EP1082447A2 - Lentiviral vectors, comprising modified major donor splice sites and major packaging singals - Google Patents
Lentiviral vectors, comprising modified major donor splice sites and major packaging singalsInfo
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- EP1082447A2 EP1082447A2 EP99957641A EP99957641A EP1082447A2 EP 1082447 A2 EP1082447 A2 EP 1082447A2 EP 99957641 A EP99957641 A EP 99957641A EP 99957641 A EP99957641 A EP 99957641A EP 1082447 A2 EP1082447 A2 EP 1082447A2
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- packaging
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- cell
- hiv
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- 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
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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
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
- C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES
Definitions
- the present invention relates to improved lentivirus- derived packaging and transducing vectors useful for the expression of genes at high levels in eukaryotic cells.
- the improved vectors are safer, yet permit increased efficiency of packaging the recombinant viral genome and increased long-term gene expression.
- Viral vectors transduce genes into target cells with high efficiencies owing to specific virus envelope-host cell receptor interaction and viral mechanisms for gene expression. Consequently, viral vectors have been used as vehicles for the transfer of genes into many different cell types including whole embryos, fertilized eggs, isolated tissue samples, and cultured cell lines. The ability to introduce and express a foreign gene in a cell is useful for the study of gene expression and the elucidation of cell lineages (J.D. Watson et al . , Recombinant DNA, 2d Ed., W.H Freeman and Co., NY [1992], pp. 256-263).
- Retroviral vectors capable of integration into the cellular chromosome, have also been used for the identification of developmentally important genes via insertional mutagenesis (J.D. Watson et al . , supra , p. 261) .
- Factors affecting viral vector usage include tissue tropism, stability of virus preparations, genome packaging capacity, and construct -dependent vector stability.
- tissue tropism a tissue tropism
- stability of virus preparations a genome packaging capacity
- construct -dependent vector stability a vector that promotes viral vectors.
- in vivo application of viral vectors is often limited by host immune responses against viral structural proteins and/or transduced gene products .
- MLV vectors have not been reported to induce cytotoxicity and do not elicit strong host immune responses
- lentiviral vectors such as HIV-1 which carry several immunostimulatory gene products have the potential to cause cytotoxicity and induce strong immune responses in vivo .
- the latter are known to induce strong cell-mediated immune responses upon transient exposure (M. Clerici et al . , J. Inf. Dis., 165:1012-1019 [1992]; M. Clerici et al . , J. Amer. Med. Assoc, 271:42-46 [1994]; L.A. Pinto etal., J. Clin.
- a final concern is the possibility of generating replication-competent , virulent virus by recombination.
- Retroviruses Retroviral Vectors
- the term "retrovirus” is used in reference to RNA viruses that utilize reverse transcriptase during their replication cycle.
- the retroviral genomic RNA is converted into double- stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a "provirus.”
- the provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.
- LTRs long terminal repeats
- the LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome .
- Retroviridae There are several genera included within the family Retroviridae, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus . Some of the retroviruses are oncogenic (i.e., tumorigenic) , while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas , and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. They are integrated into the host DNA, and are capable of transmitting sequences of host DNA from cell to cell. This has led to the development of retroviruses as vectors for various purposes including gene therapy.
- Retroviral vectors derived from the amphotropic Moloney murine leukemia virus (MLV-A) use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes.
- the amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See e . g. , E.M. Gordon and W. F. Anderson, Curr. Op. Biotechnol . , 5:611-616 [1994]; and A.D. Miller et al . , Meth. Enzymol . , 217:581-599 [1993]).
- retroviruses including human foamy virus (HFV) and human immunodeficiency virus (HIV) have gained much recent attention, as their target cells are not limited to dividing cells and their restricted host cell tropism can be readily expanded via pseudotyping with vesicular stomatitis virus G (VSV-G) envelope glycoproteins (See e . g . , J.C. Burns et al . , Proc. Natl. Acad. Sci. USA 90:8033-8037 [1993]; A.M.L. Lever, Gene Therapy. 3:470-471 [1996]; and D. Russell and A. D. Miller, J. Virol., 70:217-222 [1996]) .
- VSV-G vesicular stomatitis virus G envelope glycoproteins
- retroviral vectors derived from the amphotropic Moloney murine leukemia virus (M-MuLV) , such as pLNL6 (Genbank M63653) , see Baker, et al . , J. Virol. 61:1639 (1987), for gene transfer (see also A. D. Miller and C. Buttimore, Mol. Cell. Biol., 6:2895 [1986]).
- M-MuLV amphotropic Moloney murine leukemia virus
- pLNL6 Genebank M63653
- the MLV-A vector system is comprised of a packaging cell line expressing Gag, Pol and Env, and a vector construct containing LTRs, PBS, PPT and the packaging signal sequences. Up to 8 kbp of foreign sequences can be inserted into the MLV vector and packaged into virus particles.
- the commonly used amphotropic MLV packaging cell lines such as PA317, PG-13, i'-CRIP, GP-AM12 and FLY-A13 produce
- the M-MuLV system has several advantages: 1) this specific retrovirus can infect many different cell types; 2) established packaging cell lines are available for the production of recombinant M-MuLV viral particles; and 3) the transferred genes are permanently integrated into the target cell chromosome.
- the established M-MuLV vector systems comprise a DNA vector containing a small portion of the retroviral sequence (the viral long terminal repeat or "LTR" and the packaging or "psi" [ ⁇ ] signal) and a packaging cell line.
- the gene to be transferred is inserted into the DNA vector.
- the viral sequences present on the DNA vector provide the signals necessary for the insertion or packaging of the vector RNA into the viral particle and for the expression of the inserted gene.
- the packaging cell line provides the viral proteins required for particle assembly (D.
- the vector DNA is introduced into the packaging cell by any of a variety of techniques ( e . g. , calcium phosphate coprecipitation, lipofection, electroporation, etc.).
- the viral proteins produced by the packaging cell mediate the insertion of the vector sequences in the form of RNA into viral particles which are shed into the culture supernatant.
- the M- MuLV system has been designed to prevent the production of replication-competent virus as a safety measure.
- the recombinant viral particles produced in these systems can infect and integrate into the target cell but cannot spread to other cells.
- producer cell clones can be established to generate 10 4 -10 6 transducing units per ml. Increased transduction efficiencies may be achieved by modification of the transduction protocols through means such as repetitive infection steps, cocultivation with the producer cell line, centrifugation, and modification of the culture conditions using growth factors and fibronectin etc. (H. Kotani et al . , Human Gene Ther., 5:19-28 [1994]; and T. Moritz et al .
- M-MuLV-based retroviral vectors are limited by several intrinsic problems: 1) they do not infect non-dividing cells (D.G. Miller et al . , Mol. Cell. Biol., 10:4239 [1990]); 2) they produce only low titers of the recombinant virus (A.D. Miller and G.J. Rosman, BioTechn., 7: 980 [1989]; and A.D. Miller, Nature 357: 455 [1992]); 3) they express foreign proteins at low levels and often get "turned- off” or inactivated after integration (A. D.
- M-MuLV system e . g. , 10 6 /ml
- adenoviral system up to 10 12 /ml
- human cells are infected at a very low efficiency. This low efficiency is particularly problematic when the target cell type is represented at very low numbers in the tissue to be infected.
- the hematopoietic stem cell is a preferred target for gene therapy in a large number of disorders, these cells are present at very low frequencies. For example, totipotent embryonic stem cells have been reported to occur at a frequency of 10 "4 to 10 "6 in bone marrow (B.R. Glick and J.J. Pasternak, Molecular
- M-MuLV LTR The promoter present in the M-MuLV LTR is quite weak compared with other viral promoters such as the human cytomegalovirus immediate early (CMV-IE) enhancer/promoter.
- CMV-IE human cytomegalovirus immediate early
- the activity of the internal promoter is significantly decreased because of interference from the upstream M-MuLV promoter (i.e., transcriptional read-through interference) .
- the dual transcription-unit construct is, however, a common feature in almost all M-MuLV vectors.
- the clinically approved gene therapy vector pLNL6 has been modified to allow synthesis of high basal levels of mRNA, and increased packaging efficiency (See e . g. , co-pending U.S. Patent Appln. Ser. No. 08/336,132, and PCT/US95/14576, to Chang, herein incorporated by reference) .
- improved vector systems are urgently needed to provide a means of delivering and expressing genes efficiently in mammalian cells, particularly human cells. Improved vectors will aid the study of gene expression and development and are necessary if the promise of gene therapy is to be realized.
- the major limitation in the use of the simple retroviral vectors in gene transfer is that use of the MLV-based vector is restricted to dividing cells. This led to the development of the present invention, in which lentiviruses, capable of infecting non-dividing cells are provided.
- lentivirus refers to a group (or genus) of retroviruses that give rise to slowly developing disease.
- Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2) , the etiologic agent of the human acquired immunodeficiency syndrome (AIDS) ; visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis- encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV) , which causes immune deficiency in cats; bovine immune deficiency virus (BIV) , which causes lymphadenopathy, lymphocytosis , and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV) ,
- HIV human immuno
- viruses Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes ( i . e . , T-cells) .
- Lentivirus virions have bar-shaped nucleoids and contain genomes that are larger than other retroviruses. Lentiviruses use tRNA lys as primer for negative-strand synthesis, rather than the tRNA pro commonly used by other infectious mammalian retroviruses. The lentiviral genomes exhibit homology with each other, but not with other retroviruses (See, Davis et al . ,
- Lentiviruses including HIV, SIV, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) depend on several viral regulatory genes in addition to the simple structural gag-pol -env genes for efficient intracellular replication.
- lentiviruses use more complex strategies than classical retroviruses for gene regulation and viral replication, with the packaging signals apparently spreading across the entire viral genome.
- additional genes display a web of regulatory functions during the lentiviral life cycle. For example, upon HIV-1 infection, transcription is up-regulated by the expression of Tat through interaction with an RNA target (TAR) in the LTR.
- TAR RNA target
- HIV-1 virions contain 60 % protein and 2 % nucleic acid.
- the genome consists of two molecules of linear positive-sense single stranded RNA (held together by hydrogen bonds to form a dimer) . Even within a single virion, these molecules need not be identical. Hence, genetic variation can occur through recombination between the two viral RNAs of a single virion.
