NZ622860B2 - Use of non-subtype b gag proteins for lentiviral packaging - Google Patents
Use of non-subtype b gag proteins for lentiviral packaging Download PDFInfo
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- NZ622860B2 NZ622860B2 NZ622860A NZ62286012A NZ622860B2 NZ 622860 B2 NZ622860 B2 NZ 622860B2 NZ 622860 A NZ622860 A NZ 622860A NZ 62286012 A NZ62286012 A NZ 62286012A NZ 622860 B2 NZ622860 B2 NZ 622860B2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16051—Methods of production or purification of viral material
- C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
- C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
Abstract
Disclosed is a replication-defective lentiviral packaging vector lacking a ? site and encoding a subtype D HIV-1 Gag-Pol protein.
Description
/002363
USE OF NON-SUBTYPE B GAG NS FOR LENTIVIRAL PACKAGING
ound of the Invention
Recombinant vaccines have been developed with the progress of recombinant
DNA technology, allowing the modification of viral genomes to produce modified
viruses. In this manner, it has been possible to introduce genetic ces into non-
enic viruses, so that they encode genic proteins to be expressed in
target cells upon infection, in order to develop a specific immune response in their host.
Such es tute a major advance in vaccine technology er et al.,
Nat Rev Genet, 9(10) : 776-788, 2008). In particular, they have the advantage over
traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated
with the cture of inactivated vaccines.
Gene delivery using ed retroviruses (retroviral vectors) was introduced in
the early 1980s by Mann et al. (Cell, 33(1):153-9, 1983). The most ly used
oncogenic retroviral vectors are based on the Moloney murine leukemia virus (MLV).
They have a simple genome from which the oteins Gag, Pol and Env are
produced and are required in trans for viral replication (Breckpot et al., 2007, Gene
Ther, 14(11):847-62; He et al. 2007, Expert Rev vaccines, 6(6):913-24). Sequences
generally required in cis are the long terminal repeats (LTRs) and its vicinity: the
inverted repeats (IR or att sites) required for integration, the ing sequence LP, the
transport RNA-binding site (primer binding site, PBS), and some additional sequences
involved in reverse transcription (the repeat Rwithin the LTRs, and the polypurine
tracts, PPT, necessary for plus strand initiation). To generate replication-defective
retroviral vectors, the gag, pol, and env genes are generally entirely deleted and
replaced with an expression cassette.
Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have
emerged as promising tools for both gene therapy and immunotherapy purposes,
because they exhibit several advantages over other viral systems. In particular,
lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are
capable of transducing non-dividing cells, in ular dendritic cells (He et al. 2007,
Expert Rev vaccines, 6(6):913-24), ng antigen presentation through the
endogenous pathway.
Lentiviruses represent a genus of slow viruses of the Retroviridae family, which
includes the human immunodeficiency viruses (HIV), the simian immunodeficiency virus
(SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis
virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency
virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate uously
at variable rates during the course of the lifelong infection. Persistent replication of the
viruses in their hosts depends on their ability to circumvent host defenses.
The design of recombinant lentiviral s is based on the separation of the
cis- and acting sequences of the lentivirus. Efficient integration and replication in
viding cells requires the presence of two cis-acting sequences in the lentiviral
genome, the central polypurine tract (cPPT) and the central terminal sequence (CTS).
These lead to the formation of a triple-stranded DNA structure called the central DNA
“flap”, which maximizes the efficiency of gene import into the nuclei of viding
cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101(2) 173-85; Arhel et
al., 2007, EMBO J, :3025-37).
HIV-1 lentiviral vectors have been generated based on providing the subtype B
Gag, Pol, Tat and Rev proteins for packaging vectors in trans from a packaging
uct (Naldini et al, PNAS 15: 11382-8 (1996); Zufferey et al, Nature Biotechnology
:871-875, 1997); Dull etal, Journal of gy (1997)). These studies were
performed with subtype B Gag and Pol proteins. The effect of non-subtype B gag and
pol sequences in a HIV-1 packaging construct was not assessed.
There are many ent es of HIV-1 other than subtype B. Some
subtypes of HIV-1, such as C, E, and A, appear to be transmitted more efficiently than
HIV-1 subtype B, which is the major subtype in the United States and Europe. Essex et
al., Adv Virus Res. 1999; 53:71-88. The predominant subtype of HIV-1 that is found in
the developed Western World, clade B, differs considerably from those subtypes and
recombinants that exist in Africa and Asia, where the vast majority of fected
s reside. Spira et al., J. Antimicrobial Chemotherapy (2003) 51, 229-240. Thus,
serious discrepancies may exist between the subtype B retrovirus encountered in North
America and Europe and those viral subtypes that plague humanity on a global scale.
Id. Subtype diversity may impact on modes of HIV transmission. Homosexual and
intravenous drug abuse are the primary modes of transmission observed for clade B
s in Europe and the Americas. Id. In contrast, clades A, C, D and E predominate
in Africa and Asia where heterosexual transmission predominates. Id. In addition, some
studies suggest that AIDS ssion differs as a function of infecting subtype. Id.
Thus, it appears that HIV-1 subtype B is quite different than the other HIV-1 subtypes.
HIV phylogenic classifications are normally based either on nucleotide
sequences derived from multiple sub genomic regions (gag, pol and env) of the same
isolates, or on full-length genome sequence analysis. A phylogenic analysis of HIV-1
near-full length ces revealed that HIV-1 subtype B was most closely related
genetically to HIV subtype D (Figure 1). Phylogenic analyses of HIV-1 Gag and Pol
protein sequences also showed that HIV-1 subtype B was most closely related
genetically to HIV e D (Figure 2).
Nevertheless, HlVNDK, a subtype D virus, is significantly more cytopathic for
CD4+ lymphocytes than the HIVBRU prototype, a subtype B virus. This may be due
to enhanced nicity and infectivity of subtype D viruses. De Mareuil et al., J. Virol.
66: 6797 (1992). Phenotypic analysis of recombinant viruses indicated that 75 amino
acids from the N-terminal part of HIVNDK matrix (MA) protein, together with the HIV-
1-NDK envelope glycoprotein, are responsible for enhanced fusogenicity of HIVNDK
in CD4+ lymphocytes as well as for enhanced infectivity of HIVNDK in some CD4-
cell lines. Id.
