphavirus replicons.
Invention is credited to Goudsmit, Jaap, Koff, Wayne, Sadoff, Jerald C..
Application Number | 20030170211 10/357095 |
Document ID | / |
Family ID | 22581211 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170211 |
Kind Code |
A1 |
Goudsmit, Jaap ; et
al. |
September 11, 2003 |
Invasive bacterial vectors for expressing alphavirus replicons
Abstract
The present invention is directed to a bacterial delivery system
for delivering alphavirus replicon DNA into an animal or animal
cells with the replicon encoding one or more heterologous genes to
be expressed in the animal or the animal cells. The bacteria are
invasive bacteria or attenuated invasive bacteria engineered to
contain a DNA vector that encodes the alphavirus replicon in a
eukaryotic expression cassette. The heterologous gene can encode an
antigen, a therapeutic agent, an immunoregulatory agent, an
anti-sense RNA, a catalytic RNA, a protein, a peptide, an antibody
or an antigen-binding fragment of an antibody. In a preferred
embodiment, the heterologous gene encodes one or more antigens
useful as a vaccine for HIV. In addition to the bacterial delivery
system, the invention provides methods of introducing and
expressing the heterologous gene(s) in animal or animal cells and
methods of stimulating or inducing an immune response.
Inventors: |
Goudsmit, Jaap; (Amsterdam,
NL) ; Sadoff, Jerald C.; (Washington, DC) ;
Koff, Wayne; (Stony Brook, NY) |
Correspondence
Address: |
HALE AND DORR LLP
300 PARK AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
22581211 |
Appl. No.: |
10/357095 |
Filed: |
February 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10357095 |
Feb 3, 2003 |
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09697236 |
Oct 26, 2000 |
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6531313 |
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60161448 |
Oct 26, 1999 |
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Current U.S.
Class: |
424/93.2 ;
435/252.3; 435/320.1 |
Current CPC
Class: |
C12N 15/86 20130101;
A61P 31/04 20180101; A61P 31/22 20180101; C12N 15/63 20130101; Y02A
50/484 20180101; A61K 48/00 20130101; Y02A 50/30 20180101; C12N
2760/14034 20130101; A61K 2039/5256 20130101; A61P 31/14 20180101;
A61P 31/16 20180101; A61P 31/18 20180101; Y02A 50/476 20180101;
A61P 37/06 20180101; A61P 35/00 20180101; C12N 15/85 20130101; C12N
15/87 20130101; A61K 39/12 20130101; C12N 2740/16034 20130101; C12N
2770/36143 20130101; A61K 2039/523 20130101; A61K 39/00 20130101;
A61P 31/20 20180101 |
Class at
Publication: |
424/93.2 ;
435/252.3; 435/320.1 |
International
Class: |
A61K 048/00; C12N
001/21; A01N 063/00; C12N 001/20; C12N 015/00; C12N 015/09; C12N
015/63; C12N 015/70; C12N 015/74 |
Claims
What is claimed is:
1. A bacterial delivery system which comprises live, attenuated
invasive Salmonella bacteria containing a DNA comprising a
eukaryotic expression cassette operably linked to an alphavirus
replicon DNA capable of amplification as RNA in animal cells,
wherein the alphavirus replicon DNA comprises at least one nucleic
acid control sequence operably linked to a heterologous nucleic
acid sequence to control expression of said heterologous genes.
2. The bacterial delivery system of claim 1, wherein said
Salmonella is Salmonella typhi.
3. The bacterial delivery system of claim 2, wherein said bacteria
are attenuated via an attenuating mutation in an aro gene, an asd
gene, an htrA gene or in a combination of one or more of these
genes.
4. The bacterial delivery system of claim 3, wherein said bacteria
have an attenuating mutation in an aro gene and an asd gene.
5. The bacterial delivery system of claim 4, wherein said aro gene
is aroA and/or aroD.
6. The bacterial delivery system of claim 3, wherein said bacteria
have an attenuating mutation in an aro gene and an htrA gene.
7. The bacterial delivery system of claim 6, wherein said aro gene
is aroA and/or aroD.
8. The bacterial delivery system of claim 1, wherein said
heterologous nucleic acid sequence comprises one or more coding
regions of a gene and wherein each coding region of said
heterologous nucleic acid sequence can be expressed separately or
as an operon.
9. The bacterial delivery system of claim 8, wherein said
heterologous nucleic acid sequence encodes an antigen or an
antigenic fragment of a protein from a viral pathogen.
10. The bacterial delivery system of claim 9, wherein said viral
pathogen is HIV.
11. The bacterial delivery system of any one of claims 1-8, wherein
said heterologous nucleic acid sequence encodes one or more HIV
genes selected from the group consisting of env, gag, pol, or an
antigenic fragment of a protein encoded by any one of said genes,
wherein said genes are from an HIV isolate and/or from a consensus
sequence of HIV isolates.
12. The bacterial delivery system of claim 11, wherein said
heterologous nucleic acid sequence encodes at least one antigen or
antigenic fragment from each of the HIV genes env, gag, pol, nef,
tat, and rev.
13. A method for introducing and expressing a gene in an animal
which comprises infecting said animal with the bacterial delivery
system of claim 11, and thereby obtaining expression of a gene
product encoded by said heterologous nucleic acid sequence in said
animal.
14. The method of claim 13 wherein infecting occurs by an
intranasal delivery route.
15. A method for inducing an immune response in an animal which
comprises infecting said animal with live, attenuated invasive
Salmonella bacteria containing a DNA comprising a eukaryotic
expression cassette operably linked to an alphavirus replicon DNA
capable of amplification as RNA in animal cells, wherein the
alphavirus replicon DNA encodes at least one antigen or antigenic
fragment of a protein, and wherein said antigen or said fragment is
expressed at a level sufficient to stimulate an immune response to
said antigen or said fragment.
16. The method of claim 15, wherein said Salmonella is Salmonella
typhi.
17. The method of claim 16, wherein said bacteria are attenuated
via an attenuating mutation in an aro gene, an asd gene, an htrA
gene or in a combination of one or more of these genes.
18. The method of claim 17, wherein said bacteria have an
attenuating mutation in an aro gene and an asd gene.
19. The method of claim 18, wherein said aro gene is aroA and/or
aroD.
20. The method of claim 17, wherein said bacteria have an
attenuating mutation in an aro gene and an htrA gene.
21. The method of claim 20, wherein said aro gene is aroA and/or
aroD.
22. The method of claim 15, wherein said antigen or said antigenic
fragment is a tumor antigen, a transplantation antigen or an
autoimmune antigen.
23. The method of any one of claims 15-21, wherein said antigen or
said antigenic fragment of a protein is from a viral pathogen, a
bacterial pathogen or a parasitic pathogen.