- the HIV-1 genome is about 9.7 kb in length. Many HIV-1 proviral genome sequences have been sequenced in their entirety.
- GenBank M19921 LOCUS HIVNL43, Human immunodeficiency virus type 1, NY5/BRU (LAV-1) recombinant clone pNL4-3, 9709 bp ss-RNA, is used as a reference sequence in this discussion.
- the construction of pNL4-3 has been described in Adachi,A., Gendelman, H.E . , Koenig,S., Folks, T. , Willey,R., Rabson,A. and Martin, M.A., Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone,
- pNL4-3 is a recombinant
- the HIV-1 genome has a high mutation rate.
- a single cell can be infected by more than one viral particle simultaneously, and recombination occur between the two viral genomes.
- the HIV reverse transcriptase has a high frequency of misincorporation (:1700 to 1:4000) .
- the replication error rate for HIV is such that each newly synthesized HIV genome carries on average approximately one mutation.
- HIV-1' s are currently divided into two genetic groups based on phylogenetic reconstruction using DNA sequences. The majority of these sequences fall into the M (major) group, while a smaller, but growing, number of sequences are classified as O (outlier) .
- Most HIV-1 strains from around the world can be placed into one of nine nucleotide sequence-defined clades; these clades have been given the letter designations A through I.
- more than a dozen HIV-1 strains isolated from patients have now been shown to have chimeric genomes in that their gag and env genomic regions cluster with different clades. Interclade recombination is relatively easy to demonstrate because strains from different clades typically differ substantially in their nucleotide sequence identities.
- the env gene sequences of HIV-1 strains of different clades may differ by 20% or more.
- interclade HIV-1 recombinants have most often been detected in geographic regions where two or more clades are prevalent.
- 17 HIV clades have now been reported in humans: nine HIV-1 clades in the major grouping (A through I), three HIV-1 group O group "outlier" clades, and five HIV-2 clades.
- An additional three lentiviruses are known in nonhuman primate species (African green monkeys, mandrils, and Syke's monkeys) .
- the potential gene pool for primate lentivirus recombination is on the order of 20, e.g., 20 gag genes and 20 pol genes.
- the current HIV-1 clades may have arisen in part through past recombination between some of these genes.
- Viable recombinants between SIV and HIV (“SHIV" strains) have been genetically engineered in research laboratories..
- Each end of the DNA provirus contains the so-called long terminal repeats (LTRs) .
- LTRs long terminal repeats
- the 5' LTR and 3' LTR regions are essentially identical in the wild-type HIV-1 genome. These LTRs are 634-bp non-coding sequences, located at the extreme 5' and 3' ends of the proviral genome, that contain enhancer and promoter regions.
- the LTRs consist of three distinct coding regions, U3 , R, and U5 , which can be subdivided into the separate enhancer and promoter regions.
- the U3 region is 450, the R sequence 100 and the U5 region some 85 nt long. Transcription initiates at the first base of the R region in the 5' LTR, and polyadenylation occurs immediately after the last R region base in the 3' LTR.
- the primary transcript is thus about 600 bases shorter than the provirus.
- the U3 region includes several features of interest : the integration attachment site (att) at the far 5' end, the promoter TATA box ( a segment of DNA, located approximately 19-
- SP1 regions promoter binding site for RNA polymerase and reverse transcriptase
- GGGACTTTCC kappa-enhancer
- the R region (454-550) contains the transcription initiation site, the TAR (Tat-activating) region and the poly
- a signal (-AATAAAA-) ; the latter is significant only in the 3' LTR) .
- the primary transcript corresponds to bases 455 to
- the U5 region contains a polyA downstream element and a second integration attachment site at the 3' end. These are significant only in the 3' LTR.
- RNA cap Immediately downstream of the 5' LTR is the primer binding site (637-651) for minus-strand DNA synthesis, called the RNA cap.
- the primer binding sine is complementary to the 3' end of a Lys transfer RNA.
- the 5' leader (L) the untranslated region between the primer binding site and the initiation codon for gag, has two elements worthy of note.
- the first is the major 5' splice donor (SD) site (the splice point is at 748) which is used for the processing of full-length genomic RNA to subgenomic mRNA for the syntheses of various viral proteins.
- SD 5' splice donor
- the major splice donor site is so called because it acts as the donor site during splicing of the vif, vpr, tat, rev, vpu-env and nef subgenomic RNAs (The Gag- Pol polyprotein is translated from genomic RNA. )
- minor splice donor sites in the vicinity of the first exon of the rev gene.
- the other is the major packaging signal (psi) (651-669) which serves as a contact point for the Gag nucleocapsid (Ncp7) protein to bind the RNA and to incorporate it into virus particles. Note that one can define an extended packaging signal extending into the gag gene, to about 820.
- the 5' leader also contains a sequence which participates in the dimer- linkage structure of 70S RNA. This DLS overlaps with the major packaging signal.
- the gag gene encodes a polyprotein (55kDa) (CDS 790..2292) which is cleaved by the viral protease (see pol) to yield various core and nucelocapsid proteins .
- the gag coding region extends from the ATG initiation codon at nucleotide 337 to nucleotide 1837 relative to the RNA cap site.
- the polyprotein is translated from unspliced viral RNA.
- the precursor Gag protein is cleaved by protease to produce pl7 (the major matrix MA protein, involved in membrane anchoring, env interaction, and nuclear transport of viral core) , p24 (the core capsid CA protein) , p7 (the nucleocapsid NC protein, which binds RNA) , and p6 (which binds Vpr) .
- pl7 the major matrix MA protein, involved in membrane anchoring, env interaction, and nuclear transport of viral core
- p24 the core capsid CA protein
- p7 the nucleocapsid NC protein, which binds RNA
- p6 which binds Vpr
- gag gene is believed by some authors to contain one or more minor packaging signals.
- the pol gene (CDS est . 2085..5096) codes for a large polyprotein which is a precursor to the virion proteins providing the viral enzyme functions: protease, reverse transcriptase, and integrase .
- the gag and pol genes overlap by 241 nucleotides, and are in different reading frames.
- a slippage sequence in or upstream of the gag-pol overlap region induces an occasional ribosomal frameshift at a frequency (about 5%) which ensures that Gag proteins are made in large amounts and Pol proteins in small amounts.
- a gag- pol fusion protein (pl90) is created as a result of the ribosomal frameshift, which does not interrupt translation.
- the viral protease cleaves Gag from Pol, and further digests Gag and Pol to separate the various mature proteins .
- the cleavage products are protease (plO), reverse transcriptase (p50) , Rnase H (pl5) and integrase (p31) .
- Roughly 50% of the RT remains linked to Rnase H as a single polypeptide (p66) .
- the principal functional form of RT is actually a heterodimer of p66 and p50. All pol gene products are found within the capsid of free HIV-1 virions.
- Reverse transcriptase is responsible for the synthesis of double-stranded DNA from the viral RNA.
- Activity of RT is localized to the N-terminus. RT in HIV has an extremely high error rate, 1/1700 nucleotides.
- RT in HIV has an extremely high error rate, 1/1700 nucleotides.
- Integrase functions to integrate the proviral DNA in the host genome.
- the env gene (CDS 6221..8785) is located at the 3' end of the genome. It encodes the envelope protein gpl60, some of which is cleaved to yield the envelope proteins gpl20 and gp41. Both function in cell recognition on the outer envelope of a released virus.
- the C-terminus of gpl20 interacts with the viral receptor CD4 of human T lymphocytes to facilitate the viral entry into the host cell . Only a 12 amino acid sequence in gpl20 is necessary for binding to CD4 ; the rest of the protein is mutable.
- the gpl20 polypeptide contains nine conserved intrachain disulfide bridges and, within this scaffolding, folds into five globular domains (I-V) . There are five hypervariable regions (VI -V5) whose sequences vary especially widely among HIV-1 isolates.
- the tat gene (CDS 5830..6044, 8369..8414) encodes Tat, a trans-activating protein, the most important activator of of the LTR promoter region. Three functional domains have been identified: an amino terminal amphipathic helix, a cluster of seven cysteine residues, and a stretch of basic amino acids involved in nuclear localization. It is known that conservative mutations of the acidic amino acids of the amphipathic helix are tolerated. Tat mediates the 5' LTR by interacting with its R region, in a segment termed the "TAR" (trans-activating response) element (bases 436-497) . The "TAR" element forms a stable stem loop structure that interacts with the Tat protein to prevent premature termination of transcription initiation.
- TAR trans-activating response
- Tat is reported in the literature to be absolutely essential for HIV transcription and consequently for viral replication.
- the rev gene (CDS 5969..6044, 8369..8643) encodes Rev, another transactivator .
- Rev is phosphorylated at serine residues, but serine substitution mutants which are not phosphorylated are fully active.
- the amino terminal 20 amino acids and the carboxy terminal 25 amino acids are known to be dispensable. There are two important domains, a stretch of basic amino acids, which is involved in nuclear localization and in interaction with RRE RNA, and a leucine-rich region, presumed to be involved in transactivation, whose leucines are intolerant of mutation.
- Rev is a protein whose target is termed RRE (Rev-response element) , on the env protein coding region of the mRNA. Interaction of Rev with the RRE region apparently allows for transport of unspliced RNA from the nucleus to the cytoplasm.
- RRE (7758-7992) is an RNA secondary structure element. Proviruses lacking Rev function remain transcriptionally active but fail to generate new viral particles .
- the nef gene (CDS 8787..9407) encodes Nef, and overlaps the env gene and the 3' LTR. Nef may be involved in signal transduction, although this is controversial. There has also been speculation that Nef down-regulates viral expression.
- Nef protein does not appear to be essential to the HIV life cycle in tissue culture.
- the vif gene (CDS 5041..5619) encodes Vif, the virion infectivity factor. Vif -deficient mutants are typically much less efficient than wild type HIV at cell-free (as opposed to cell-to-cell) virus transmission. It is not a virion component and the mechanism by which it affects infectivity is unclear.
- the vpr gene (CDS 5559..5849) encodes Vpr, a virion protein which accelerates the replication and cytopathic effect of HIV-1 in CD4+ T-cells. About 100 copies of Vpr are associated with each virion.
- the vpu gene (CDS 6061..6306) encodes Vpu.
- the vpu gene encodes part of a polycistronic transcript which also includes the env gene .
- Vpu is a cytoplasmic protein which is thought to facilitate assembly and/or release of viral particles.