There is a need in the art for lentiviral ing constructs producing higher
titers of packaged lentiviral vectors, in order to reduce injection volumes, increase
dosages, reduce the cost of vaccination, and se the number of patients that could
be treated with one batch. The current invention fulfills this need.
Brief Summary of the Invention
The gag-pol gene of e B of HIV-1 in a lentiviral packaging d
(construct p8.74) was replaced by the gag-pol gene of a subtype D HIV-1 to generate
construct pThV-GP-N. The ucts were used for iral vector production.
Approximately 2—fold higher titers were obtained using the pThV-GP-N d as
compared to uct p8.74. Thus, the l ofa subtype D virus increases the titer
of lentiviral vector particles relative to a Gag-Pol of a subtype B virus.
The invention encompasses a lentiviral ing vector comprising a subtype D
gag-pol sequence, particularly from HIV-1 NDK. In a preferred embodiment, the
lentiviral packaging vector comprises the nucleotide sequence ofSEQ ID NO:1. In a
preferred embodiment, the lentiviral packaging vector encodes the amino acid
sequence of SEQ ID N02.
The nucleotide sequence of SEQ ID NO 1 is:
atgggtgcgagagcgtcagtattaagcgggggaaaa“Laga,acanggaaagaattcggttac
ggccaggaggaaagaaaaaatatgcactaaaacaLLogaLa,gggcaagcagggagctagaacg
ac ,LaaLchggch ,LLagagaca:cagaaggc vgvaaacaaataataggacagcta
caacca,c,a ,Lcaaacagga :cagaagaaa ,LagaLcaL vagaLaaLacagLagcaaccctct
aLLng,aca ,gaaaggatagaggtaaaagacaccaaagaagctgtagaaaagatggaggaaga
acaaaacaaaagtaagaaaaagacacagcaagcagcagctgatagcagccagg':cagccaaaat
taccc:atagtgcagaacctacaggggcaaatggtacatcaggccaLa ,cacc,agaac:ttga
acgca:gggtaaaagtaatagaagaaaaggccttcagcccggaagtaa :acccatgttttcagc
agaaggagccaccccacaagat :taaacaccatgc:aaacacagtggggggacatcaa
gcagctatgcaaatgc :aaaagagacca:caatgacgaagc:gcagaatgggacagattacatc
cagvgcavgcagggcc:gttgcaccaggccaaatgagagaaccaaggggaagtgata :agcagg
aac,ac,agLacch “caggaacaaaLagca vgga “gacaagcaacccacc,a,cccag':agga
gaaa,c,aLaaaaga ,ggaLaaLchgggaL ,aaa ,aaaaLagLaagaaLgva,agccc,g,ca
gca vggacataagacagggaccaaaggaacct :ttagagac:atgtagaccgg,Lc,a,aa
aanC ,aagagccgagcaagcttcacagga :gtaaaaaac acagaaach,g ,gch
caaaa:gcaaacccagattgtaaaactatC :taaaagcat:gggaccacaggc ,acac vagaag
tgacagcatgccagggag':gggggggcccggcca :aaagcaagagvv vggcvgaggc
aatgagccaag':aacagg vcagc ,acLgcagLaaLgaLgcagagaggcaa ccca
agLaL,aang ,caac “gngcaaggaagggcacacagcaaaaaa vgcagggccc
ctagaaaaaagggcng vggaaa:gcggaagggaaggacaccaaatgaaagat :gcactgaaag
acaggctaattttt :agggaaga:ttggccttcccacaagggaaggccggggaat :ttcttcag
agcagaccagagccaacagccccaccagcagagagcL “cgggL vggggaggaga :aaccccct
ctcagaaacaggagcagaaagacaaggaacLgLaLcc “LLagc,,cchcaaaLcachLLng
caacgacccctcgtcacaataaagatagggggacagc :aaaggaagctctattagatacaggag
cagaLgaLacagva ,LagaagaaaLaaa vL vgccaggaaaa:ggaagccaaaaatgataggggg
aa:tggaggLLL “a ,caaagtaagacag “a ,gaLcaaaLac ,ca,agaaaLchvgga,aLaaa
gcvavgggvacagva “Lag vaggacctacacctg':caacaLaa ,ggaagaaaL,Lg ,gaccc
gc,gcach vaaa ,,,LccaaLvachc ,aLLgaaac:gtaccagtaaaattaaagcc
aggaa:ggatggcccaaaag vaaacaatggcca vgacgaagaaaaaataaaagcattaacag
aaa,L ,gLacagaaatggaaaaggaaggaaaaa ,,caagaattgggcc:gaaaatccatataa
Lac,ccaa,aLLLgcca aaaaagacag':accaag':ggagaaaa,Lag,agaLL,caga
gaacttaataagagaac :caagaL,Lc ,gggaggttcaat:aggaataccgcatcctgcagggc
:gaaaaagaaaaaatcag':aacagvac vggaLnggnga vgcaLa,L,chagLLcchLaga
:gaagattttaggaaa “a ,accgcaLL ,accaLachag ,aLaaacaatgagacaccagggatt
agaLaLcag,acaaLg ,chcccacagggatggaaagga gcaatattccaaagtagca
tgacaaaaa:cttagagccctttagaaaacaaaatccagaaaLagLLaLcLa ,caaLacanga
LgaLL,g,a ,gLaggaLc Lgac tagggcagcatagaacaaaaatagaggaattaaga
gaaca,cea “gagnggggaL ,accacaccagataaaaaacatcagaaagaacctccatttc
LLngangg ,LaLgaac ,cca,chgaLaaa ,ggacagtacagcctataaacctgccagaaaa
agaaagctggactgtcaa :gatatacagaagt :agtggggaaattaaactgggcaagccagatt
tatgcaggaattaaagtaaagcaatta :gtaaactccttaggggaaccaaagcactaacagaag
:agtaccactaacagaagaagcagaat :agaactggcagaaaacagggaaattctaaaagaacc
agtaca:ggagtgtattatgacccatcaaaagac cagaactacagaaacaaggggac
ggccaa:ggacataccaaa:ttatcaagaaccaL ,aaaaatctaaaaacaggaaagtatgcaa
gaacgaggggtgcccacac ,aaLgaLg aa aggcagtgcaaaaaa:agccac