24. The method of claim 23, wherein said viral pathogen is HIV.
25. The method of any one of claims 15-21, wherein said antigen or
antigenic fragment is encoded by one or more HIV genes selected
from the group consisting of env, gag, pol, nef, tat, or rev,
wherein said HIV genes are from an HIV isolate and/or from a
consensus sequence of HIV isolates.
26. The method of claim 25, wherein said alphavirus replicon DNA
encodes at least one antigen or antigenic fragment from each of the
HIV genes env, gag, pol, nef, tat, and rev.
27. The method of any one of claims 15-21, wherein infecting occurs
by an intranasal delivery route.
28. The method of claim 25, wherein infecting occurs by an
intranasal delivery route.
29. The method of claim 26, wherein infecting occurs by an
intranasal delivery route.
30. A bacterial delivery system which comprises live, attenuated
invasive Shigella bacteria containing a DNA comprising a eukaryotic
expression cassette operably linked to an alphavirus replicon DNA
capable of amplification as RNA in animal cells, wherein the
alphavirus replicon DNA comprises at least one nucleic acid control
sequence operably linked to a heterologous nucleic acid sequence to
control expression of said heterologous genes.
31. The bacterial delivery system of claim 30, wherein said
Shigella is Shigella flexneri or Shigella flexneri 2a
32. The bacterial delivery system of claim 31, wherein said
bacteria are attenuated via an attenuating mutation in an aro gene,
a gua gene, a virG gene or in a combination of one or more of these
genes.
33. The bacterial delivery system of claim 32, wherein said
bacteria have an attenuating mutation in an aro gene and a virg
gene.
34. The bacterial delivery system of claim 33, wherein said aro
gene is aroA and/or aroD.
35. The bacterial delivery system of claim 32, wherein said
bacteria have an attenuating mutation in a gua gene and a virG
gene.
36. The bacterial delivery system of claim 35, wherein said aro
gene is aroA and/or aroD.
37. The bacterial delivery system of claim 30, wherein said
heterologous nucleic acid sequence comprises one or more coding
regions of a gene and wherein each coding region of said
heterologous nucleic acid sequence can be expressed separately or
as an operon.
38. The bacterial delivery system of claim 37, wherein said
heterologous nucleic acid sequence encodes an antigen or an
antigenic fragment of a protein from a viral pathogen, a bacterial
pathogen or a parasitic pathogen.
39. The bacterial delivery system of claim 38, wherein said viral
pathogen is HIV.
40. The bacterial delivery system of any one of claims 30-37,
wherein said heterologous nucleic acid sequence encodes one or more
HIV genes selected from the group consisting of env, gag, pol, or
an antigenic fragment of a protein encoded by any one of said
genes, wherein said genes are from an HIV isolate AND/or from a
consensus sequence of HIV isolates.
41. The bacterial delivery system of claim 40, wherein said
heterologous nucleic acid sequence encodes at least one antigen or
antigenic fragment from each of the HIV genes env, gag, pol, nef,
tat, and rev.
42. A method for introducing and expressing a gene in an animal
which comprises infecting said animal with the bacterial delivery
system of claim 40, and thereby obtaining expression of a gene
product encoded by said heterologous nucleic acid sequence in said
animal.
43. The method of claim 42, wherein infecting occurs by an
intranasal delivery route.
44. A method for inducing an immune response in an animal which
comprises infecting said animal with live, attenuated invasive
Shigella bacteria containing a DNA comprising a eukaryotic
expression cassette operably linked to an alphavirus replicon DNA
capable of amplification as RNA in animal cells, wherein the
alphavirus replicon DNA encodes at least one antigen or antigenic
fragment of a protein, and wherein said antigen or said fragment is
expressed at a level sufficient to stimulate an immune response to
said antigen or said fragment.
45. The method of claim 44, wherein said Shigella is Shigella
flexneri or Shigella flexneri 2a.
46. The method of claim 45, wherein said bacteria are attenuated
via an attenuating mutation in an aro gene, a gua gene, a virG gene
or in a combination of one or more of these genes.
47. The method of claim 46, wherein said bacteria have an
attenuating mutation in an aro gene and a virG gene.
48. The method of claim 47, wherein said aro gene is aroA and/or
aroD.
49. The method of claim 48, wherein said bacteria have an
attenuating mutation in a gua gene and a virG gene.
50. The method of claim 49, wherein said aro gene is aroA and/or
aroD.
51. The method of claim 44, wherein said antigen or said antigenic
fragment is a tumor antigen, a transplantation antigen or an
autoimmune antigen.
52. The method of any one of claims 44-50, wherein said antigen or
said antigenic fragment of a protein is from a viral pathogen, a
bacterial pathogen or a parasitic pathogen.
53. The method of claim 52, wherein said viral pathogen is HIV.
54. The method of claim of any one of claims 44-50, wherein said
antigen or antigenic fragment is encoded by one or more HIV genes
selected from the group consisting of env, gag, pol, nef, tat, or
rev, wherein said HIV genes are from an HIV isolate and/or from a
consensus sequence of HIV isolates.
55. The method of claim 54, wherein said alphavirus replicon DNA
encodes at least one antigen or antigenic fragment from each of the
HIV genes env, gag, pol, nef, tat, and rev.
56. The method of any one of claims 44-50, wherein infecting occurs
by an intranasal delivery route.
57. The method of claim 54, wherein infecting occurs by an
intranasal delivery route.
58. The method of claim 55, wherein infecting occurs by an
intranasal delivery route.
Description
[0001] This application is a continuation of U.S. Ser. No.
09/697,236 filed Oct. 26, 2000.
FIELD OF THE INVENTION
[0002] The present invention is directed to a bacterial delivery
system for delivering alphavirus replicon DNA into an animal or
animal cells, the replicon encoding a heterologous gene to be
expressed in the animal or the animal cells. The bacteria are
invasive bacteria or attenuated invasive bacteria engineered to
contain a DNA vector that encodes the alphavirus replicon in a
eukaryotic expression cassette. Upon bacterial infection, primary
transcription of the DNA vector is driven by the eukaryotic
expression vector and produces an alphavirus replicon RNA which is
transported the cytoplasm. That RNA is transcribed and translated
to express the heterologous gene encoded in the alphavirus
replicon. The heterologous gene may encode an antigen, a
therapeutic agent, an immunoregulatory agent, an anti-sense RNA, a
catalytic RNA, a protein, a peptide or any other molecule desired
for delivery to an animal or animal cell. In a preferred
embodiment, the heterologous gene encodes an antigen useful as a
vaccine for HIV. In addition to the bacterial delivery system, the
invention provides methods of introducing and expressing the
heterologous gene, methods of stimulating or inducing an immune
response and vaccines therefor.
BACKGROUND OF THE INVENTION
[0003] There are many applications for delivering DNA to animals or
animal cells including for gene therapy of acquired or inherited
diseases or conditions, for DNA-based vaccination, for
understanding genetic structure and for studying the molecular
mechanisms underlying gene expression.