- the 3' LTR is identical to the 5' LTR, but is significantly mainly by virtue of its poly-A signal (9602..9607) , and the "R" repeat sequence (9529..9626) allowing RT jumping during DNA synthesis.
- HIV-1 infects activated and resting lymphocytes, terminally differentiated monocytes and neuronal cells through cellular receptors and co-receptors such as CD4 , chemokine receptors and galactosyl ceramide (J.M. Harouse et al . , Science
- the restricted lentiviral host cell tropism can be expanded by pseudotyping the virus particles with broadly tropic viral envelope proteins from human T cell leukemia virus type I (HTLV-I) , amphotropic MLV envelope protein or the vesicular stomatitis virus G glycoprotein (J.C. Burns et al . , Proc. Natl. Acad. Sci. USA. 90:8033-8037 [1993]; N.R. Landau et al . , J. Virol., 65:162-169 [1991]; K.A. Page et al . , J. Virol., 64:5270-5276 [1990]; and D. H.
- a CD4 receptor can be introduced into target cells by adenovirus transduction before HIV vector transduction in a two-step transduction protocol (K. Miyake et al . , Human Gene Ther., 7:2281-2286 [1996]). Naldini et aJ . have demonstrated that HIV-1 vectors pseudotyped with MLV-A or VSV-G envelope could produce up to
- One difficulty related to HIV vector development encountered during the development of the present invention is the cytotoxicity of many HIV gene products to human cells.
- Cell lines expressing Tat, Rev, Nef have been established.
- expression of Gag, Rev and Vpr has been shown to induce cytopathology, cell death and cell cycle arrest in human cells (See, M. Emerman, Curr. Biol., 6:1096-1103 [1996]; G. Miele and A. M. L. Lever, Gene Ther., 3:357-361 [1995]; and T. Nosaka et al . , Exp . Cell. Res,.
- HIV-1 Vpr also induces apoptosis in human cells.
- the expression of VSV-G protein induces syncytium formation which again is problematic for establishing a packaging cell line.
- lentiviral vectors have the potential to overcome this limitation of prior vectors systems.
- the foremost safety consideration is the risk that either packaging vector and transducing vector will recombine, either with themselves or with defective virus endogenous to the host cell genome, to produce a replication-competent, infectious lentivirus, in particular, replication-competent HIV (RC-HIV) .
- RC-HIV replication-competent HIV
- the vector constructs are replication-defective, the risk of generating RC-HIV is increased with the DNA co-transfection procedure, when a high frequency of recombination events can occur at both DNA and RNA levels.
- the packaging constructs and the transducing vectors of lentiviruses could potentially recombine and generate replication-competent viruses (RCV) as do the MLV vectors during co-transfection.
- RCV replication-competent viruses
- lentiviral vectors were developed that use a pan-tropic envelope gene such as amphotropic MLV env or
- VSV-Gs This reduced the possibility of producing a wild-type lentiviral RCV (e . g. , an HIV-1 Env-trophic virus). However, it is still possible that an RCV could be generated via recombination with these pan-tropic env genes or endogenous retrotransposon env genes.
- RCV retroviral sequences
- a member of the HERV family encodes a protein resembling the lentivirus rev gene product with a nucleolar localization signal, a putative RNA binding domain, and a sequence similar to the Rev effector domain consensus sequence (R. Lower et al . , J. Virol., 69:141-149 [1995]).
- Some human tissues and cell lines such as the placenta, syncytiotrophoblasts, brain, differentiated U-937 cells, teratocarcinomas, and the mammary carcinoma T47D cells have been shown to express complete human endogenous retrovirus env gene and release retrovirus-like particles. These endogenous retroviruses may form defective particles which lack infectivity.
- Shimada, et al . , J. Clin. Investig. , 88: 1043-7 (1991) describes a recombinant HIV-1 gene transfer system employing two vectors.
- the packaging vector has a CMV promoter, and an insertion mutation in the packaging signal.
- the transducing vector replaces part of gag, and all of pol, with a reporter gene cassette.
- the vector system uses wild type HIV-1 Env proteins to target CD cells. It is worth noting that Shimada et al . state that sequences upstream of gag AUG are important for gag expression, implying that they cannot be modified.
- the first 500 nt of the gag gene may be directly or indirectly involved in the binding of the viral RNA to the nucleocapsid of the virion, and that a stretch within the env gene, including the RRE, also contains a packaging signal.
- Markowitz et al Placed the gag and pol genes of MLV on one plasmid, and the env gene on the other.
- the plasmids had deletions of the 3 ' LTRs and the packaging signal as well. Hence, to generate intact retrovirus, there would need to be several recombination events. Markowitz' strategy was adapted to HIV-1 by Naldini et al . , as described below. Naldini, et al .
- the first packaging vector (pCMV ⁇ R9) provides the HIV gag, pro, pol, vif, nef, tat, rev, and vpr genes, but the env and vpu genes, and the packaging signal, were inactivated.
- the human cytomegalovirus (CMV) immediate early promoter was substituted for the 5' LTR, while the 3' LTR was replaced with a polyA site from the human insulin gene. The major splice donor site was preserved.
- a second packaging vector was used to broaden the tropism of the vector system. In one variant, this vector expressed the amphotropic envelope of Moloney leukemia virus
- the final element of the system was a transducing vector (pHR' ) , providing, m order, the 5'LTR, the major splice donor site, the major packaging signal, nearly 350 base pairs of gag, the env sequence encompassing the RRE element, a splice acceptor site, an internal CMV promoter, a reporter gene (luciferase or beta-galactosidase) , and a 3' LTR.
- pHR' transducing vector
- Miyoshi, et al . , J. Virol., 72:8150-57 created a self -inactivating HIV-1 vector.
- the U3 region of the 5' LTR was replaced with the CMV promoter, and self-mactivation was accomplished by deleting 133 bp m the U3 region of the 3' LTR, including the TATA box and binding sites for transcription factors Spl and NF-kappa B. The deletion is transferred to the 5' LTR after reverse transcription and integration m infected cells, resulting m the transcriptional mactivation of the LTR m the proviruses .
- the vector system comprised packaging vector pEV53 (gag+pol+tat+rev+env-) , pseudotypmg vector pCI-VSV-G, and transducing vector pECX .
- pEV53 there were partial deletions m env (736 nt from SU coding region and 168 nt from TM coding region) .
- the PPT and 3' LTR were also deleted, but a BGH polyA was provided.
- the CMV enhancer/promoter was substituted for the U3 domain of the 5' LTR.
- the vector provided all cis-acting sequences required to support reverse transcription and integration, and a cloning site for inserting foreign genes such as beta- galactosidase gene (lacZ) or puromycin-N-acetyl transferase dominant selectable marker gene (puro) .
- EIAV has a relatively simple genome organization. Hence, it was not clear that his results could be extrapolated to HIV, SIV or FIV. Also, Olsen' s vectors exhibited a level of transfection about 100-fold lower than with MLV vectors .
- Sodroski USP 5,654,195 (1997) describes a hybrid virus in which the 5' DNA segment encodes functional SIV or HIV-2 gag, pol, pro, vif, and vpx proteins, and the 3' DNA segment encodes functional HIV-1 env, tat and rev proteins, and a functional SIV or HIV-2 nef protein.
- the 5' and 3' LTRs are from SIV or HIV-2.
- Sodroski USP 5,665,577 (1997) discloses an HIV vector which comprises the gag, pol and env genes but lacks the HIV major packaging signal, identified therein as AAAAATTTTGACTAGCGGA . When introduced into a eukaryotic host cell, these express the structural proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication-competent HIV virion.
- the present invention contemplates attenuated lentiviruses, and improved viral packaging and transducing vectors derived from lentiviruses, especially HIV-1, and useful for the delivery of nonlentiviral genes to target cells. It also contemplates the use of these vectors in delivering transgenes to target cells, especially nondividing cells, in organisms, especially humans.
- the packaging vectors (HP) of the present invention differ from those known previously in that they contain less in the way of lentiviral sequences from a single lentivirus, and hence present a reduced risk of recombination.
- the packaging vectors of the present invention are characterized by either the use of a modified but functional major splice donor site, substantially incapable of serving as a site for homologous recombination, or by the complete omission of the major splice donor site.
- the modified major splice donor site is modified so that it is substantially identical to the major splice donor site of a non-lentiviral retrovirus, especially that of Rous Sarcoma Virus (RSV) .
- RSV Rous Sarcoma Virus
- non-essential sequences such as the accessory genes, of the source lentivirus are also deleted in the course of the construction of the packaging vector.
- the wild-type promoter and enhancer are replaced with a nonhomologous promoter (and, optionally, a nonhomologous enhancer) .
- These changes likewise serve to reduce the risk of generating replication-competent virus through recombination with the transducing vector or a defective provirus endogenous to the host or target cell .
- the 5' LTR promoter is an tightly inducible promoter, so that expression of Gag, Pol and Env proteins is under the control of the biologist. This, together with the inactivation of certain accessory genes, tends to reduce cytotoxicity.
- the Gag and Pol functions are encoded by one vector and the Env functions (preferably, a non HIV-1 -like envelope protein) by another vector.
- gag expression is enhanced by the operable linking of the gag gene to a Kozak sequence.
- the transducing vector (FV) likewise is characterized by a functional major splice donor site which differs from that of its source lentivirus.
- its major splice donor site need not be identical to that of the packaging vector (s) .
- the modification should leave a functional packaging signal, too.
- it likewise has a strong nonlentiviral promoter/enhancer in place of the normal 5' LTR.
- the gag (except for packaging signals) and pol sequences are deleted.
- the env sequences are deleted to the extent that this can be done without a substantial loss in yield. While there may still be regions of sequence identity between the packaging and transducing vectors which are sufficiently long to present a meaningul risk of homologous recombination, a characteristic of the preferred vector system is that homologous recombination alone, among only the packaging and transducing vectors, cannot create a recombinant virus which possesses, simultaneously, a functional packaging signal, a functional major splice donor site, and a gag AUG, even if the recombined virus possesses a 5' promoter/enhancer and genes otherwise encoding equivalents of the Gag, Pol and Env proteins.
- the first region of significant homology is in the gag gene, after the initiation codon.
- the recombinant virus derives a functional packaging signal and a functional major splice donor site from the transducing vector, it will lack the gag AUG, since it can crossover to the packaging vector only after the AUG.