catagtgatatggggaaagac ,chaaaL ,aaac,acccatacaaaaggaaacatgg
gaaacaegg ,ggaLagag La ,ngcaagccachggaLLcc “gag ,gggaatttgtcaataccc
CLCCLL,ag ,aaaaL ,ang ,accagt:agagaaggaacccataa :aggagcagaaactttcta
eggggcagc ,aa eagagagac:aaattaggaaaagcaggaLa ,g,Lachacagagga
agacagaaagLLg ,CCCL ,LcacLgacacgacaaatcagaagac “gag,Lacaagcaattaatc
,agCLL eacagga:tcgggattagaag':aaacatagtaacagat :cacaatatgcactaggaat
cattcaagcacaaccagataagag egaa ,cagagLLachachaaa ,aa,agagcagctaata
aaaaaggaaaaggLLLachggca eggg eaccagcacacaaaggaa eggaggaaatgaacaag
LagaLaaaL ,achag “cagggaa:caggaaagLacLa ,LLL,gga,ggaa,agaLaaggctca
ggaagaaca:gagaaa:atcacaacaa:tggagagcaa ,ggc,angaLLL,aachaccacct
gtggtagcgaaagaaa ,agLachagc ,nga ,cagcLaaaaggagaagccatgcatg
gacaagLagac ,gLag eccaggaa:atggcaa ,LagaL,gLacacatC':ggaaggae aagttat
cctggtagcag ecaeg eagccag ,ggc,aLa ,agaagcagaagttattccagcageaacgggg
caagaaacagca ,aceL ,CLCL ,aaaaL ,agcaggaagatggccagtaaaagtagte catacag
ataa:ggcagcaatt:caccag ,chacag ,Laaggccgcc :gttggtgggcagggetcaaaca
ggaa ,LngaaLLcchacaaLccccaaag':caaggag ,ag ,agaaLcLaLgaaLae agaatta
aagaaaattataggacaggtaagagatcaagctgaaca ,CL,aagacagcagtacaeatggcag
tccacaattttaaaagaaaaggggggat :gggggatacagtgcaggggaae gaataa
agacataa:agcaacagacatacaaactagagaa ,acaaaaacaaatcataaaaattcaaaa
tttcgggt:tattacagggacagcagagaLccaa ,ngaaaggaccagcaaagct:ctctgga
aaggtgaaggggcag wag ,aaLacaagacaaLag egacaeaaagg:agtaccaagaagaaaag'
aaaga:cattaggga ,La ,ggaaaacagatggcagnga egaLLg,nggcaagtagacagga
gagga:taac (SEQ ID NO 1).
The amino acid sequence of SEQ ID NO 2 is:
WO 46034
VLSGGKLDAWERIRLRPGGKKKYALKHLIWASRELERIALNPGLLETSEGCK
Q|IGQLQPSIQTGSEELRSLYNTIATLYCVHERIEVKDTKEAVEKMEEEQNKSKKKTQQ
AAADSSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEG
ATPQDLNTMLNTVGGHQAAMQMLKETINDEAAEWDRLHPVHAGPVAPGQMREPRG
SDIAGTTSTLQEQIAWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFR
DWDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPQATLEEMMTACQ
HKARVLAEAMSQVTGSVTAVMMQRGNFKGPRKSIKCFNCGKEGHTAKNC
RAPRKKGCWKCGREGHQMKDCSERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPA
ESFGFGEEITPSQKQEQKDKELYPLASLKSLFGNDPSSQFFREDLAFPQGKAGEFSSE
QTRANSPTSRELRVWGGDNPLSETGAEGQGTVSFSFPQITLWQRPLVTIKIGGQLKEA
LLDTGADDTVLEEMNLPGKWKPKMIGGIGGFIKVRQYDQIL|E|CGYKAMGTVLVGPTP
VNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPWKQWPLTEEKIKALTEICTEMEK
EGKISRIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKK
KKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQ
SSMTKILEPFRKQNPEIVIYQYMDDLWGSDLEIGQHRTKIEELREHLLRWGF'I'I'PDKK
HQKEPPFLWMGYELHPDKWTVQPIKLPEKESWTVNDIQKLVGKLNWASQIYAGIKVK
QLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAELQKQGDGQ
WTYQIYQEPFKNLKTGKYARTRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQK
ETWE'IWWIEYWQA'IWIPEWER/NTPPLVKLWYQLEKEPIIGAETFWDGAANRETKL
GKAGWTDRGRQKVVPFTDTTNQKTELQAINLALQDSGLEVNIVTDSQYALGIIQAQPD
KSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSQGIRKVLFLDGIDKAQEE
HEKYHNNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTH
LEGKV|LVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKVVHTDNGSNFTSATV
KAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHN
FKRKGGIGGYSAGERIIDIIATDIQTRELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKG
EGAVVIQDNSDIKVVPRRKVKIIRDYGKQMAGDDCVASRQDED (SEQ ID N02).
The invention encompasses a lentiviral packaging vector comprising a subtype D
MA sequence, particularly from HIV-1 NDK. In a preferred embodiment, the |entivira|
packaging vector encodes an MA protein comprising the amino acid ce ofSEQ
ID NO:3.
The amino acid sequence of SEQ ID NO 3 is:
MGARASVLSGGKLD'IWERIRLRPGGKKKYALKHLIWASRELERFTLNPGLLETSEGCK
Q|IGQLQPSIQTGSEEIRSLYNTVATLYCVHERIEVKDTKEAVEKMEEEQNKSKKKTQQ
AAADSSQVSQNY (SEQ ID NO 3).
Preferably, the lentiviral packaging vector encoding the HIV Gag MA protein
generates at least a 1.5 fold increase, or at least a 2-fold increase, in the titer of a
packaged lentiviral vector as compared to the lentiviral packaging vector ng an
HIV Gag MA protein of HIV-1 BRU, e.g., p8.74. Preferably, the lentiviral packaging
vector is replication-defective and lacks a LIJ site.