[0004] Successful delivery of DNA to animal tissue has been
achieved by cationic liposomes (Watanabe et al., Mol. Reprod. Dev.,
38:268-274 (1994)), direct injection of naked DNA into animal
muscle tissue (Robinson et al., Vacc., 11:957-960 (1993); Hoffman
et al., Vacc., 12:1529-1533; (1994); Xiang et al., Virol.,
199:132-140 (1994); Webster et al., Vacc., 12:1495-1498 (1994);
Davis et al., Vacc., 12:1503-1509 (1994); and Davis et al., Hum.
Molec. Gen., 2:1847-1851 (1993)), and embryos (Naito et al., Mol.
Reprod. Dev., 39:153-161 (1994); and Burdon et al., Mol. Reprod.
Dev., 33:436-442 (1992)), or intradermal injection of DNA using
"gene gun" technology (Johnston et al., supra). A limitation of
these techniques is that they only efficiently deliver DNA to
parenteral sites. At present, effective delivery of eukaryotic
expression cassettes to mucosal tissue has been met with limited
success. This is presumably due to poor access to these sites,
toxicity of the delivery vehicles or instability of the delivery
vehicles when delivered orally.
[0005] For DNA-based vaccination, delivery by injection of naked
plasmid DNA has shown potential in mouse models for inducing both
humoral and cellular immune responses. However, in larger animals,
using DNA delivery for vaccination has been hampered by requiring
large amounts of DNA or inducing persistent expression of an
antigen with the potential for developing tolerance to the antigen.
Berglund reported a strategy for inducing or enhancing an immune
response by injecting mice with plasmid DNA containing an
alphavirus DNA expression vector having a recombinant Semliki
Forest Virus (SFV) replicon in a eukaryotic expression cassette
[Berglund et al.. (1998) Nature Biotechnology 16:562-565]. The
eukaryotic expression cassette controlled expression of the primary
nuclear transcription of the SFV replicon. This SFV replicon
transcript, encoding the heterologous antigen, was transported to
the cytoplasm and amplified by the self-encoded SFV replicase
complex. The amplified RNA replicon lead to high level production
of an mRNA encoding the heterologous antigen. Similar results were
described by Polo and his group [Polo et al.. (1998) Nature
Biotechnology 16:517-518; Hariharan et al.. (1998) J. Virol.
72:950-958]. Both groups found strong immune responses could be
induced using small amounts of input plasmid DNA. Although this
method allows greater expression from the input DNA vector, the
method still has the disadvantages associated with parenteral
delivery.
[0006] Alternatively, a method to deliver DNA to animals that
overcomes the disadvantages of conventional delivery methods is by
administering attenuated, invasive bacteria containing a bacterial
DNA vector having a eukaryotic expression cassette encoding the
gene to be expressed. For example, U.S. Pat. No. 5,877,159 to
Powell et al.. describes live bacteria that can invade animal cells
without establishing a productive infection or causing disease to
thereby introduce a eukaryotic expression cassette encoding an
antigen capable of being expressed by the animal cells. While this
method allows delivery of the DNA vaccine to mucosal surfaces,
including easy administration, a concern for vaccine delivery in
developing countries, it does not have the advantage of providing
amplifiable mRNA encoding the gene of interest.
[0007] Accordingly, the present invention combines use of a live
attenuated invasive bacteria with eukaryotic expression cassettes
encoding an alphavirus replicon to provide improved bacterial
delivery systems to deliver a heterologous gene, and preferably a
gene encoding an antigen, to an animal. Such systems have the
advantages of both bacterial delivery systems and alphavirus
replicon vectors and are efficacious, cost effective, and safe. The
bacterial delivery systems of the invention are particularly useful
for delivering DNA for gene therapy and vaccinations.
[0008] All cited references and patents are incorporated herein in
their entirety by reference.
SUMMARY OF THE INVENTION
[0009] In accordance with the invention, one embodiment is directed
to a bacterial delivery system which comprises a live invasive
bacteria or attenuated, invasive bacteria containing a DNA
comprising a eukaryotic expression cassette operably linked to an
alphavirus replicon DNA capable of amplification in animal cells,
wherein the alpha virus replicon DNA comprises an alphavirus
replicon comprises nucleic acid sequences operably linked to a
heterologous nucleic acid sequence to control expression thereof.
The heterologous nucleic acid sequence can encode an antigen, an
antigenic fragment of a protein, a therapeutic agent, an
immunoregulatory agent, an anti-sense RNA, a catalytic RNA, a
protein, a peptide or any other molecule encodable by DNA and
desired for delivery to an animal or animal cell. The heterologous
nucleic acid sequences can be obtained from a virus selected from
the group consisting of influenza virus, respiratory syncytial
virus, HPV, HBV, HCV, HIV, HSV, FeLV, FIV, HTLV-I, HTLV-II, and
CMV. Such viral sequences can encode one or more viral genes or
antigenic fragments thereof. The heterologous nucleotide sequence
can encode a cytokine, an interleukin, erythropoietin or other
immunostimulatory or immunoregulatory protein.
[0010] Another embodiment of this invention is directed to a method
for introducing and expressing a gene in an animal comprising
infecting said animal with live invasive bacteria, wherein said
bacteria contain a DNA comprising a eukaryotic expression cassette,
wherein said cassette expresses an alphavirus replicon RNA capable
of amplification in cells of said animal, wherein said RNA encodes
a heterologous gene product, and wherein said gene product is
expressed in said animal. The method is applicable to deliver genes
encoding an antigen, an antigenic protein fragment, a therapeutic
agent, an immunoregulatory agent, an anti-sense RNA, a catalytic
RNA, a protein, a peptide or any other molecule encodable by DNA
and desired for delivery to an animal or an animal cell.
[0011] Another aspect of the invention provides a method for
inducing an immune response in an animal which comprises infecting
said animal live attenuated invasive bacteria, wherein said
bacteria contain one or more DNAs comprising a eukaryotic
expression cassette, wherein said cassette expresses an alphavirus
replicon RNA capable of amplification in said animal, wherein said
RNA encodes an antigen, and wherein said antigen is expressed at a
level sufficient to stimulate an immune response to said antigen.
In a preferred embodiment the antigen is derived from a virus
[0012] Yet another aspect of the invention relates to a method for
introducing and expressing a gene in animal cells comprising (a)
infecting said animal cells with live invasive bacteria, wherein
the bacteria contain one or more DNAs comprising a eukaryotic
expression cassette, wherein said cassette expresses an alphavirus
replicon RNA capable of amplification in said animal cells and
wherein said RNA encodes a heterologous gene product; and (b)
culturing those cells under conditions sufficient to express the
gene product.