- it has the 5' sequence of the packaging vector through the gag AUG, it will lack a functional packaging signal and a functional major splice donor site.
- a replication-competent virus could still be generated by nonhomologous recombination, or by further recombination with a defective endogenous retrovirus .
- Figure 1A is a simplified schematic illustration showing the HIV-l genomic structure.
- Figure IB is a simplified schematic illustration of the HIV-1 LTR.
- Figure IC provides simplified schematic illustrations of three HIV-1 LTR deletion constructs.
- Figure ID provides simplified schematic illustrations of three heterologous enhancer/promoter inserts (human CMV IE (a) , human CMV IE (b) , and Mo-MLV) .
- Figure 2 is a graph showing the reverse transcriptase activity of a representative attenuated recombinant HIV-1 tat mutants over time (days post-infection) .
- Figures 3A-3C show the organization of the HIV-1 genome and a series of HIV-1 mutants containing LTR, tat, and nef mutations .
- Figure 4 shows replication efficiencies of several HIV-1 recombinants carrying heterologous genes.
- Figure 5 shows an HIV-1 transducing vector diagram for the HIV packaging construct 1-del.env (pHP-ldl).
- Figure 6 shows cell-free RT activity (cpm/ ⁇ L) measured in duplicate using supernatant of cell cultures transfected with various packaging vectors .
- Figure 7 shows seven pHP-1 -derived packaging vector constructs.
- Figure 8 shows six pTV-derived transducing vector constructs .
- Figure 9A shows a pTVi//-derived construct.
- Figure 9B shows a pTV ⁇ -derived construct.
- Figure 10 shows the Gag processing rates of wild-type HIV- infected MT4 compared with tat-C HIV chronic high producing cells .
- Figure 11 shows kinetics of retro- and lentiviral transgene expression in three different human cell lines.
- Celss were transduced with 10 s tu of pTV ⁇ CMV-nlacZ or pMFGnlacZ and propagated for long-term study. At different passage times as indicated, cells were collected and stained for j ⁇ -galactosidase activity to determine the percentage of positive cells.
- Figure 12 illustrates the possible cross-over to generate RCV from co-transfection of pHP-dl.28 and pTV-dl . CMVnlacZ .
- Fig. 12A similarly illustrates possible crossover with the same packaging vector and a different transducing vector, pTV ⁇ .
- Figures 13A provides a schematic showing a portion of the wild-type HIV-1 sequence, as well as the tat-B (wild-type sequence provided in SEQ ID NO: 4; the tat-B sequence is provided in SEQ ID NO: 20) .
- Figure 13B provides a schematic showing a portion of the wild-type HIV-1 sequence, as well as the nef-A mutations and nef-B mutations (wild-type sequence provided in SEQ ID NOS : 5 and 6) .
- the nef-B mutations are shown in SEQ ID NOS: 18 and 19) .
- the nef -A sequence is the same as the wildtype sequence for the sequence shown starting at base 9001 ( i . e .
- SEQ ID NO : 6 represents the sequences for both wild-type and nef-A) .
- the nef-A sequence is the same as the nef-B sequence shown in SEQ ID NO: 5 ( i . e .
- SEQ ID NO: 5 represents the sequences for both nef-A and nef-B in the sequence shown starting at base 8781) .
- Figure 14 compares (A) HIV genome structure with that one embodiment (B) of an HP/TV vector system. Att, integrase attachment site; SD, splice donor; ⁇ , packaging signal; ppt , polypurine tract. In pHPdl20, 28 nucleotides in env was deleted as elsewhere described.
- Figure 15 Mutagenesis and PCR primers and their relative locations on pTV. Various primers for PCR mutagenesis are set on the pTV map as shown at the top. The location and direction of these primers are also depicted. Figure 16.
- Experimental approach diagram For vector production and titration, five plasmids were co-transfected into TE671 cells as shown to the left. Human growth hormone plasmid pXGH5 was used as transfection control. To examine RNA expression of pTV vectors, pCMVrev, pCEP4tat, and pXGH5 were co-transfected.
- Figure 17 provides the sequence of a portion of the wild- type HIV-1 sequence, as well as the tat-B (wild-type sequence provided in SEQ ID NO : 4 ) , and tat-A (SEQ ID NO:16), tat-B (SEQ ID NO : 20 ) , and ta t- C ( SEQ ID NO : 17 ) .
- Figure 18 compares the strucutres of pHP, wt HIV-1 and pTV ⁇ .
- Figures 19A-19J show the structures of HIV-1 and numerous transducing vector variants, together with the viral titers relative to pTV ⁇ set at unity. The locations of the SD, the gagAUG, and various known or potential packaging signals (stem- loops) are indicated.
- HIV-1 and its packaging signals (A) compared to (A) pTV ⁇ CMVnlacZ and derivatives: (B) gag-env deletion mutants, (C) packaging signal mutants ⁇ AUG, ⁇ SD, ⁇ SD/AUG, (D) env mutants,
- the actual titer vaule of the wild type pTV construct is 7.3 x 10 5 +/-0.2.
- Fig. 19(1) The 3' U5 was deleted by PCR mutagenesis as described in Materials and Methods (pTVDCMVnlacZdl3 ' U3#1U5) .
- the actual titer value of the wild type pTV construct was 7.3 x 10 5 +/- 0.2.
- Figure 20 is a table setting forth the relative titers for the transducing vectors of Figs. 19A-19C, but further indicating the number of constructs tested in each sample group, the standard error, and the paired P value.
- Figure 21 is a table comparing wild-type HIV-1, pHP and pTV 5' sequences.
- Figure 22 schematically presents the detection of proviral DNA after lentiviral vector transduction.
- FIG 23 Analyses of 5' splice site and SL2 deletion mutants. Schematic illustration of the four stem- loop structure of the HIV packaging signal, SD mutations, and relative vector efficiencies. The relative vector titer of each mutant was determined by normalizing against that of pTV which was 7.3 + 0.2 x 10 5 tu/ml and is arbitrarily set at 1.00.
- B Quantitative analyses of viral titer, cytoplasmic full- length viral RNA, virion RNA, and packaging efficiency of SD3 , SD4 , and SD1* mutants vs. wt pTV. For easy comparison, the vector titer, cytoplasmic full-length RNA, packaged virion RNA, and packaging efficiency are all normalized against those of pTV which are set at 1.00.
- Figure 24 Analyses of gag AUG and SL4 mutants.
- A Schematic diagram of gag AUG and 5' gag mutants in comparison to a previously reported mutant, gag/env.dl5* (4) , and relative vector titers.
- B Quantitative comparison of viral titer, cytoplasmic unspliced RNA, packaged virion RNA, and packaging efficiency.
- Figure 25 Analyses of vector functions of combination mutations in SD, SA, gag AUG, gag, and env.
- A Schematic diagram of pTV mutant constructs and their relative titers.
- Figure 26 Currently preferred HP/TV lentiviral vector system (a) the wild type HIV-1 diagram and the known locations of packaging signals and CRS/INS regulatory elements, (b) pHP has the entire 5' LTR deleted except for some sequences in the U3 (HIV-1, pNLA-3, nt.288-318) plus the TAR sequence, and the rest of HIV-1 sequence starts from gag AUG to nt.8784 at the AUG of nef gene. The most important safety feature is two deletion regions in the env gene, dl.695 and dl.28. We have also made mutations in vpu, vpr, vif in pHP .
- the most advanced pTVdl.SIN transducing construct has similar 5' U3 sequence to pHP 5' U3 , and dl.35 in the 5' U5 , the intact 5' untranslated leader containing packaging signals, and 40 nt of gag sequence but with mutations in the splice donor site (SDl) and gag AUG (TAG) .
- SDl splice donor site
- TAG gag AUG
- Figure 27 An advanced (theoretical) HP/TV vector system.
- the packaging vector we have replaced the 5' LTR and TAR sequences completely with EF1 alpha enhancer/promoter and HIV sequence starts from gag AUG and ends at the end of pol.
- EF1 alpha enhancer/promoter we will need to insert a CTE-like element to ensure gag-pol expression in different types of cell lines.
- Certain cell types, such as TE671 cells may not need CTE .
- the pTV construct has only about 550 nt of HIV sequence left, including TAR (5'R), 35 nt deleted U5, PBS, leader sequence containing SL1-SL4 packaging signal and 40 nt of gag sequence, the 3' end PPT, 24 nt att L of 3' U3 , and R sequence.
- the payload of this vector is more than 9 kp in theory.
- Figure 28 Possible cross-over between pHP and pTV of our Fig. 26 version.
- the 5' cross-over may occur in the 40 nt of gag sequence through homologous recombination or, legitimate recombination as shown in solid arrow, (or in the 5' TAR region, but must go back from the 40 nt of gag) to pick up gag- pol and the rest of the genome sequence, and the 3' (dashed arrow) cross-over lacks sequence homology; so it has to be non- homologous recombination (illegitimate recombination) .
- the result is a non-functional defective RCV.
- the titers reported in various examples and figures for the same construct may vary depending on which runs are averaged together. Generally, the most inclusive titer data is on the figures . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
- the present invention relates to attenuated and/or replication-defective lentiviruses, and to packaging and transducing vectors derived in whole or in part from a lentivirus .
- this invention relates to a recombinant HIV-1 vector system with multiple safety features based on a packaging/helper construct (pHP) and a transducing vector construct (pTV) .
- pHP packaging/helper construct
- pTV transducing vector construct
- the possibility of producing replication competent virus (RCV) was carefully examined.
- the transduction efficiency of the HP/TV vector and a conventional MLV vector was studied using different human cell types including TE671 (muscle) , 293T (kidney) , HepG2 (liver) , neuronal stem cells and primary CD34 hematopoietic progenitor cells and nonhuman primary rat neural and muscle cells. Transduction efficiency was assayed over short and long duration in tissue culture.
- the safety, expression kinetics, duration and integration status of various lentiviral HP/TV vector systems are presented.
- a "source” or “original” lentivirus is a wild-type lentivirus from which an attenuated and/or replication- defective lentivirus is derived, or which is used as a starting point, during construction of the packaging or transducing vector, for the preparation of one or more of the genetic elements of the vector.
- the genetic element may be employed unchanged, or it may be mutated (but not beyond the point where it lacks a statistically significant sequence similarity to the original element) .