In a preferred embodiment, the lentiviral packaging vector encodes an HIV Gag
MA protein having an amino acid at position 12 that is not a glutamic acid and an amino
acid at position 15 that is not an arginine. Preferably, the iral packaging vector
does not have both a valine at on 46 and a leucine at on 61.
In a preferred embodiment, the amino acid at position 12 of the MA protein is a
lysine. In a preferred embodiment, the amino acid at on 15 is a threonine. In a
preferred embodiment, the amino acid at position 15 is an alanine. In a preferred
embodiment, the amino acid at position 46 is a leucine. In a red embodiment, the
amino acid at position 61 is an isoleucine. In a preferred ment, the amino acid at
position 61 is a nine.
In one embodiment, the vector does not encode a functional Env protein.
The invention also encompasses methods for making the above lentiviral
packaging s and methods for using these lentiviral packaging vectors.
Brief Description of the Drawings
The invention is more fully understood through reference to the drawings.
Fig. 1 depicts phylogenic trees of HIV viruses.
Fig. 2A and B depict phylogenic trees of HIV GAG (A) and POL (B) proteins.
GAG and POL protein sequences were obtained from:
http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.htm|. For each known HIV
clade, one patients (for clade B) or two ts’ virus sequences were randomly
chosen and GAG and POL protein sequences were compared to the reference clade B
proteins (B.FR.83.HXBZ_LAI_IIIB_BRU.KO3455). Alignments were performed using
Vector NTI advance 11 (Invitrogen).
Fig. 3 depicts titers obtained using the p8.74 and the pThV-GP-N plasmids for
vector production. Lentiviral particles were produced using the proviral plasmid -
CMV-GFP), the pseudotyping plasmid (pTHV-VSV.G) and either the commonly used
packaging plasmid ed from the BRU strain, p8,74) or an NDK-derived packaging
plasmid (pTHV-GP-N). With each packaging plasmid, 18 independent transfections
were performed and the particles titers were ed by FACS analysis. Similar
results were also obtained using a vector employing a proviral plasmid containing the
B2 microglobulin promoter driving HIV antigen expression.
Fig. 4 depicts production ofwild type BRU and NDK viruses and tion of
their respective early phase efficiency. 293T cells were transfected either with plasmid
encoding for the wild type BRU (pBRU) or NDK (pNDK-N) virus. Viral supernatants
were collected after 48 hours and diluted to infect P4-CCR5 cells passing a
stable luciferase reporting gene which expression is driven by the HIV LTR, allowing a
luciferase production in presence of the TAT protein (brought by the virus). Serial
dilution of either BRU or NDK viruses were used to infect P4-CCR5 cells and the
luciferase expression (A) or the luciferase/P24 ratio (B) were measured.
Fig. 5 s vector production using different ratios of BRU ) and NDK
(pThV GP-N) derived packaging plasmids. For each conditions (from Oug NDK + 10ug
BRU to 10ug NDK + Oug BRU), the titer (gray bars) and the P24 level (black squares)
were measured.
Fig. 6 depicts a Western blot of vector atants produced using either BRU
(8,74) or NDK (pThV GP-N) derived packaging plasmids. The P24 protein and
precursors detection was performed using the NIH anti-P24 MAB 12-5C).
Fig. 7 depicts a sequence alignment of the N-terminal MA sequences of a clade
B virus (BRU; SEQ ID NO:3) with a clade D virus (NDK; SEQ ID NO:4).
Fig. 8 depicts a sequence alignment of the N-terminal MA sequences of clade B
quses.
Fig. 9 depicts a sequence alignment of the N-terminal MA sequences of clade D
quses.
Fig. 10 s a sequence alignment of the N-terminal MA sequences of a clade
B virus (BRU) with viruses of other clades.
Detailed Description of the Invention
Subtype B HIV-1 viruses differ from other HIV-1 subtypes in that e B
viruses appear to be transmitted less efficiently and have a different mode of
transmission. Nevertheless, e B viruses have been used extensively in the
generation of HIV-1 lentiviral vectors and lentiviral packaging s. To determine
whether the Gag and Pol proteins of btype B viruses could be used for the
generation of lentiviral packaging vectors, the gag-pol gene of HIV-1 subtype B in a
lentiviral packaging plasmid (construct p8.74)was replaced by the gag-pol gene ofa
subtype D HIV-1 to generate uct pThV-GP-N. The constructs were used for
lentiviral vector production. Approximately 2-fold higher titers were obtained using the
pThV-GP-N plasmid as ed to construct p8.74 (Figure 3). Thus, the subtype D
HIV-1 Gag-Pol in the packaging vector increased the titer of lentiviral vector over the
titer seen with a subtype B HIV-1 Gag-Pol.
Increased titer of the lentiviral vector is beneficial in allowing the reduction in
contaminants in a given dose of lentiviral . This facilitates a reduction in injection
volumes, and an increase in possible dosages. It further facilitates ng the cost of
vaccination by reducing the quantity of materials and labor necessary to achieve a
particular dose, and by increasing the number of patients that could be treated with a
single batch of lentival vector.
Serial dilutions of HIV-1 BRU and HIV-1 NDK viruses indicated that the HIV-1
NDK virus was more nt in the early phases of the virus life cycle e 4). By
mixing different amounts of the subtype B and subtype D packaging vectors, it was
demonstrated that the subtype D packaging vector increased both the titer and the level
of p24 in the lentiviral vector preparations (Figure 5). The p24 in the lentiviral vector
preparations using the subtype D packaging vector was also observed to be less
completely processed, showing higher levels of p24 precursors (Figure 6). Thus, the
Gag protein in the subtype D packaging vector was exhibiting various differences from
the subtype B packaging vectors.
It is known that, with HIV-1 Env, the 75 amino acids from the N-terminal part of
NDK matrix (MA) protein is sible for ed fusogenicity of HlVNDK
in CD4+ lymphocytes as well as for enhanced infectivity of HlVNDK in some CD4-
cell lines. De Mareuil et al., J. Virol. 66: 6797 (1992). Since the Env protein used for
generation of lentiviral s with the subtype B and subtype D ing vectors is
the same (i.e., VSV), only the differences in HIV-1 MA are present in the subtype B and
subtype D packaging vectors.