DETAILED DESCRIPTION OF THE INVENTION
[0013] As used herein, "invasive bacteria" are bacteria that are
capable of delivering eukaryotic expression cassettes to animal
cells or animal tissue. "Invasive bacteria" include bacteria that
are naturally capable of entering the cytoplasm or nucleus of
animal cells, as well as bacteria that are genetically engineered
to enter the cytoplasm or nucleus of animal cells or cells in
animal tissue. As used herein, "attenuated, invasive bacteria" are
invasive bacteria as defined herein which are capable of infecting
an animal host without establishing a productive infection and/or
causing disease in the infected host. Thus, at most an attenuated
bacterial strain may cause a self-limiting,
clinically-insignificant infection.
[0014] Attenuated bacteria of the invention can be prepared by
methods known in the art. For example, attenuating mutations can be
introduced into bacterial pathogens using non-specific mutagenesis
either chemically, using agents such as
N-methyl-N'-nitro-N-nitrosoguanidine, or using recombinant DNA
techniques; classic genetic techniques, such as Tn10 mutagenesis,
P22-mediated transduction, lambda-phage mediated crossover, and
conjugational transfer; or site-directed mutagenesis using
recombinant DNA techniques. Recombinant DNA techniques are
preferable. Examples of such attenuating mutations include, but are
not limited to:
[0015] (i) auxotrophic mutations, such as aro (Hoiseth et al.,
Nature, 291:238-239 (1981)), gua (McFarland et al., Microbiol.
Path., 3:129-141 (1987)), and (Park et al.., J. Bact.,
170:3725-3730 (1988), thy (Nnalue et al., Infect. Immun.,
55:955-962 (1987)), and asd (Curtiss, supra) mutations;
[0016] (ii) mutations that inactivate global regulatory functions,
such as cya (Curtiss et al., Infect. Immun., 55:3035-3043 (1987) ),
crp (Curtiss et al. (1987), supra), phoP/phoQ (Groisman et al.,
Proc. Natl. Acad. Sci., USA, 86:7077-7081 (1989); and Miller et
al., Proc. Natl. Acad. Sci., USA, 86:5054-5058 (1989)), phoP.sup.c
(Miller et al., J. Bact., 172:2485-2490 (1990)) or ompR (Dorman et
al., Infect. Immun., 57:2136-2140 (1989)) mutations;
[0017] (iii) mutations that modify the stress response, such as
recA (Buchmeier et al., Mol. Micro., 7:933-936 (1993)), htrA
(Johnson et al., Mol. Micro., 5:401-407 (1991)), htpR (Neidhardt et
al., Biochem. Biophys. Res. Com., 100:894-900 (1981)), hsp
(Neidhardt et al., Ann. Rev. Genet., 18:295-329 (1984)) and groEL
(Buchmeier et al., Sci., 248:730-732 (1990)) mutations;
[0018] (iv) mutations in specific virulence factors, such as lsyA
(Libby et al., Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag
or prg (Miller et al. (1990), supra; and Miller et al. (1989),
supra), iscA or virG (d'Hauteville et al., Mol. Micro., 6:833-841
(1992)), plcA (Mengaud et al., Mol. Microbiol., 5:367-72 (1991);
Camilli et al., J. Exp. Med, 173:751-754 (1991)), and act (Brundage
et al., Proc. Natl. Acad. Sci., USA, 90:11890-11894 (1993))
mutations;
[0019] (v) mutations that affect DNA topology, such as topA (Galan
et al., Infect. Immun., 58:1879-1885 (1990)) mutation;
[0020] (vi) mutations that block biogenesis of surface
polysaccharides, such as rfb, galE (Hone et al., J. Infect. Dis.,
156:164-167 (1987)) or via (Popoff et al., J. Gen. Microbiol.,
138:297-304 (1992)) mutations;
[0021] (vii) mutations that modify suicide systems, such as sacB
(Recorbet et al., App. Environ. Micro., 59:1361-1366 (1993); Quandt
et al., Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al., App.
Environ. Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA
(Molin et al., Ann. Rev. Microbiol., 47:139-166 (1993))
mutations;
[0022] (viii) mutations that introduce suicide systems, such as
lysogens encoded by P22 (Rennell et al., Virol., 143:280-289
(1985)), lambda murein transglycosylase (Bienkowska-Szewczyk et
al., Mol. Gen. Genet., 184:111-114 (1981)) or S-gene (Reader et
al., Virol., 43:623-628 (1971)); and
[0023] (ix) mutations that disrupt or modify the correct cell
cycle, such as minB (de Boer et al., Cell, 56:641-649 (1989))
mutation.
[0024] The attenuating mutations can be either constitutively
expressed or under the control of inducible promoters, such as the
temperature sensitive heat shock family of promoters (Neidhardt et
al., 1984, supra), or the anaerobically induced nirB promoter
(Harborne et al., Mol. Micro., 6:2805-2813 (1992)) or repressible
promoters, such as uapA (Gorfinkiel et al., J. Biol. Chem.,
268:23376-23381 (1993)) or gcv (Stauffer et al., J. Bact.,
176:6159-6164 (1994)).
[0025] The particular naturally occurring invasive bacteria (or
attenuated, invasive bacteria) employed in the present invention is
not critical thereto. One of ordinary skill in the art can readily
determine which bacterial strains are appropriate for use with the
animal or animal cells intended to be infected based on the
animal's or cells' susceptibility to infection by different
bacterial species. Examples of such naturally-occurring invasive
bacteria include, but are not limited to, Salmonella spp. Shigella
spp., Listeria spp., Rickettsia spp. and enteroinvasive Escherichia
coli. Any of these strains can be attenuated if needed using known
methods.
[0026] Examples of Shigella strains which can be employed in the
present invention include, but are not limited to, Shigella
flexneri 2a (ATCC No. 29903), Shigella sonnei (ATCC No. 29930), and
Shigella disenteriae (ATCC No. 13313). An attenuated Shigella
strain, such as Shigella flexneri 2a 2457T .DELTA. aroA .DELTA.
virG mutant CVD 1203 (Noriega et al., supra), Shigella flexneri
M90T .DELTA. icsA mutant (Goldberg et al., Infect. Immun.,
62:5664-5668 (1994)), Shigella flexneri Y SFL114 aroD mutant
(Karnell et al., Vacc., 10:167-174 (1992)), and Shigella flexneri
.DELTA. aroA .DELTA. aroD mutant (Verma et al., Vacc., 9:6-9
(1991)) are preferably employed in the present invention.
Alternatively, new attenuated Shigella spp. strains can be
constructed by introducing an attenuating mutation either
singularly or in conjunction with one or more additional
attenuating mutations.
[0027] Examples of Listeria strains which can be employed in the
present invention include Listeria monocytogenes (ATCC No. 15313).
Attenuated Listeria strains, such as L. monocytogenes .DELTA. actA
mutant (Brundage et al., supra) or L. monocytogenes .DELTA. plcA
(Camilli et al., J. Exp. Med., 173:751-754 (1991)) are preferably
used in the present invention. Alternatively, new attenuated
Listeria strains can be constructed by introducing one or more
attenuating mutations as described for Shigella spp. above.