- a vector may have more than one source lentivirus, and the different source lentiviruses may be, e.g., HIV-1 and HIV-2, or HIV and SIV, and so forth.
- genetic element includes but is not limited to a gene.
- the cognate lentivirus is the wild-type lentivirus with which the vector in question has the greatest percentage sequence identity at the nucleic acid level. Normally, this will be the same as the source lentivirus. However, if a source lentivirus is extensively mutated, it is conceivable that the vector will then more closely resemble some other lentivirus. It is not necessary that the cognate lentivirus be the physical starting point for the construction; one may choose to synthesize a genetic element, especially a mutant element, directly, rather than to first obtain the original element and then modify it.
- cognate protein e.g., splice donor site or packaging signal
- percentage sequence identities are of course determined at the amino acid level.
- the term "cognate” lentivirus may be difficult to interpret in the extreme case, i.e., if all lentiviral genetic elements have been replaced with surrogate non-lentiviral genetic elements. In this case, the preferred source HIV-1 strain mentioned previously is arbitrarily considered to be the cognate lentivirus.
- HIV type 2 HIV-2
- HIV-2 infection is associated with natural protection against HIV-1 infection.
- Simian immunodeficiency virus (SIV) also infects human cells; however, it is unclear whether it can cause AIDS in humans.
- both HIV-2 and SIV may be better candidates than HIV-1 for developing lentiviral vectors. It may be advantageous to derive both the packaging and transducing vectors from a lentivirus other than HIV-1, or to derive one from HIV-1 and the other from a lentivirus other than HIV-1.
- replication refers not to the normal replication of proviral DNA in a chromosome as a consequence of cell reproduction, or the autonomous replication of a plasmid DNA as a result of the presence of a functional origin of replication, but rather to the completion of a complete viral life cycle wherein infectious viral particles containing viral RNA enter a cell, the RNA is reverse transcribed into DNA, the DNA integrates into the host chromosome as a provirus, the infected cell produces virion proteins and assembles them with full length viral genomic RNA into new, equally infectious particles .
- replication-competent refers to a wild-type virus or mutant virus that is capable of replication, such that replication of the virus in an infected cell result in the production of infectious virions which, after infecting another, previously uninfected cell, causes the latter cell to likewise produce such infectious virions .
- the present invention contemplates the use of replication-defective virus.
- the term "attenuated virus” refers to any virus (e . g. , an attenuated lentivirus) that has been modified so that its pathogenicity in the intended subject is substantially reduced.
- the virus is attenuated to the point it is nonpathogenic from a clinical standpoint, i.e., that subjects exposed to the virus do not exhibit a statistically significant increased level of pathology relative to control subjects.
- the present invention contemplates the preparation and use of an attenuated lentivirus.
- the attenuated lentivirus is selected from the group consisting of attenuated mutants of human immunodeficiency virus type 1, human immunodeficiency virus type 2, feline immunodeficiency virus, simian immunodeficiency virus, visna-maedi, caprine arthritis-encephalitis virus, equine infectious anemia virus, and bovine immune deficiency virus.
- the attenuated virus may be an attenuated HIV-1, attenuated HIV-2, attenuated SIV, or a virus comprised of portions of more than one lentiviral species ( e . g. , a hybrid, comprised of portions of HIV-1 and HIV-2, or HIV-1 and SIV, etc.)
- a reference virus is a virus whose genome is used in describing the components of a mutant virus.
- a particular genetic element of the mutant virus may be said to differ from the cognate element of the reference virus by various substitutions, deletions or insertions. It is not necessary that the mutant virus actually be derived from the reference virus .
- the preferred reference HIV-1 was mentioned previously.
- HIV-2 see LOCUS HIV2ROD, 9671 bp ss-RNA, Human immunodeficiency virus type 2, isolate ROD, complete proviral genome, ACCESSION M15390, see Clavel,F., Guyader,M., Guetard,D., Salle,M., Montagnier , . and Alizon,M, Molecular cloning and polymorphism of the human immunodeficiency virus type 2, Nature 324, 691-695 (1986) .
- the preferred reference SIV sequence is LOCUS SIVMM239, 13068 bp ss-RNA, a Simian immunodeficiency virus isolated from a macaque, isolate 239 (Macaca mulatta Mm239-82) ; complete proviral genome and flanking sequence, GenBank ACCESSION
- the present invention contemplates a gene amplification and transfer system comprising a transducing vector (TV) , one or more compatible packaging vectors (HP) , and a suitable host cell, the transducing vector and at least one packaging vector being derived from a lentivirus, which allow (1) transfection of the packaging vectors into the host cell to form a packaging cell line which produces essentially (packaging vector RNA) - free viral particles, (2) transfection of the transducing vector into the packaging cell line, (3) the packaging of the transducing vector RNA by the packaging cell line into infectious viral particles, and (4) the administration of the particles to target cells so that such cells are transduced and subsequently express a transgene carried by the transducing vector .
- TV transducing vector
- HP compatible packaging vectors
- Either the particles are administered directly to the subject, in vivo, or the subject's cells are removed, infected in vitro with the particles, and returned to the body of the subject .
- the 5 ' LTR and 3 'LTR R regions must be sufficiently iden ti cal so tha t ssDNA jumping by the reverse transcriptase will occur.
- one vector can encode gag and pol functions , and another vector, env functions .
- the packaging vectors and transducing vectors of the present invention are each replication-incompetent viruses.
- the vectors chosen for incorporation into a given vector system of the present invention are such that it is not possible, without further mutation of the packaging vector (s) or transducing vector, for the cotransfected cells to generate a replication-competent virus by homologous recombination of the packaging vector (s) and transducing vector alone.
- the two vector constructs, pHP, (which directs the synthesis of necessary viral proteins for virion assembly) , and pTV, (which serves as a gene transducing vehicle for foreign gene delivery) were derived from a LTR- modified recombinant HIV-1 plasmid pNL4-3.
- pHP-1 contains a recombinant cytomegalovirus immediate early (CMV- IE) enhancer/promoter HIV-TAR element which replaces the 5' LTR of pNL4-3.
- CMV- IE cytomegalovirus immediate early
- the entire untranslated 5' leader sequence, nef, and the 3' LTR of pNL4-3 were also deleted in pHP-1.
- the HIV-1 5' untranslated leader sequence was replaced with an artificial 59 bp Rous sarcoma virus (RSV) major splice donor sequence containing a mutated RSV gag AUG and a preferred eukaryotic translation initiation sequence (- CCACCATG-) for the HIV-1 Gag synthesis.
- RSV Rous sarcoma virus
- the nef gene in pNL4-3 was replaced by the bacterial g t selective marker gene, and the 3' LTR was replaced by the SV40 polyadenylation signal.
- the env gene was deleted by Bal31 exonuclease digestion at the Nhel site. Two variants of env- deleted pHP-1 were generated and analyzed, pHP-ldl.2 (with a 2-nt deletion) and pHP-ldl.28 (with a 28-nt deletion).
- pHP-VSVG An alternative pHP construct, pHP-VSVG, was generated by inserting the VSV-G envelope gene between the env AUG and the Nhel site ' in gpl20, and introducing mutations in vpr, ta t, and vpu .
- the transducing constructs were made from p ⁇ L4-3 by deleting sequence extending from the middle of gag to the middle of env.
- a reporter gene cassette containing a heterologous enhancer/promoter directing a reporter gene was inserted in the nef region of the pTV vector.
- the HP/TV vector system has gone through extensive RCV testing and has been approved for use in a biosafety level II laboratory employing a level III standard operating protocol.
- the level III protocol was approved by the Biosafety Committees at the University of Alberta and the University of Florida.
- the lentiviral vector tissue culture and animal studies were performed in a level II+/III laboratory using protocols approved by the Animal Warfare Committees and the Biosafety Committees at the University of Alberta and at the University of Florida.
- the most preferred system includes a transducing vector featuring the dl . SDl/gag/env/RRE deletion in combination with the 3' U3 mutation of pTVdl .
- HIV sequnce deleted from the pTV transducing vector which include the 5' major splice site (Splice donor site, SD) all env sequence (including RRE deletion) deleted, most of gag deleted except for the first 40 nucleotide, most of 5' U3 , 3' U3 and 3' U5 deleted, and part of the 5' U5 deleted (35 nucleotide deletion) .
- This will generate a vector with less than 550 nucleotides of HIV sequence.
- the entire HIV genome is about 9.5 to lOkb.
- the vector system to be able to accomodate more than 9 kb of insert (i.e., a payload of 9 kb or so) .
- This is a major improvement on both safety and capacity of the lentiviral vector system.
- the advantages are 1. improved safety for less HIV sequence homology; 2. increased payload; 3. SIN feature; 4. Rev and possibly Tat independent (Tat may be needed for high efficiency gag-pol synthesis.
- the transducing vector with least homology but with optimal titer i.e. no less than 50% of the reference transducing vector pTV ⁇ CMVnlacZ titer
- the 5' U5 can be deleted further as shown in (dl.62) which drop the titer down to 28%, and the gag/env.dl.7 construct with deletion of SL4 in the packaging signal including deletion of gag AUG and the first 40 nucleotide, which drops the titer down to 26%.
- Our preferred packaging vector is one without all HIV sequences except gag-pol ORF and regulatory elements (such as CTE replacing RRE/Rev function) necessary for gag-pol synthesis .
- Our preferred pseudotypmg vector is pHEF-VSVG, but we have interest in envelope proteins from other viruses, such as ebola virus.
- packaging signal or “packaging sequence” refers to sequences located within the retroviral genome or a vector which are required for, or at least facilitate, insertion of the viral or vector RNA into the viral capsid or particle.
- the packaging signals in an RNA identify that RNA as one which is to be packaged into a virion.
- the term “packaging signal” is also used for convenience to refer to a vector DNA sequence which is transcribed into a functional packaging signal. Certain packaging signals may be part of a gene, but are recognized in the form of RNA, rather than as a peptide moiety of the encoded protein.
- the major packaging signal is the signal having the predominant effect on whether viral RNA is inserted into the particle. This signal is located in the 5' leader region
- HIV-1 packaging signals There are also minor packaging signals with a lesser effect on packaging efficiency.