The conservation and divergence of amino acids in the inal 75 amino
acids of M from various clades of HIV-1 viruses was examined. HIV-1 NDK showed 10
differences in amino acids from HIV-1 BRU (Figure 7). However, only eight of these
ences were seen when HIV-1 NDK was compared to an assortment of 20 ent
HIV-1 subtype B viruses (Figure 8). When other subtype D viruses were included in the
comparison, only 4 of these differences remained e 9). These differences are the
absence of a glutamic acid at amino acid position 12, the absence of an arginine at
amino acid position 15, the absence of a valine at amino acid on 46, and the
absence of a leucine at amino acid position 61 in subtype D viruses.
When other subtype viruses were included in the comparison, the other subtypes
appeared to align with subtype D, preserving nearly all of these differences (Figure 10).
Nearly all of the non-subtype B viruses exhibited a lysine at position 12. Many of the
non-subtype B viruses exhibited an alanine at position 15. Nearly all of the non-subtype
B viruses exhibited a leucine at on 46. Nearly all of the non-subtype B viruses
exhibited a methionine or isoleucine at position 61. The non-subtype B viruses
ted the consensus of a lysine at position 12, an amino acid other than arginine at
position 15, a e at position 46, and an isoleucine or methionine at position 61.
The invention encompasses ing vectors encoding non-subtype B Gag
and/or Pol proteins and host cells comprising these s. The invention further
encompasses methods for making packaging vectors encoding non-subtype B Gag
and/or Pol proteins. The invention also encompasses methods for using these
ing vectors to generate lentiviral s, and lentiviral vectors comprising non-
subtype B Gag and/or Pol proteins.
Packaging Vectors
The invention encompasses packaging vectors encoding non-subtype B Gag
and/or Pol proteins. A lentiviral “packaging vector” is defined herein as a nucleic acid
sequence not encoding functional HIV-1 Env and lacking a LIJ site, but capable of
expressing lentiviral Gag and/or Pol proteins that can be incorporated into viral particles
when cotransfected with a vector containing appropriate lentiviral cis-acting signals for
packaging. The lentiviral ing vector of the invention is unable to replicate itself by
ing and reverse ribing its own sequence.
The packaging vector can be an RNA or DNA vector. The non-subtype B Gag
and Pol ns can be selected from subtype A, subtype C, subtype D, subtype E,
subtype F1, subtype F2, subtype G, subtype H, and subtype J ns, and
recombinants thereof. A preferred packaging vector comprises SEQ ID NO:1 or
encodes SEQ ID N02.
The invention encompasses packaging vectors encoding non-subtype B Gag
ns and host cells comprising these vectors. The non-subtype B Gag proteins can
be selected from subtype A, e C, subtype D, subtype E, e F1, subtype F2,
subtype G, subtype H, and e J proteins, and recombinants thereof. A preferred
packaging vector encodes the Gag protein portion of SEQ ID NO:2.
The ion encompasses packaging vectors encoding non-subtype B MA
proteins and host cells comprising these vectors. The non-subtype B MA ns can
be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2,
subtype G, subtype H, and subtype J ns, and recombinants thereof. A preferred
packaging vector encodes SEQ ID NO:3 or the MA protein portion ofSEQ ID NO:2.
In various embodiments, the packaging vector comprises SEQ ID NO:1 or a
nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical with SEQ ID
NO:1. In various embodiments, the ing vector s SEQ ID NO:2, SEQ ID
NO:3, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical
with SEQ ID NO:2 or SEQ ID NO:3.
As used herein, the t identity of two nucleic acid sequences can be
determined by comparing sequence information using the GAP computer program,
version 6.0 described by DevereuX et al. (Nucl. Acids Res. 12:387, 1984) and ble
from the University ofWisconsin Genetics Computer Group (UWGCG), using the
default ters for the GAP program including: (1) a unary ison matrix
(containing a value of 1 for identities and 0 for entities) for nucleotides, and the
weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986,
as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,
National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no
penalty for end gaps.
In various embodiments, the vector comprises a sequence encoding an HIV-1
MA protein having one or more of the following features:
the absence of a glutamic acid at amino acid position 12;
the absence of an arginine at amino acid position 15;
the absence of a valine at amino acid position 46; and
the absence of a leucine at amino acid position 61.
In various embodiments, the vector comprises a sequence encoding an HIV-1
MA protein having one or more of the following features:
the amino acid at position 12 is a ;
the amino acid at position 15 is a threonine;
the amino acid at position 15 is an alanine;
the amino acid at on 46 is a leucine;
the amino acid at position 61 is an isoleucine; and/or
the amino acid at position 61 is a methionine.
The packaging vector preferably encodes HIV-1 Gag and Pol. Most preferably,
the packaging vector encodes an HIV-1 Gag MA protein.
The packaging vector can contain viral or non-viral sequences for expression of
Gag and Pol. The ing vector can contain an HIV-1 LTR or the U3 region of an
HIV-1 LTR. In other embodiments, the packaging vector does not contain HIV-1 LTRs.
Any promoter can be used to drive expression of Gag and Pol. Preferably, the promoter
is a strong promoter in human cells. Most preferably, the packaging vector contains a
Cytomegalovirus (CMV) promoter, a CMV early er/chicken B actin (CAG)
promoter, a Rous Sarcoma Virus (RSV) promoter, a human oglycerate kinase
(hPGK) promoter, or a U3 from an LTR (e.g., roliferative sarcoma virus (MPSV)
U3) promoter driving expression of the encoded genes, e.g. gag and pol.
Preferably, the packaging vector contains a polyadenylation signal. Any
polyadenylation signal can be. Preferably, polyadenylation signal is a strong signal in
human cells. Most preferably, the polyadenylation signal is a human (12 globin or a
Bovine Growth e (BGH) polyadenylation signal.
Preferably, the packaging vector ns a Rev-responsive element (RRE). In a
preferred embodiment, the packaging vector expresses an HIV-1 Rev protein. In a
preferred embodiment, the packaging vector contains at least one splice donor site and
at least one splice acceptor site. In one embodiment, the packaging vector ses
an HIV-1 Tat protein.