[0028] Examples of Rickettsia strains which can be employed in the
present invention include Ricketsia rickettsiae (ATCC Nos. VR149
and VR891), Ricketsia prowaseckii (ATCC No. VR233), Ricketsia
tsutsugamuchi (ATCC Nos. VR312, VR150 and VR609), Ricketsia mooseri
(ATCC No. VR144), Ricketsia sibirica (ATCC No. VR151), and
Rochalimaea quitana (ATCC No. VR358). Attenuated Ricketsia strains
are preferably used in the present invention and can be constructed
by introducing one or more attenuating mutations as described for
Shigella spp. above.
[0029] Examples of enteroinvasive Escherichia strains which can be
employed in the present invention include Escherichia coli strains
4608-58, 1184-68, 53638-C-17, 13-80, and 6-81 (Sansonetti et aL,
Ann. Microbiol. (Inst. Pasteur), 132A:351-355 (1982)). Attenuated
enteroinvasive Escherichia strains are preferably used in the
present invention and can be constructed by introducing one or more
attenuating mutations as described for Shigella spp. above.
[0030] Examples of Salmonella strains which can be employed in the
present invention include Salmonella typhi (ATCC No. 7251) and S.
typhimurium (ATCC No. 13311). Attenuated Salmonella strains are
preferably used in the present invention and include S. typhi
aroAaroD (Hone et al., Vacc., 9:810-816 (1991)) and S. typhimurium
aroA mutant (Mastroeni et al., Micro. Pathol., 13:477-491 (1992))).
Alternatively, new attenuated Salmonella strains can be constructed
by introducing one or more attenuating mutations as described for
Shigella spp. above.
[0031] Examples of additional bacteria which can be genetically
engineered to be invasive include, but are not limited to, Yersinia
spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria
spp., Aeromonas spp., Franciesella spp., Corynebacterium spp.,
Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp.,
Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas
spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus
spp., and Erysipelothrix spp. These organisms can be engineered to
mimic the invasion properties of Shigella spp., Listeria spp.,
Rickettsia spp., or enteroinvasive E. coli spp. by inserting genes
that enable them to access the cytoplasm of an animal cell.
Examples of useful strains from these bacteria are found in U.S.
Pat. No. 5,877,159 and also include Mycobacterium bovis BCG.
[0032] Examples of such genes include the invasive proteins of
Shigella, hemolysin or the invasion plasmid of Escherichia, or
listeriolysin O of Listeria, as such techniques are known to result
in strains that are capable of entering the cytoplasm of infected
animal cells (Formal etal., Infect. immun., 46:465 (1984); Bielecke
etal., Nature, 345:175-176 (1990); Small et al., In:
Microbiology-1986, pages 121-124, Levine et al., Eds., American
Society for Microbiology, Washington, D.C. (1986); and Zychlinsky
et al., Molec. Micro., 11:619-627 (1994)). Any gene or combination
of genes, from one or more sources, that mediates entry into the
cytoplasm of animal cells will suffice. Thus, such genes are not
limited to bacterial genes, and include viral genes, such as
influenza virus hemagglutinin HA-2 which promotes endosmolysis
(Plank et al., J. Biol. Chem., 269:12918-12924 (1994)).
[0033] The above invasive genes can be introduced into the target
strain using chromosome or plasmid mobilization (Miller, A Short
Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1992); Bothwell et al., supra; and
Ausubel et al., supra), bacteriophage-mediated transduction (de
Boer, supra; Miller, supra; and Ausubel et al., supra), or chemical
(Bothwell et al., supra; Ausubel et al., supra; Felgner et al.,
supra; and Farhood, supra), electroporation (Bothwel et al., supra;
Ausubel et al., supra; and Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.) and physical transformation techniques (Johnston et
al., supra; and Bothwell, supra). The genes can be incorporated on
bacteriophage (de Boer et al., Cell, 56:641-649 (1989)), plasmids
vectors (Curtiss et al., supra) or spliced into the chromosome
(Hone et al., supra) of the target strain.
[0034] Furthermore, the bacteria for use in the invention can be
modified to increase their ability to infect mucosal surfaces and
tissues in an animal Such modifications permit the bacteria to
circumvent natural host barriers. Methods for constructing such
bacteria are described in U.S. Pat. No. 5,877,159.
[0035] In accordance with the invention the invasive or attenuated
invasive bacteria contain a DNA comprising a eukaryotic expression
cassette operably linked to an alphavirus replicon DNA. A
eukaryotic expression cassette is usually in the form of a plasmid
which contains elements needed for transcription of the alphavirus
replicon DNA and transport from the nucleus into the cytoplasm. For
example, RNA polymerase II cassettes provide the needed control and
regulatory elements. Hence, the elements for transcription include
but are not limited to promoters active in eukaryotic cells,
enhancers, transcription termination signals including
polyadenylation signals or polyA tracts, elements to facilitate
nucleocytoplasmic transport, elements to facilitate processing of
the 3' alphavirus replicon RNA into an authentic virus-like RNA 3'
ends and the like.
[0036] Hence, the particular eukaryotic cassette employed in the
present invention is not critical thereto, and can be selected
from, e.g., any of the many commercially available cassettes, such
as pCEP4 or pRc/RSV obtained from Invitrogen Corporation (San
Diego, Calif.), pXT1, pSG5, pPbac or pMbac obtained from Stratagene
(La Jolla, Calif.), pPUR or pMAM obtained from ClonTech (Palo Alto,
Calif.), and pSV.beta.-gal obtained from Promega Corporation
(Madison, Wis.), or synthesized either de novo or by adaptation of
a publically or commercially available eukaryotic expression
system.
[0037] The individual elements within the eukaryotic expression
cassette can be derived from multiple sources and may be selected
to confer specificity in sites of action or longevity of the
cassettes in the recipient cell. Such manipulation of the
eukaryotic expression cassette can be done by any standard
molecular biology approach.
[0038] Various promoters well-known to be useful for driving
expression of genes in animal cells, such as the viral-derived
SV40, CMV immediate early and, RSV promoters or eukaryotic derived
.beta.-casein, uteroglobin, .beta.-actin or tyrosinase promoters.
The particular promoter is not critical to the invention, unless
the object is to obtain tissue-specific expression. In this case,
the promoter can be selected which is only active in the desired
tissue or selected cell type. Examples of tissue-specific promoters
include, but are not limited to, .alpha.S1- and .beta.-casein
promoters which are specific for mammary tissue (Platenburg et al.,
Trans. Res., 3:99-108 (1994); and Maga et al., Trans. Res., 3:36-42
(1994)); the phosphoenolpyruvate carboxykinase promoter which is
active in liver, kidney, adipose, jejunum and mammary tissue
(McGrane et al., J. Reprod. Fert., 41:17-23 (1990)); the tyrosinase
promoter which is active in lung and spleen cells, but not testes,
brain, heart, liver or kidney (Vile et al., Canc. Res.,
54:6228-6234 (1994)); the involucerin promoter which is only active
in differentiating keratinocytes of the squamous epithelia (Carroll
et al., J. Cell Sci., 103:925-930 (1992)); and the uteroglobin
promoter which is active in lung and endometrium (Helftenbein et
al., Annal. N.Y. Acad. Sci., 622:69-79 (1991)).