- Several studies have shown that many sequences in HIV-1, including LTR, TAR, RRE, and in the 5' and 3' gag ORF, the pol ORF, and in the sequences flanking the RRE, contribute to efficient genome packaging, pointing to the complex nature of HIV-1 packaging signals (See e . g. , A. Aldovini and R. A. Young, J. Virol., 64:1920-1926 [1990]; J.F. Kaye et al . , J. Virol., 69:6593-6599 [1995]; A. Lever et al . , J. Virol., 63:4085-4087 [1989]; J. Richardson et al . , J. Virol., 67:3997-4005 [1993]).
- the human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures.
- Parolin et al demonstrated that up to 653 nt in the gag coding region can enhancer RNA packaging efficiency. See Parolin C, Dorfman T, Palu G, Gottlinger H, Sodroski J.
- HIV-1 packagng signal requires the very 5' edge of the RNA and sequences downstream of the 170th nt of gag or sequences in pol, see Berkowitz RD, Hammarskjold M-L, Helga-Maria C, Rekosh D, Goff SP. 5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal. Virology 1995; 212:718-723. Their studies indicate that for efficient packaging function, the four stem-loop structure may not be sufficient. Instead, the packaging signal as well as its sequence context consist of the entire packaging signal. This is consistent with the study of Kaye et al .
- a further reason for including the major packaging signal in a transducing vector is because it overlaps with the dimer linkage sequence (DLS) which is also essential for genome packaging ( See, J.L. Clever et al . , J. Virol., 70:5902-5908 [1996] ; J.-C. Paillart et al . , J. Virol., 70:8348-8354 [1996]; and J.-C. Paillart et al . , Proc. Natl. Acad. Sci. USA. 93:5572-5577 [1996]).
- DLS dimer linkage sequence
- a packaging vector and a transducing vector The key distinction between a packaging vector and a transducing vector is that in the packaging vector, the major packaging signal is inactivated, and, in the transducing vector, the major packaging signal is functional. Ideally, in the packaging vector, all packaging signals would be inactivated, and, in the transducing vector, all packaging signals would be functional. However, countervailing considerations, such as maximizing viral titer, or inhibiting homologous recombination, may render such constructs less desirable.
- Packaging Sys tem Packaging Vectors ; Packaging Cell Line
- a packaging system is a a vector, or a plurality of vectors, which collectively provide in expressible form all of the genetic information required to produce a virion which can encapsidate suitable RNA, transport it from the virion- producing cell, transmit it to a target cell, and, in the target cell, cause the RNA to be reverse transcribed and integrated into the host genome in a such a manner that a transgene incorporated into the aforementioned RNA can be expressed.
- the packaging system must be substantially incapable of packaging itself. Rather, it packages a separate transducing vector which is described below.
- the general abbreviation for a packaging vector in this specification is HP or pHP.
- the packaging system will provide functional equivalents of the gag, pol and env genes as discussed below.
- GPE gag-pol genes
- E an "E” vector
- a three vector system is possible if one is willing to construct distinct gag and pol genes on separate vectors, and operably link them to different regulatable promoters (or one to a regulatable and the other to a constitutive promoter) such that their relative levels of expression can be adjusted appropriately.
- the vector or vectors which together compose the packaging system are called the packaging and pseudotyping vectors.
- a pseudotyping vector is one which encodes an Env- equivalent function, e.g., VSV-G, but not a lentiviral env.
- a packaging vector is any vector providing at least one of Gag, Pol or Env, or an equivalent of at least one of Gag or Pol.
- a vector may be both a pseudotyping vector (providing, e.g., VSV-G) and a packaging vector (providing Gag and Pol) , but normally these functions are separated.
- a packaging system need not include a pseudotyping vector but must include at least one packaging vector.
- a packaging cell line is a suitable host cell transfected by a packaging system which, under achievable conditions, produces viral particles.
- packaging cell lines is typically used in reference to cell lines that express viral structural proteins (e . g. , gag, pol and env) , but do not contain a packaging signal.
- a cell line has been genetically engineered to carry at one chromosomal site within its genome, a 5 ' -LTR-grag-pol -3 ' -LTR fragment that lacks a functional psi + sequence (designated as ⁇ psi) , and a 5' -LTR- env- 3' -LTR fragment which is also ⁇ psi located at another chromosomal site. While both of these segments are transcribed constitutively, because the psi + region is missing and the viral RNA molecules produced are less than full-size, empty viral particles are formed.
- a host cell is transfected by the packaging vector (s) alone, it produces substantially only viral particles without the full-length packaging vector
- the packaging vector lacks a functional primer binding site, even if these particles infect a new cell, the packaging vector RNA will not be reverse transcribed back into DNA and therefore the new cell will not produce virion.
- the packaging vector is a replication-incompetent virus.
- Preferred packaging vectors are identical or at least substantially identical, to one or more of packaging vectors disclosed in the examples, such as pHP-1, pHP-dl.2 and pHP- dl.28, pHP-VSVG, pHP-CMV, pHP-CMVdel . TAR/SD, pHP-CMV-EFl ⁇ intron, and pHP-EF, or are hybrids thereof.
- the pHP construct was made by first replacing the 5' LTR with the CMV-TATA-TAR chimeric promoter, obtained from the BbrPI to Hindlll fragment of the chimeric LTR containing CMV IE promoter-TATA box and TAR seqence, which was derived from a recombinant HIV-1 LTR as described previously, see Chang L-J, Zhang C. Infection and replication of Tat -minus human immunodeficiency viruses: genetic analyses of LTR and tat mutants in primary and long-term human lymphoid cells. Virology 1995; 211:157-169; Chang L-J, McNulty E, Martin M.
- Human immunodeficiency viruses containing heterologous enhancer/promoters are replication competent and exhibit different lymphocyte tropisms . J Virol 1993; 67:743-752, then deleting the rest of the 5' leader sequence extending from the Hindlll site in the end of TAR region to the gag AUG using a synthetic oligonucleotide containing a splice donor site of Rous sarcoma virus and a conserved Kozak sequence -CCACC- adj acent to the gag AUG. The Kozak sequence serves to increase the translational efficiency. The gag-pol coding sequence is kept intact.
- the conserved reverse transcriptase (RT) domain of the pol sequence is replaced with RSV RT domain by PCR amplification and cloning.
- the vif, vpr, vpu and env genes were mutated by site-specific mutagenesis to eliminate the AUG initiation codon and some of the coding sequence but not affecting Gag-Pol or Ta /Rev syntheses.
- the tat coding sequence can also be mutated as described below either by inserting multiple stop codons (e.g. tat-B mutant) or by deleting the initiation AUG codon and part or all of the coding sequence (e.g. tat-C mutant) because the pHP can be tat - independent .
- a rev independent gag-pol construct can also be made by mutating the nuclear retention signals in the gag-pol coding region as indicated by Schneider R, Campbell M, Nasioulas G, Felber BK, Pavlakis GN. Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev- independent expression of Gag and Gag/protease and particle formation. J. Virol. 1997; 71:4892-4903, and in the env coding region including the RRE element. In this rev- independent pHP construct, the rev open reading frame is mutated by removing the intiation codon AUG and deleting the coding sequence.
- the packaging cell and/or cell line contains a transducing vector.
- the packaging cell line will package the transducing vector into infectious particles. Such a cell line is referred to herein as a "transgenic virion production cell line" .
- packaging may be inducible, as well as non- inducible .
- inducible packaging cells and packaging cell lines lentiviral particles are produced in response to at least one inducer.
- no inducer is required in order for lentiviral particle production to occur.
- the packaging vectors necessarily differ from wild-type, replication-competent lentiviral genomes by virtue of the inactivation of at least one packaging signal of the cognate wild-type genome. More than one packaging signal may be inactivated.
- the only lentiviral genes provided by the packaging vector are those encoding structural, or essential regulatory, proteins.
- nonvirion proteins like tat, but are not packaging, transducing or pseudotyping vectors.
- a transducing vector is an expression vector which bears an expressible nonlentiviral gene of interest and includes at least one functional lentiviral packaging signal, so that, after said transducing vector is transfected into a packaging cell line, the transducing vector is transcribed into RNA, and this RNA is packaged into an infectious viral particle. These particles, in turn, infect target cells, their RNA is reverse transcribed into DNA, and the DNA is incorporated into the host cell genome as a proviral element, thereby transmitting the gene of interest to the target cells.
- transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of infection rather than by transfection.
- retroviral vectors are transduced.
- an anti -HIV gene carried by a retroviral vector can be transduced into a cell through infection and provirus integration.
- a "transduced gene” is a gene that has been introduced into the cell via lentiviral or vector infection and provirus integration.
- viral vectors e . g. , "transducing vectors” transduce genes into "target cells” or host cells) .
- transducing vectors It may be convenient to classify transducing vectors as follows :
- Generation 0 pTV pTV vectors containing non-replication essential genes or genetic elements. (e.g vectors previously reported by Naldini et al . and Shimada et al .
- Generation 1 pTV pTV vectors with deletions of all the accessory genes and non-replication essential genetic elements (e.g. vif, vpr, vpu, nef, NF-kB/Spl)
- Generation 2 pTV pTV vectors with deletions of replication-essential genetic elements (e.g., gag AUG, SDsite, env sequences, RRE, TAR, such elements are also missing on pHP)
- replication-essential genetic elements e.g., gag AUG, SDsite, env sequences, RRE, TAR, such elements are also missing on pHP
- Generation 3 pTV pTV vectors with substitutions of vector-essential genetic elements (complementary substitutions are also present on pHP) .
- Preferred transducing vectors are identical or substantially identical to the transducing vectors disclosed in the examples, such as the GO and Gl vectors, including pTVi/', pTVi/ oO, pTV ⁇ / 40, pTV. ⁇ . nlacZ, and pTV ⁇ //CMV-nlacZ-hyg-dl .
- the present invention encompasses transducing vectors that are suitable for use in the present invention that are linked to any gene of interest (or a "marker gene” or “reporter gene,” used to indicate infection or expression of a gene) .
- One preferred transducing vector pTV is made of a chimeric CMV-TATA-TAR-U5/att-PBS-packaging signal-mutated SD-portion of gag-portion of env-mutated nef-PPT-U3/att-R-U5 which exhibits packaging function like the wild type HIV.
- the U5 sequence was mutated such that all of it was deleted except for the 3' 24 nt att site.