In preferred embodiments, the packaging vector lacks sequences encoding HIV-
1 Vif, Vpr, Vpu, and/or Nef. The packaging vector can comprise a sequence encoding
an HIV-1 MA n having one or more of the features discussed herein.
In one embodiment, the packaging vector encodes only 1 HIV-1 protein, Gag. In
one embodiment, the packaging vector encodes only 2 HIV-1 proteins, Gag and Pol. In
one embodiment, the packaging vector encodes only 3 HIV-1 proteins, selected from
Gag, Pol, Rev, and Tat. In one embodiment, the ing vector encodes only 4 HIV-
1 proteins, Gag, Pol, Rev, and Tat.
In one embodiment, the vector ses (from 5’ to 3’) a CMV promoter, a
nucleic acid sequence encoding HIV-1 Gag-Pol, an exon encoding part of Tat and Rev,
a splice donor site, an intron containing an RRE, a splice or site, an exon
encoding part of Tat and Rev, and a polyadenylation signal. Preferably, the HIV-1 Gag-
Pol is a subtype D HIV-1 Gag-Pol.
2012/002363
The packaging vector may further contain an origin for ation in bacteria or
eukaryotic cells. The packaging vector may contain a selectable marker gene for
ion in bacteria or eukaryotic cells.
The invention encompasses host cells comprising the packaging vectors of the
invention. The host cells can be transiently ected with the packaging vectors of
the invention. The host cells can be cell lines with the packaging vectors of the
invention integrated into the genome of the host cell. Many different cells are suitable
host cells Preferably, the cells are human cells, most preferably an immortalized human
cell line. In one embodiment, the cells are HEK 293T cells. In one embodiment, the
cells are HeLA, HT1080 or PER C6 cells (Delenda et al, Cells for Gene Therapy and
vector Production, from Methods in Biotechnology, Vol 24 l Cell Biotechnology :
Methods and Protocols, 2nOI Ed. Edited by R. Portner, Humana Press Inc, Totowa, NJ).
Packaging Systems
The invention asses lentiviral packaging systems sing cells
expressing non-subtype B Gag and/or Pol proteins. A lentiviral “packaging system” is
defined herein as a cell-based system comprising cells expressing at least lentiviral
Gag and Pol proteins in the absence of a LIJ site, and capable of packaging and reverse
ribing an exogenous nucleic acid containing a LIJ site. The cells of the lentiviral
packaging system can also express other viral proteins. Preferably, the lentiviral
packaging system expresses an envelope protein. The envelope protein can be a
lentiviral (e.g., HIV-1 Env) or non-lentiviral (e.g., VSV, Sindbis virus, Rabies virus)
envelope protein. In various embodiments, the lentiviral packaging system expresses
an HIV-1 Tat and/or Rev n.
In various embodiments, the cells of the lentiviral packaging system contain
sequences encoding HIV-1 Gag and/or Pol stably integrated into their . In
s embodiments, the cells of the lentiviral packaging system contain sequences
encoding an envelope n stably integrated into their genome. In various
embodiments, the cells of the lentiviral packaging system contain sequences encoding
HIV-1 Tat and/or Rev stably integrated into their genome.
In various embodiments, the cells of the lentiviral packaging system transiently
express HIV-1 Gag and/or Pol ns. In various embodiments, the cells of the
lentiviral packaging system transiently express an envelope protein. In various
embodiments, the cells of the lentiviral packaging system transiently express HIV-1 Tat
and/or Rev proteins.
wo 2013/046034
The cells of the lentiviral packaging system can express non-subtype B Gag and
Pol proteins selected from subtype A, e C, subtype D, subtype E, subtype F1,
subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof.
In one embodiment, the cells of the lentiviral ing system comprise SEQ ID NO:1
or express SEQ ID NO:2.
In various embodiments, the cells of the lentiviral packaging system express
non-subtype B Gag proteins. The non-subtype B Gag proteins can be selected from
subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, e G,
subtype H, and subtype J proteins, and recombinants thereof.
In various embodiments, the cells of the iral packaging system express
non-subtype B MA proteins. The non-subtype B MA proteins can be selected from
subtype A, subtype C, subtype D, subtype E, subtype F1, e F2, subtype G,
subtype H, and subtype J proteins, and inants thereof. A preferred packaging
vector encodes SEQ ID NO:3 or the MA protein portion ofSEQ ID NO:2.
In various embodiments, the cells of the lentiviral packaging system can contain
any of the lentiviral vectors of the invention.
In various embodiments, the cells of the lentiviral packaging system contain SEQ
ID NO:1 or a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical
with SEQ ID NO:1. In various embodiments, the cells of the lentiviral packaging system
express a protein with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or an
amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% cal with SEQ ID
N02 or SEQ ID NO:3.
Methods of Producing Packaging Vectors
The ion encompasses methods for making packaging vectors encoding
non-subtype B Gag and/or Pol proteins. The packaging vector can comprise any of the
features discussed herein.
In one embodiment, the method comprises inserting a Gag and/or Pol sequence
from a non-subtype B HIV-1 virus into a plasmid under the control of a V
promoter (e.g., CMV promoter) to generate a packaging . In one embodiment, the
ing vector s only 1 HIV-1 protein. In one embodiment, the packaging
vector encodes only 2 HIV-1 proteins. In one ment, the packaging vector
encodes only 3 HIV-1 proteins, selected from Gag, Pol, Rev, and Tat. In one
embodiment, the packaging vector encodes only 4 HIV-1 proteins, Gag, Pol, Rev, and
Tat.
In various embodiments, the plasmid comprises one or more of a CMV promoter,
an exon encoding part of Tat and Rev, a splice donor site, an intron containing an RRE,
a splice acceptor site, an exon encoding part of Tat and Rev, and a polyadenylation
signal.
In various embodiments, the packaging vector comprises a CMV promoter, an
exon ng part of Tat and Rev, a splice donor site, an intron containing an RRE, a
splice or site, an exon ng part of Tat and Rev, and a polyadenylation
signal.
The non-subtype B HIV-1 virus can be ed from e A, subtype C,
subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J
viruses, and recombinants f.
In one embodiment, the method comprises providing a packaging vector
comprising a Gag sequence of an HIV-1 subtype B virus and replacing Gag sequences
in the vector with ces from an HIV-1 non-subtype B virus.