[0039] Alternatively, cell specific enhancer sequences can be used
to control expression, for example human neurotropic papovirus JCV
enhancer regulates viral transcription in glial cells alone
(Remenick et al., J. Virol., 65:5641-5646 (1991)). Yet another way
to control tissue specific expression is to use a hormone
responsive element (HRE) to specify which cell lineages a promoter
will be active in, for example, the MMTV promoter requires the
binding of a hormone receptor, such as progesterone receptor, to an
upstream HRE before it is activated (Beato, FASEB J., 5:2044-2051
(1991); and Truss et al., J. Steroid Biochem. Mol. Biol.,
41:241-248 (1992)).
[0040] Suitable transcription termination elements include the SV
40 transcription termination region and terminators derived
therefrom.
[0041] Additional examples of eukaryotic expression cassettes
and/or regulatory elements suitable for expressing alphavirus
replicon DNA are described in U.S. Pat. Nos. 5,824,538 and
5,877,159.
[0042] The bacteria of the bacterial delivery systems can contain
one or more eukaryotic expression cassettes operably linked to an
alphavirus replicon. Such cassettes can be provided on the same or
different plasmids or DNA molecules contained in the bacteria. For
example, in some instances it may be desirable for the eukaryotic
expression cassette to be integrated into the bacterial chromosome
or other episomal DNA.
[0043] Alphavirus are from the Togavirus family and are well known
in the art. There are 26 known viruses and virus subtype classified
using the hemagglutination assay. See, e.g., U.S. Pat. No.
5,843,723 for list of the many of the alphaviruses. The commonly
studied alphaviruses include Sindbis, SFV, Venezuelan equine
encephalitis virus (VEE) and Ross River virus. The morphogenesis of
the viruses is fairly uniform and the virions are small enveloped
60-65 nm particles of positive strand RNA. The genomic RNA (49S
RNA) of alphaviruses is approximately 11-12 kb in length, and
contains a 5' cap and a 3' polyadenylate tail. Infectious enveloped
virus is produced by assembly of the viral nucleocapsid proteins
onto genomic RNA in the cytoplasm, and budding through the cell
membrane embedded with viral-encoded glycoproteins. During viral
replication, the genomic 49S RNA serves as template for synthesis
of a complementary negative strand. The negative strand in turn
serves as template for full-length genomic RNA and for an
internally initiated positive-strand 26S subgenomic RNA. The
nonstructural proteins are translated from the genomic RNA.
Alphaviral structural proteins are translated from the subgenomic
26S RNA. All viral genes are expressed as polyproteins and
processed into individual proteins by proteolytic cleavage
post-translation.
[0044] As used herein, an "alphavirus replicon" of the present
invention is used interchangeably to refer to RNA or DNA comprising
those portions of the alphavirus genome RNA essential for
transcription and export of a primary RNA transcript from the cell
nucleus to the cytoplasm, for cytoplasmic amplification of the
transported RNA and for subgenomic RNA expression of a heterologous
nucleic acid sequence. Thus, the replicon encodes and expresses
those non-structural proteins needed for cytoplasmic amplification
of the alphavirus RNA and expression of the subgenomic RNA. It is
further preferable that the alphavirus replicon can not be
encapsidated to produce alphavirus particles or virions. This is
achieved in replicons which lack one or more of the alphavirus
structural genes, and preferably all of the structural genes. In a
preferred embodiment, alphavirus replicons of the invention are
capable of being transcribed from a eukaryotic expression cassette
and processed into RNA molecules with authentic alphavirus-like 5'
and 3' ends.
[0045] Alphavirus replicons and expression vectors containing them
are well known in the art and many vectors containing a wide range
of alphavirus replicons have been described. Examples of such
replicons can be found, e.g., in U.S. Pat. Nos. 5,739,026;
5,766,602; 5,789,245; 5,792,462; 5,814,482; and 5,843,723 and in
Polo, supra, and Berglund, supra. While many of the features of
these alphavirus replicons are useful for the present invention not
all of them are essential for the reasons set forth above. So long
as a portion of the alphavirus replicon does not interfere with
production of the primary RNA, cytoplasmic amplification thereof
and expression of the heterologous nucleic acid sequence, such
portions can remain as part of the replicon. Those skilled in the
art can readily determine the nature of and remove any unnecessary
or interfering sequences.
[0046] The patents and references set forth above also describe
representative methods for constructing and producing the
alphavirus replicons of the inventions. Alphavirus replicons can be
prepared from any alphavirus or any mixture of alphavirus nucleic
acid sequences. In this regard the preferred alphavirus replicons
are derived from Sindbis virus, SFV, VEE or Ross River virus.
[0047] The alphavirus replicons can be incorporated as DNA into
eukaryotic expression cassettes using recombinant DNA techniques
conventional in the art.
[0048] In accordance with the invention, the alphavirus replicon
comprises a nucleic acid sequence operably linked to a heterologous
nucleic acid sequence to control expression thereof. The
heterologous nucleic acid sequence can encode an antigen, an
antigenic fragment of a protein, a therapeutic agent, an
immunoregulatory agent, an anti-sense RNA, a catalytic RNA, a
protein, a peptide or any other molecule encodable by DNA and
desired for delivery to an animal or animal cell. The heterologous
nucleic acid sequences can be obtained from a virus selected from
the group consisting of influenza virus, respiratory syncytial
virus, HPV, HBV, HCV, HIV, HSV, FeLV, FIV, HTLV-I, HTLV-II, and
CMV. Such viral sequences can encode one or more viral genes or
antigenic fragments thereof. The heterologous nucleotide sequence
can also encode a cytokine, an interleukin, erythropoietin or other
immunostimulatory or immunoregulatory protein.
[0049] As used herein, heterologous refers to the relationship
between the source of the alphavirus replicon and the source of the
heterologous nucleic acid sequence. Thus, the heterologous nucleic
acid gene will not be encode an alphavirus gene but could encode a
gene that is either foreign or endogenous to the animal cells that
have been infected with the bacterial delivery system of the
invention. As used herein, "foreign gene or nucleic acid sequence"
means a gene or a nucleic acid sequence encoding a protein or
fragment thereof or anti-sense RNA or catalytic RNA, which is
foreign to the recipient animal cell or tissue, such as a vaccine
antigen, immunoregulatory agent, or therapeutic agent. An
"endogenous gene or nucleic acid sequence" means a gene or a
nucleic acid sequence encoding a protein or part thereof or
anti-sense RNA or catalytic RNA which is naturally present in the
recipient animal cell or tissue.