- the 5' chimeric promoter is derived from the NF-kB/Spl deleted CMV-TATA construct of the HIV LTR mutant described previously which directs transcription at the native HIV transcriptional initiation site.
- the TAR is in the R region which can be mutated at both ends to maintain the repetitive function of the R but significantly different from the wild type HIV R.
- the R sequence can be replaced with RSV R so it is completely different from HIV R sequence.
- the PBS can be modified to become RSV
- the packaging signal will have conserved stem-loop secondary structure as described by McBride et al . as SL1 to SL4 but with mutations in SD (GGTG to GCAG or GGGG) and gag AUG (replaced with ACC or UAG) We showed that the latter mutations have minimal effect on packaging efficiency.
- the mutant SD/gagAUG pTV RNA genome is packaged into transducing particles at near 70% wild type efficiency.
- the vectors of the present invention are capable of "high efficiency transduction.” This is intended to encompass transducing vectors capable of transduction at a level of at least 10 5 /ml, although in particularly preferred embodiments, the vectors are capable of transduction levels of up to 10 10 /ml.
- the term “low efficiency transduction” refers to transducing vectors capable of transduction at levels less than or equal to 10 3 /ml.
- the term “long-term transduction” refers to vectors that are capable of remaining transduced in host or target cells for time periods that are longer than those observed with other vectors.
- the present invention provides lentiviral vectors that are capable of remaining transduced for at least 120 days, more preferably at least one year, most preferably for the life of the subject or the necessary time course of treatment.
- Long-term gene transduction and high efficiencies of transduction of human cells by the HIV vectors of the present invention were compared with the conventional MLV vector ( See, Table 5) .
- the duration of expression is a function of the choice of promoter and the target cell type, more so than the choice of vector.
- stable transduction or “stably transduced” refers to the introduction and integration of foreign DNA into the genome of the transducted cell.
- stable transductant refers to a cell which has stably integrated foreign DNA into the genomic DNA.
- transient transduction or “transiently transduced” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transducted cell.
- the foreign DNA persists in the nucleus of the transducted cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
- transient transductant refers to cells which have taken up foreign DNA but have failed to integrate this DNA.
- the target and/or host cells of the present invention are "non-dividin ⁇ " cells. These cells include cells such as neuronal cells that do not normally divide. However, it is not intended that the present invention be limited to non-dividing cells (including, but not limited to muscle cells, white blood cells, spleen cells, liver cells, eye cells, epithelial cells, etc.).
- the vector and the vector progeny are capable of transducing a plurality of target cells so as to achieve vector titers of at least 10 5 cfu/ml .
- the preferred multiplicity of infection would be at least one (i.e., one hit on average per cell), more preferably at least two.
- vector titers are at least 10%, more preferably at least 25%, still more preferably at least 50%, of (a) wild-type reference lentivirus, and/or (b) the titer of pTV ⁇ CMVnlacZ.
- HIV-2 and SIV vector systems may be developed (pH2P and pSIVP) .
- the 5' LTR and the untranslated leader sequences of HIV-2ROD and SIVmac239 may be replaced with the recombinant HP-1 enhancer/promoter and a synthetic leader sequence with or without a splice donor site, both obtainable from the pHP vectors.
- the 3' LTR may be replaced by the SV40 polyadenylation signal.
- the nef and env genes may both be deleted from the vector.
- vpx is preferably included in the HIV-2/SIV packaging cells because it has been shown that the HIV-2/SIV vpx (or SIVagm vpr) is necessary and sufficient for nuclear import function and does not inhibit cell cycle progression as does vpr.
- the VSV-G envelope gene is preferably expressed from a separate expression vector.
- HIV-2 genomes can be assembled into the HIV-1 particles, indicating that the packaging signals of SIV or HIV-2 can be recognized by HIV-1 nucleocapsids.
- a hybrid vector which is essentially an HIV-1 derived vector with SIV or HIV-2 packaging signals (from 3' of the PBS to the extended gag sequences) .
- These HIV-2 and SIV transducing vectors may be tested in co-transfection experiments using pH2P or pSIVP.
- transducing vectors wherein the lentiviral genetic elements are derived solely from HIV-2 (pTV2) and SIV (pTVS) .
- a strong heterologous promoter is preferably used and the transcription initiation site is placed at the beginning of the R-U5 sequence . Sequences in gag-pol and env genes are deleted and the major SD and the gag AUG are mutated.
- a CMV-driven reporter gene cassette such as the CMV-IE-nlacZ-IRES-hyg from the pTV ⁇ -nlacZ-hyg vector may be inserted in the nef ORF of the HIV-2 and the SIV vectors.
- the 3' LTR resembles the native LTR but with a deletion in the U3 except for the 5 'att site.
- a splice donor site is a sequence which directs the splicing of one exon to another exon.
- the first exon lies 5' of the second exon, and the splice donor site overlapping and flanking the first exon on its 3' side recognizes a splice acceptor site flanking the second exon on its 5' side.
- the first three bases of the splice donor consensus are the last three bases of the exon.
- a splice acceptor site is a sequence which acts in conjunction with a splice donor site, so that the intron separating the two sites is removed.
- the HIV-1 major SD is replaced with the cognate RSV SD , or a mutated form thereof .
- the preferred mutant synthetic RSV SD sequence is split into two parts with an Age l site (ACCGGT ) inserted in place of the RSV gag AUG site :
- Synthetic RSV SD (sequence derived from RSV, Gene Bank ACCESSION # AF052428. is underlined and in bold) 5 ' -AGCTTGGTCGCCCGGTGGATCAAGACCGGTAGCCGTCATAAAGGTGATTTCGTCGGATC-3
- HIV-1 LTR consensus A 5' leader sequences (5'sj is in bold and underlined, the construct was made from HIV-1NL4-3, Access # M19921):
- HIV-2 and SIV sites are as follows :
- HIV- 2ROD 5 ' splice j unction (ACCESSION # M15390)
- the artificially engineered splice donor ( SD) site from Rous sarcoma virus (RSV) in the pHP- 1 construct a site that is unrelated to HIV sequences , was found to work like the wild- type SD site ( i . e . , allowing part ition of spliced ta t and rev, and unspliced gag-pol mRNAs into the cytoplasm) .
- the splice donor site in the packaging constructs is used solely for the expression of tat and rev genes downstream and serves to stabilize the gag-pol transcript. It is possible that tat and rev functions can be provided in trans and the 5' splice donor site can be totally eliminated.
- an SV40 promoter with a replication origin can be used in the packaging constructs and the DNA can be transfected into a SV40 large T antigen expressing cell lines such as C0S7 cells (African green monkey kidney cells expressing SV40 T Ag) .
- the packaging signal is of course inactivated in the packaging vectors.
- a functional packaging signal is required, but need not be identical to the source signal.
- the packaging signals have a secondary structure; they may be mutated so as to alter the primary sequence while substantially retaining the secondary structure. Applicant has found it possible to mutate the HIV-1 major packaging signal by replacing GGTG with GCAG or GGGG.
- Lentiviral packaging signals may be replaced with nonlentiviral packaging signals, or functional mutants thereof, such as the cognate packaging signal of another virus, such as RSV or MLV. If so, it will generally be necessary to make corresponding mutations in the Gag nucleocapsid protein so that it recognizes the new packaging signal. Thus, one could make a chimera of the Gag nucleocapsid protein and the cognate nucleocapsid protein of the other virus.
- Gag protein and “Gag proteins” refer to any or all proteins, respectively, encoded by the gag gene, including both the ultimate virion proteins and their precursors (i.e., proteins which are processed intracellularly into the ultimate virion proteins.)
- Poly protein (s) and “Env protein(s)” are analogously defined. These terms can be further modified by "-like” or “-equivalent” as elsewhere defined.
- the structural virion genes are the gag, pol and env genes. At least one, and preferably all of these genes is inactivated in the transducing vector. The only part of gag or env necessary to keep is the part that play essential roles in packaging.
- the only part of gag or env necessary to keep is the part that play essential roles in packaging.
- site- specific mutagenesis can be performed to further change these sequences to introduce stop codons in the gag gene and in the env gene and to kill the RRE function of interacting with Rev. This latter changes can further improve the safety of the HP/TV vector system.
- packaging vectors must collectively provide genes encoding the functions of the gag, pol and env genes in order to provide a functional virion. Nonetheless, these genes may differ from the source genes by silent and other functional mutations.
- Silent Mutations may be made almost freely throughout the gene. The only areas where caution is required is where the choice of sequence has regulatory significance, e.g., the slippage region in gag-pol, or the RRE region in env. In some instances, such as in the case of an INS element, it may actually be desirable to inactivate the regulatory element. In other instances, the regulatory element may be useful, and only silent mutations which leave it functional are desirable.
- the accuracy of these predictions is dependent in part on whether a 3-D structure for the protein is known, whether homologous proteins (i.e., functional mutants, naturally occurring or otherwise) have been sequenced, and whether the biologically relevant binding sites of the protein have been identified.
- a general source of 3D structures is the Protein Data Bank, which is searchable on the Internet.
- Gag The overall sequence variability of Gag proteins in HIV-1 isolates is more than 20%. With deliberate mutations, it is likely that a higher degree of sequence variation can be tolerated.
- Env For the Env proteins gpl20 and gp41, the overall sequence variability among HIV-1 isolates exceeds 60%. For 3D structures, see PDB entries laik (gp41) and lacy (gpl20 fragment) For discussion of functional domains, see Moulard M, Challoin L, Canarelli S, Mabrouk K, Darbon H, Retroviral envelope glycoprotein processing: structural investigation of the cleavage site, Biochemistry 1998 Mar 3 1 ; 3 7 ( 13 ) : 4 510 - 4517 .
- the envelope proteins encoded by the packaging vector may be lentiviral or non-lentiviral proteins.
- the advantage of a non-lentiviral protein is that it can confer on the produced particles the ability to bind to a cell surface receptor of a class of cells not normally infected by the lentivirus.
- An example of a non-lentiviral envelope protein of interest is the vesicular stomatitis virus (VSV) G protein. VSV-G pseudotyped particles are rigid and can be concentrated more than 1000- fold.
- the packaging vectors encodes a nonlentiviral envelope protein
- it is referred to as an envelope pseudotyping vector.
- the pseudotyping vector is selected from the group consisting of pHEF-VSVG, pHEF.A-env, Gibbon ape leukemia virus env, and MLV- Amphotropic env.