The non-subtype B HIV-1 virus can be selected from subtype A, subtype C,
subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J
viruses, and recombinants thereof. In a preferred embodiment, non-subtype B HIV-1
virus is HIV-1 NDK.
Methods of Producing Lentiviral s
The invention also encompasses methods for using packaging vectors encoding
HIV-1 non-subtype B Gag and/or Pol proteins to generate lentiviral vectors. In one
embodiment, the invention encompasses administering a packaging vector encoding an
HIV-1 non-subtype B Gag or Pol n to a cell with a lentiviral vector. The packaging
vector can comprise any of the features discussed herein.
The lentiviral vector ses ting sequences for packaging and reverse
transcription, including a LIJ site and primer binding site. Preferably, the lentiviral vector
comprises two HIV-1 LTR sequences. In one embodiment, one of the LTRs is deleted
for U3 and R sequences. Preferably, the lentiviral vector comprises a central polypurine
tract (cPPT) and a central al sequence (CTS). The lentiviral vector preferably
encodes a lentiviral or non-lentiviral protein, such as a selectable marker or tumor
In one embodiment, the lentiviral vector comprises one or more HIV antigen,
preferably an HIV-1 antigen. Most preferably, the antigen is a Gag, Pol, Env, Vif, Vpr,
Vpu, Nef, Tat, or Rev antigen. The n can be a single antigen, a mix of antigens,
an antigenic polypeptide, or a mix of antigenic polypeptides from these proteins. In a
preferred embodiment, the lentiviral vector comprises an HIV-1 p24 Gag antigen.
In one embodiment, the invention encompasses a lentiviral vector comprising an
NlS-containing promoter. An “NlS-containing promoter” comprises an NF-Kb binding
site, an interferon stimulated se element (ISRE), and an SXY module (SXY).
es of naturally occurring ntaining promoters are the [52m promoter and
the MHC class I gene promoters. These naturally occurring NlS-containing promoters
are generally cloned or reproduced from the promoter region of a gene encoding a
protein [52m or a MHC class I protein, or referred to as vely encoding such
proteins in genome databases (ex: NCBI polynucleotide database
http://www.ncbi.nlm.nih.gov/guide/dna-rna). Both [52m and class I MHC proteins enter
the Major ompatibility Complex (MHC). [52m and class I MHC promoter
sequences are also usually referred to as such in genome databases - i.e. annotated as
being [52m and class I MHC promoter sequences.
MHC class I and BZ-microglobulin ers contain the shared structural
homologies of NlS—containing promoters. These promoters also share the ability to be
strongly activated in dendritic cells, as well as, to lower intensity, in the ty of the
other human body tissues.
In one ment, the packaging vector and the lentiviral vector are introduced
er into a cell to allow the formation of iral vector les containing the Gag
protein produced by the packaging vector and the nucleic acid produced by the
lentiviral vector. Preferably, this is achieved by cotransfection of the cells with the
packaging vector and the lentiviral vector. The cells can also be transfected with a
nucleic acid encoding an Env protein, preferably a VSV Glycoprotein G. Preferably, the
lentiviral vector particles are capable of entry, reverse transcription, and expression in
an appropriate host cell.
In one embodiment, the packaging vector or the lentiviral vector is stably
integrated into cells, and the non-integrated vector is transfected into the cells to allow
the formation of iral vector particles.
In one embodiment, the method further comprises collecting the lentiviral vector
produced by the cells.
In one embodiment, the method further ses selecting for a packaging
vector that packages a higher titer of the lentiviral vector than a same packaging vector
encoding a subtype B Gag or Pol protein. Preferably, the titer is increased at least 1.5
or 2-fold relative to the packaging vector encoding a subtype B Gag or Pol protein.
Lentiviral Vector Particles
The invention also encompasses lentiviral vector particles comprising HIV-1 non-
subtype B Gag and/or Pol proteins. The non-subtype B Gag and Pol proteins can
comprise any of the features discussed herein.
The lentiviral vector particle comprises a nucleic acid sing cis-acting
sequences for packaging and e transcription, including a LIJ site and primer
binding site in association with Gag, Pol and Env proteins. ably, the nucleic acid
comprises two HIV-1 LTR sequences. In one embodiment, one of the LTRs is deleted
for U3 and R ces. Preferably, the nucleic acid of the lentiviral vector particle
comprises a central polypurine tract (cPPT) and a l terminal sequence (CTS).
The nucleic acid preferably encodes a lentiviral or non-lentiviral protein, such as a
selectable marker or tumor antigen. Preferably, the lentiviral vector particle comprises a
VSV Glycoprotein.
Preferably, the lentiviral vector comprises an NlS-containing er. In one
embodiment, the promoter is a [52m promoter.
In one embodiment, the lentiviral vector comprises one or more HIV antigen,
preferably an HIV-1 antigen. Most preferably, the antigen is a Gag, Pol, Env, Vif, Vpr,
Vpu, Nef, Tat, or Rev antigen.
The lentiviral vectors of the ion can be stered to a host cell,
including a human host.
The lentiviral vector particle can contain a targeting mechanism for specific cell
types. See, e.g., Yang et al., Targeting lentiviral vectors to specific cell types in vivo,
PNAS 113(31):11479-11484 (2006), which is hereby incorporated by reference.
Targeting can be achieved through an antibody that binds to a cell surface n on a
cell. The targeted cell type is preferably a tic cell, a T cell, a B cell. Targeting to
tic cell type is preferred and can be accomplished through pe proteins that
specifically bind to a DC surface protein. See, e.g., Yang et al., Engineered Lentivector
Targeting of Dendritic Cells for In Vivo, Nat Biotechnol. 2008 March ; 26(3): 326—334,
which is hereby incorporated by reference.
The present ion further relates to the use of the iral s according
to the invention, especially in the form of lentiviral vector particles, for the preparation of
therapeutic compositions or vaccines which are capable of inducing or contributing to
the occurrence or ement of an immunogical reaction against epitopes, more
particularly those encoded by the transgene present in the vectors.
Examples
e 1. Plasmid construction
The l gene was amplified by PCR, using two primers and , a
clone of HIV-1 NDK, as template. In order to obtain pThV-GP-N plasmid, the PCR
product was digested with Eagl /Sa|| and inserted in packaging construct p8.74, also
digested by Eagl /Sa||.