[0050] The antigen may be a protein or antigenic fragment thereof
from viral pathogens, bacterial pathogens, and parasitic pathogens.
Alternatively, the antigen may be a synthetic gene, constructed
using recombinant DNA methods, which encode antigens or parts
thereof from viral, bacterial, parasitic pathogens. These pathogens
can be infectious in humans, domestic animals or wild animal
hosts.
[0051] The antigen can be any molecule that is expressed by any
viral, bacterial, parasitic pathogen prior to or during entry into,
colonization of, or replication in their animal host.
[0052] Single or multiple eukaryotic expression cassettes can be
delivered that express any combination of viral, bacterial,
parasitic antigens, or synthetic genes encoding all or parts or any
combination of viral, bacterial, parasitic antigens.
[0053] The viral pathogens, from which the viral antigens are
derived, include, but are not limited to, Orthomyxoviruses, such as
influenza virus; Retroviruses, such as RSV and SIV, Herpesviruses,
such as EBV; CMV or herpes simplex virus; Lentiviruses, such as
human immunodeficiency virus; Rhabdoviruses, such as rabies;
Picornoviruses, such as poliovirus; Poxviruses, such as vaccinia;
Rotavirus; and Parvoviruses. Examples of protective antigens of
viral pathogens include the HIV antigens Nef, p24, gp 120, gp41, gp
160, Tat, Rev, and Pol et al., Nature, 313:277-280 (1985)) and T
cell and B cell epitopes of gp120 (Palker et al., J. Immunol.,
142:3612-3619 (1989)); the hepatitis B surface antigen (Wu et al.,
Proc. Natl. Acad. Sci., USA, 86:4726-4730 (1989)); rotavirus
antigens, such as VP4 (Mackow et al., Proc. Natl. Acad. Sci., USA,
87:518-522 (1990)) and VP7 (Green et al., J. Virol., 62:1819-1823
(1988)), influenza virus antigens such as hemagglutinin or
nucleoprotein (Robinson et al.., Supra; Webster et al., Supra) and
herpes simplex virus thymidine kinase (Whitley et al., In: New
Generation Vaccines, pages 825-854). In the case of HIV the
antigens can be from any structural, accessory or regulatory gene,
and includes combinations or chimeras of such genes in multiple or
single alphavirus replicons.
[0054] The bacterial pathogens, from which the bacterial antigens
are derived, include but are not limited to, Mycobacterium spp.,
Helicobacter pylori, Salmonella spp., Shigella spp., E. coli,
Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas
spp., Vibrio spp., and Borellia burgdorferi.
[0055] Examples of protective antigens of bacterial pathogens
include the Shigella sonnei form 1 antigen (Formal et al., Infect.
Immun., 34:746-750 (1981)); the O-antigen of V. cholerae Inaba
strain 569B (Forrest et al., J. Infect. Dis., 159:145-146 (1989);
protective antigens of enterotoxigenic E. coli, such as the CFA/I
fimbrial antigen (Yamamoto et al., Infect. Immun., 50:925-928
(1985)) and the nontoxic B-subunit of the heat-labile toxin
(Clements et al., 46:564-569 (1984)); pertactin of Bordetella
pertussis (Roberts et al., Vacc., 10:43-48 (1992)), adenylate
cyclase-hemolysin of B. pertussis (Guiso et al., Micro. Path.,
11:423-431 (1991)), and fragment C of tetanus toxin of Clostridium
tetani (Fairweather et al., Infect. Immun., 58:1323-1326
(1990)).
[0056] The parasitic pathogens, from which the parasitic antigens
are derived, include but are not limited to, Plasmodium spp.,
Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp.,
Entamoeba spp., Eimeria spp., Leishmania spp., Schistosome spp.,
Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and
Onchocerea spp.
[0057] Examples of protective antigens of parasitic pathogens
include the circumsporozoite antigens of Plasmodium spp. (Sadoff et
al., Science, 240:336-337 (1988)), such as the circumsporozoite
antigen of P. bergerii or the circumsporozoite antigen of P.
falciparum; the merozoite surface antigen of Plasmodium spp.
(Spetzler et al., Int. J. Pept. Prot. Res., 43:351-358 (1994)); the
galactose specific lectin of Entamoeba histolytica (Mann et al.,
Proc. Natl. Acad. Sci., USA, 88:3248-3252 (1991)), gp63 of
Leishmania spp. (Russell et al., J. Immunol., 140:1274-1278
(1988)), paramyosin of Brugia malayi (Li et al., Mol. Biochem.
Parasitol., 49:315-323 (1991)), the triose-phosphate isomerase of
Schistosoma mansoni (Shoemaker et al., Proc. Natl. Acad. Sci., USA,
89:1842-1846 (1992)); the secreted globin-like protein of
Trichostrongylus colubriformis (Frenkel et al., Mol. Biochem.
Parasitol., 50:27-36 (1992)); the glutathione-S-transferase's of
Frasciola hepatica (Hillyer et al., Exp. Parasitol., 75:176-186
(1992)), Schistosoma bovis and S. japonicum (Bashir et al., Trop.
Geog. Med., 46:255-258 (1994)); and KLH of Schistosoma bovis and S.
japonicum (Bashir et al., supra).
[0058] In the present invention, the live invasive bacteria can
also deliver eukaryotic expression cassettes encoding a therapeutic
agent to animal cells or animal tissue. For example, the eukaryotic
expression cassettes can encode tumor-specific, transplant, or
autoimmune antigens or parts thereof. Alternatively, the eukaryotic
expression cassettes can encode synthetic genes, which encode
tumor-specific, transplant, or autoimmune antigens or parts
thereof. Examples of tumor specific antigens include prostate
specific antigen (Gattuso et al., Human Pathol., 26:123-126
(1995)), TAG-72 and CEA (Guadagni et al., Int. J. Biol. Markers,
9:53-60 (1994)), MAGE-1 and tyrosinase (Coulie et al., J.
Immunothera., 14:104-109 (1993)). Recently it has been shown in
mice that immunization with non-malignant cells expressing a tumor
antigen provides a vaccine effect, and also helps the animal mount
an immune response to clear malignant tumor cells displaying the
same antigen (Koeppen et al., Anal. N.Y. Acad. Sci., 690:244-255
(1993)). Examples of transplant antigens include the CD3 receptor
on T cells (Alegre et al., Digest. Dis. Sci., 40:58-64 (1995)).
Treatment with an antibody to CD3 receptor has been shown to
rapidly clear circulating T cells and reverse most rejection
episodes (Alegre et al., supra). Examples of autoimmune antigens
include IAS .beta. chain (Topham et al., Proc. Natl. Acad. Sci.,
USA, 91:8005-8009 (1994)). Vaccination of mice with an 18 amino
acid peptide from IAS beta. chain has been demonstrated to provide
protection and treatment to mice with experimental autoimmune
encephalomyelitis (Topham et al., supra).
[0059] Alternatively, in the present invention, live invasive
bacteria can deliver eukaryotic expression cassettes encoding
immunoregulatory molecules. These immunoregulatory molecules
include, but are not limited to, growth factors, such as M-CSF,
GM-CSF; and cytokines, such as IL-2, IL-4, L-5, IL-6, IL-10, IL-12
or IFN-gamma. Recently, delivery of cytokines expression cassettes
to tumor tissue has been shown to stimulate potent systemic
immunity and enhanced tumor antigen presentation without producing
a systemic cytokine toxicity (Golumbek et al., Canc. Res.,
53:5841-5844 (1993); Golumbek et al., Immun. Res., 12:183-192
(1993); Pardoll, Curr. Opin. Oncol., 4:1124-1129 (1992); and
Pardoll, Curr. Opin. Immunol., 4:619-623 (1992)).
[0060] The antisense RNA and catalytic RNA species delivered to
animal cells can be targeted against any molecule present within
the recipient cell or likely to be present within the recipient
cell. These include but are not limited to RNA species encoding
cell regulatory molecules, such as interleukin-6 (Mahieu et al.,
Blood, 84:3758-3765 (1994)), oncogenes such as ras (Kashani-Sabet
et al., Antisen. Res. Devel., 2:3-15 (1992)), causative agents of
cancer such as human papillomavirus (Steele et al., Canc. Res.,
52:4706-4711 (1992)), enzymes, viral RNA's and pathogen derived
RNA's such as HIV-1 (Meyer et al., Gene, 129:263-268 (1993);
Chatterjee et al., Sci., 258:1485-1488 (1992); and Yamada et al.,
Virol., 205:121-126 (1994)). The RNAs can also be targeted at
non-transcribed DNA sequences, such as promoter or enhancer
regions, or to any other molecule present in the recipient cells,
such as but not limited to, enzymes involved in DNA synthesis or
tRNA molecules (Scanlon et al., Proc. Natl. Acad. Sci. USA,
88:10591-10595 (1991); and Baier et al., Mol. Immunol., 31:923-932
(1994)).
[0061] In the present invention, live invasive bacteria can also
deliver eukaryotic expression cassettes encoding proteins to animal
tissue from which they can later be harvested or purified. An
example is the delivery of a eukaryotic expression cassette under
the control of a mammary specific viral promoter, such as derived
from mouse mammary tumor virus (ATCC No. VR731), encoding
.alpha.-antitrypsin to mammary tissue of a goat or sheep.
[0062] Alternatively an invasive bacteria carrying a eukaryotic
expression cassette can be introduced to a tissue site such that it
would not spread from such a site. This could be accomplished by
any of several methods including delivery of a very limited dose,
delivery of a severely attenuated auxotrophic strain, such as an
asd mutant (Curtiss et al., supra) that will be rapidly inactivated
or die, or delivery of a bacterial strain that contains attenuating
lesions, such as a suicide systems (Rennell et al., supra; and
Reader et al., supra) under the control of a strong promoter, such
as the anaerobic nirB promoter (Harborne et al., supra) which will
be switched on within the recipient host tissue. Additionally,
through use of different species and/or serotypes multiple doses of
invasive bacteria, the eukaryotic expression cassette of interest
can be given to an animal so as to manipulate expression levels or
product type. This approach obviates the need for specially raised
transgenic animals containing tissue specific promoters and having
tight control of expression, as is currently the case (Janne et
al., Int. J. Biochem., 26:859-870 (1994); Mullins et al.,
Hyperten., 22:630-633 (1993); and Persuy et al., Eur. J. Bichem.,
205:887-893 (1992)).
[0063] As a further alternative, single or multiple eukaryotic
expression cassettes encoding tumor-specific, transplant, and/or
autoimmune antigens, can be delivered in any single or multiple
combination with eukaryotic expression cassettes encoding
immunoregulatory molecules or other proteins.
[0064] The invasive bacteria containing the eukaryotic expression
cassette can be used to infect animal cells that are cultured in
vitro. The animal cells can be further cultured in vitro, and the
cells carrying the desired genetic trait can be enriched by
selection for or against any selectable marker introduced to the
recipient cell at the time of bactofection. Such markers may
include antibiotic resistance genes, e.g., hygromycin, or neomycin,
selectable cell surface markers, or any other phenotypic or
genotypic element introduced or altered by bactofection. These in
vitro-infected cells or the in vitro-enriched cells can then be
introduced into animals intravenously, intramuscularly,
intradermally, or intraperitoneally, or by any inoculation route
that allows the cells to enter the host tissue.
[0065] Alternatively, the invasive (or invasive, attenuated)
bacteria containing the eukaryotic expression cassettes can be
introduced to infect the animal by intravenous, intramuscular,
intradermal, intraperitoneally, peroral, intranasal, intraocular,
intrarectal, intravaginal, oral, immersion and intraurethral
inoculation routes.
[0066] The amount of the live invasive (or invasive, attenuated)
bacteria of the present invention to be administered will vary
depending on the species of the subject, as well as the disease or
condition that is being treated. Generally, the dosage employed
will be about 10.sup.3 to 10.sup.11 viable organisms, preferably
about 10.sup.5 to 10.sup.9 viable organisms. Alternatively, when
bactofecting individual cells, the dosage of viable organisms to
administered will be at a multiplicity of infection ranging from
about 0.1 to 10.sup.6, preferably about 10.sup.2 to 10.sup.4.
[0067] The invasive bacteria of the present invention are generally
administered along with a pharmaceutically acceptable carrier or
diluent.
[0068] The particular pharmaceutically acceptable carrier or
diluent employed is not critical to the present invention. Examples
of diluents include a phosphate buffered saline, buffer for
buffering against gastric acid in the stomach, such as citrate
buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0)
alone (Levine et al., J. Clin. Invest., 79:888-902 (1987); and
Black et al. J. Infect. Dis., 155:1260-1265 (1987)), or bicarbonate
buffer (pH 7.0) containing ascorbic acid, lactose, and optionally
aspartame (Levine et al., Lancet, II:467-470 (1988)). Examples of
carriers include proteins, e.g., as found in skim milk, sugars,
e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers
would be used at a concentration of about 0.1-90% (w/v) but
preferably at a range of 1-10% (w/v).
[0069] When infecting animal cells, the methods of the invention
can be used in mammalian, avian, insect cells and the like.
Preferably the mammalian cells are selected from the group
consisting of human, bovine, ovine, porcine, feline, buffalo,
canine, goat, equine, donkey, deer, and primate cells.
[0070] When infecting animals, the methods of the invention are
preferably used in mammals and birds. The preferred mammal is a
human.
* * * * *