Example 2. Production of lentiviral vector by transfection
Lentiviral vector stock was produced using pFLAP CMV GFP bis and pTHV-
VSV.G CO)bis in combination with p8.74 or pThV-GP-N. 36 transfections were
done, 18 with p8.74 and 18 with pThV-GP-N. All the supernatant were stored at -80°C.
The plasmid pFLAP-CMV GFP bis encoded for the Green Fluorescence protein
(GFP), which expression can be ed by flow cytometry.
Example 3. Titration of lentiviral vector production
Vector titer was determined by the frequency of GFP sion in 293T cells.
Cells were cultivated in 24-well plates, in DMEM containing 10% FBS, until they
d a density of 1X105 cells per well. The cells were then transduced with different
volume of vector supernatant in a final volume of 300 uL. After 2 hours, 700 pl of fresh
medium containing 10% FBS was added in each well. 72 hours after uction, the
medium was then removed and the cells washed in Dulbecco’s phosphate-buffered
saline (DPBS; Gibco). Cells were removed with 0.05% Trypsin-EDTA (Gibco).
Trypsinization was stopped by the addition of 300 pl complete DMEM, and the cells
were transferred to a tube for FACS, after which the number of GFP-expressing cells
was counted with a FACSCaIibur (BD Biosciences) using an excitation wavelength of
509 nm. Only the t of GFP positive cells under 30% was considered.
The results are shown in Figure 3. A significant difference between the pThV-
GP-N and the p8.74 vectors productions was seen, with higher titers ed using the
pThV-GP-N plasmid. Indeed, the vector titer obtained with the packaging plasmid
pThV-GP-N was 2 fold higher than the vector titer obtained with the classically used
plasmid p8.74 (p<0.001 according to Student test).
Example 4. Increased titer of lentiviral vector with pThV-GP-N
HIV-1 BRU and HIV-1 NDK viruses were made on 293T cells and used to
transduced P4 CCR5 cells. These cells encompass a stable |uciferase gene under the
control of the HIV LTR: If they are transduced with TAT protein (which is the case when
they are infected with a WT HIV), the LacZ gene is expressed and a luciferase
expression can be ed. HIV-1 Gag p24 and luciferase expression were
measured. The results are shown in Figure 4, and confirm thatwild type NDK virus has
a higher transduction rate than the wild type BRU one.
e 5. Increased p24 and titer with pThV-GP-N
Different ratios of p8,74 and pSD GP NDK packaging vectors were used to
produce lentiviral vector particles. For each ratio, the titer and the P24 level were
ed. The results are shown in Figure 5 and demonstrate that it is the presence of
the NDK packaging plasmid that is responsible for the enhancement of the production
titers and of the P24 level .
Example 6. Decreased p24 processing with pThV-GP-N
ing vectors p8.74 and pTHV-GP-N were used to produce supernatants
containing lentiviral vector particles. Western blots were performed on the supernatants
using the NIH anti-P24 MAB 12—5C). The results are shown in Figure 6, and
confirm that the difference between the NDK and BRU packaging plasmids relies on the
production of P24 protein and precursor, as the NDK seems to generate a higher P24
sis (presence of P24 precursor in the viral supernatant) when the BRU shows
only P24 in viral supernatant.
Claims
We
Claims (19)
1. A replication-defective lentiviral packaging vector lacking a Ψ site and encoding a subtype D HIV-1 Gag-Pol protein.
2. The vector of claim 1, wherein the HIV-1 l protein is the HIV-1 Gag-Pol protein of HIV-1 NDK.
3. The vector of claim 2, wherein the vector encodes the amino acid sequence of SEQ ID NO:2.
4. The vector of any one of claims 1 to 3, n the vector is a plasmid comprising a nucleotide sequence encoding a subtype D HIV-1 Gag-Pol protein.
5. A method for generating a packaging vector, the method comprising inserting a tide sequence encoding a subtype D HIV-1 Gag-Pol protein into a plasmid under the control of a non-HIV promoter to generate the packaging .
6. The method of claim 5, wherein the HIV-1 Gag-Pol protein is the HIV-1 Gag- Pol protein of HIV-1 NDK.
7. The method of claim 6, wherein the vector encodes the amino acid sequence of SEQ ID NO:2.
8. An in vitro or ex vivo method for generating a lentiviral vector particle, the method sing administering a packaging vector encoding a subtype D HIV-1 Gag- Pol protein to a cell with a lentiviral .
9. The method of claim 8, wherein the packaging vector and the lentiviral vector are plasmids.
10. The method of claim 8, wherein the cell ently expresses subtype D HIV-1 Gag and Pol proteins.
11. The method of claim 8, wherein the packaging vector is integrated into the genome of the cell.
12. The method of claim 8, wherein the HIV-1 Gag-Pol protein is the HIV-1 Gag- Pol protein of HIV-1 NDK.
13. The method of claim 12, wherein the packaging vector encodes the amino acid sequence of SEQ ID NO:2.
14. The method of claim 8, further comprising selecting for a packaging vector that packages a higher titer of the lentiviral vector than a same ing vector ng a subtype B HIV-1 Gag-Pol n.
15. A isolated cell comprising the vector of any one of claims 1-3.
16. The cell of claim 15, wherein the vector is integrated into the genome of the cell.
17. A lentiviral vector particle comprising subtype D HIV-1 Gag and Pol proteins.
18. The lentiviral vector particle of claim 17, wherein the Gag and Pol proteins are HIV-1 NDK Gag and Pol proteins.
19. The method of claim 8, wherein the Gag and Pol proteins are Gag and Pol proteins produced by expressing a nucleic acid sequence that s the amino acid sequence of SEQ ID NO:2.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11306222 | 2011-09-26 | ||
EP11306222 | 2011-09-26 | ||
PCT/IB2012/002363 WO2013046034A2 (en) | 2011-09-26 | 2012-09-25 | Use of non-subtype b gag proteins for lentiviral packaging |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ622860A NZ622860A (en) | 2016-05-27 |
NZ622860B2 true NZ622860B2 (en) | 2016-08-30 |
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