- Env proteins of HIV-1 may be replaced with Env proteins of other lentiviruses, of nonlentiviral retroviruses, of nonretroviral viruses, or with chimeras of these proteins with other peptides or proteins.
- Env proteins of VSV G protein
- the hemagglutinin protein of influenza virus the surface antigen (S and preS) of hepatitis B virus
- Env protein of RSV the Env protein of RSV.
- Respiratory syncytium virus (RSV) ... lung and respiratory tracts Herpes simplex virus.... central nerves system
- HCV.... liver, spleen We used HN and F of Sendai virus by PCR amplification of the viral RNA and cloned it into expression vector pHEFla. Upon co-transfection of this pseudotyping vector with pHP and pTV, we obtained vector titer on TE671 cells about 10% of the VSV-G pseudotyped vector system. It is possible that this pseudotyped virus may have increased efficiency on lung epithelial cells.
- a chimera may be constructed of an env protein and of a ligand that binds to a specific cell surface receptor in order to target the vector to cells expressing that receptor.
- Examples are chimeras including FLA16 (a 6 a. a. peptide which binds integrin receptors), erythropoietin (which binds the erythropoietin receptor) , human heregulin (which binds the EGF and related receptors) , and stromal cell derived-factor (SDF-1) (which binds to CXCR4 chemokine receptor of CD34 cells) .
- FLA16 a 6 a. a. peptide which binds integrin receptors
- erythropoietin which binds the erythropoietin receptor
- human heregulin which binds the EGF and related receptors
- SDF-1 stromal cell derived-factor
- the chimera could include an antibody variable light or heavy domain, or both domains joined by suitable peptide linker (a so-called single chain antibody) .
- an antibody domain could target any desired cell surface molecule, such as a tumor antigen, the human low-desnity lipoprotein receptor, or a determinant on human MHC Class I molecules .
- Virions may be chemically, enzymatically or physically modified after production in order to alter their cell specificity. Examples of modifications include chemical or enzymatic addition of a ligand which would be recognized by a cell surface receptor (e.g., addition of lactose so that the virions will transduce human hepatoma cells which express asialoglycoprotein receptors), or incubation of the virus with a biotinylated antibody directed against the vector's Env protein, followed by addition of a streptavidin- linked ligand recognized by the cell -surface receptor. A heterobispecific antibody could be used to link the virion' s Env protein to such a ligand, too.
- the vector system may provide the regulatory proteins, or surrogates therefor, or wholly omit them. If Tat or Rev equivalents are provided, the corresponding genes may be placed on the transducing vector, or on the same or different helper vector (s) . These genes need not be placed on the packaging vectors. Again, silent mutations may be made almost freely. Functional mutation of Tat and Rev should be feasible. For the 3D structure of Rev, see PDB entry lrpv; for RRE, see letf and letg; for Tat, see ltiv; for TAR, see lkis.
- Tat is a transcriptional factor which acts to bind the polll transcription elongation complex and increases the processivity of transcription.
- the amino acid sequence of Tat is highly conserved amongst different HIV-1 strains with more than 80% homology. Mutational analysis has shown that the functional domain is in the first coding exon because deletion of the second exon does not affect its transactivation function. The N-terminal domain is highly charged and contains a long stretch of basic amino acids such as arginine which is the characteristic of RNA binding domain.
- Tat has been shown to bind to the TAR sequence at the loop of a stem-loop structure in the 5' end of the genome. In addition to its transcriptional activation function, Tat has also been shown to enhance reverse transcription and in our laboratory, we have shown that Tat can enhance gag protein precursor processing. Therefore, the multiple functions of Tat may indicate that it may be required for high titer vector production. However, Tat may be substituted with different lentiviral transactivators to avoid recombination of HIV sequences .
- Rev is also a transcriptional regulator which acts at a post transcriptional step in the nucleus to enhance the export of RRE-containing RNA to the cytoplasm. Its amino acid sequence is highly conserved amongst different HIV-1 strains. Human T cell leukemia virus type 1 (HTLV-1) encodes a similar protein named Rex. Rex and Rev share low sequence homology (less than 40%) but have similar functions. Mutational analysis have shown that rev function requires both coding exons. Rev binds to RRE in env and interacts with cellular proteins in the nucleus to mediate the nuclear export of the RRE-containing transcripts. The function of Rev is dispensable if RRE and the inhibitory sequences in the gag-pol and env are mutated.
- TAR and RRE are known for their functions in mediating Tat and Rev interaction with the viral RNA, these two RNA elements may have other functions unrelated to Tat and Rev interaction which may be important for gene transfer vector function. It is possible that RRE or TAR may contain minor packaging signal to enhance viral RNA encapsidation. The example of RRE mutation on vector function is presented later.
- Tat and Rev have been reported to be absolutely required for viral replication in vitro or in vivo Vaishnav YN, Wong-Staal F. The biochemistry of AIDS. Ann Rev Biochem 1991; 60:577-630; Greene WC. Regulation of HIV-1 gene expression. Annu Rev Immunol 1990; 8:453-475.
- a small element from the Mason-Pfizer monkey virus genome can make human immunodeficiency virus type 1 expression and replication Rev- independent , Bray M, Prasad S, Dubay JW, et al .
- a small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev- independent .
- Proc Natl Acad Sci Usa 1994; 91:1256-1260 and this strategy has been used to develop a rev-independent HIV vector system, see Srinivasakumar N, Chazal N, Helga-Maria C, Prasad S, Hammarskjold M-L, Rekosh D.
- the accessory proteins of HIV-1 may have important functions in viral pathogenesis, see Trono D. HIV accessory proteins: leading roles for the supporting cast. Cell 1995; 82:189-192; but they are dispensible for viral replication in tissue culture.
- the accessory genes are not essential to the creation of functional packaging and transducing vectors, i.e., they may be completely deleted. Hence, it is unnecessary to consider in detail the guidance offered by the art as to which mutations of the accessory proteins might be functional. Of course, if one chooses to retain an accessory gene, such guidance can be found in the literature on, e.g., sequences of HIV-1 isolates.
- lentiviral accessory genes when constructing the transducing vector, in order to reduce the risk of homologous recombination to form RCV.
- certain accessory genes such as vpr or vpx, may increase transduction efficiency of nondividing cells, in which case there is a countervailing advantage to retaining them in a form in which they encode functional protein. If so, silent mutations, and other functional mutations, may be introduced to reduce the risk of homologous recombination without loss of gene function.
- the 5' LTR can be totally eliminated but a functional promoter will be needed to drive RNA transcription and gag-pol gene expression.
- a strong enhancer/promoter e.g., one at least 50% as strong as the wild-type 5' LTR, will be used to replace the 5 ' LTR .
- Tat may be needed for high efficiency of Gag-Pol synthesis.
- HIV-1 TAR sequence may be retained in the 5' end for enhanced promoter function.
- the necessary functions for vector production in the 5' LTR are the repetitive sequence R, which serves as annealing sequence for minus-strand DNA jumpting to the 3' R, and the attachment site (att) in the 3' end of U5 adjacent to the PBS for provirus integration.
- the R can be made different from the native HIV R but have the same mutated R in the 3' end.
- the att site is necessary for integrase recognition and binding and therefore cannot be changed (unless one can be coordinated with a corresponding change in the gag-encoded integrase) .
- the lentiviral promoter /enhancer elements of the 5' LTR are replaced with a nonlentiviral promoter/enhancer in at least one (a) the packaging vectors or (b) the transducing vector.
- Both the HP 5' LTR and TV 5 ' LTR promoter/enhancers may be replaced with the same or with different promoter/enhancers, e.g., CMV IE in one and EF-lo; in the other.
- CMV IE promoter/enhancers
- no HIV functional elements are necessary for the packaging construct.
- the transducing vector several elements are needed, in an order from 5' to 3 ' including PBS, packaging signal, and dimer linkage sequence (DLS) .
- DLS dimer linkage sequence
- HIV uses lysine tRNA PBS which may be mutated to a different retroviral PBS such as histidine tRNA or proline tRNA of RSV or MLV.
- a coupled change in the RT domain which recognizes the corresponding PBS will also be needed.
- the packaging signal for HIV RNA has been shown to include different areas in the genome. It is possible that site-specific mutations can be made to change the primary sequence but maintain the secondary structure .
- the major 5' splice donor site and the gag AUG have been shown by others to be essential for genome packaging. However, we have demonstrated that both the SD and the gag AUG can be mutated and the modified transducing vector can still be packaged in high efficiency (see examples below) .
- the DLS is not well defined. However, both primary sequence and secondary structure may be necessary for a functional DLS which overlaps the packaging signal between SD and the gag AUG.
- the packaging vector replaces the HIV- 1 SD with an RSV SD .
- the splicing junction sequences have been previously studied. Ezzell C. Eukaryotic mRNA processing. The Journal of NIH Research 1995; 7:101-104; Mount SM. AT-AC introns: an ATtACk on dogma. Science 1996; 271:1690-1692.
- the first tat coding exon contains positive and negative splicing regulatory elements and the splicing signals can be hundreds of nucleotides away from the splice junction sites.
- Amendt BA, Hesslein D, Chang LJ, Stoltzfus CM Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1. Mol Cell Biol 1994; 14:3960-3970.
- the R regions of the transducing vector may be replaced with a functional R from another source, e.g., RSV.
- RSV a functional R from another source
- the MLV R is longer than that of RSV and therefore is less desirable. It is believed that almost any sequence of similar length to the HIV R region would work if it appears in both ends of the vector genome, allowing RT to jump and anneal.
- a mutation of one genetic element is preferably complemented by a mutation of another genetic element in the same or a different vector:
- vector tat and vector TAR (d) vector tat and vector TAR, and (e) transducing vector att site and packaging vector gag integrase protein.
- wild-type X implies that the protein is identical to a naturally occurring form of protein X.
- X "-like" protein implies that the protein is either identical to X, or is a mutant as described in (b) above. The precise scope of this term will vary depending on how narrowly X is defined. If the reference is to an "HIV-1 gpl20-like protein", the amino acid sequence and biological activity of the X-like protein will be compared to that of the HIV-1 gpl20 proteins. If it is to a "lentiviral Env-like protein” , it will be compared to that of the most similar of the lentiviral envelope proteins. And so forth.
- a mutant is more likely to be considered substantially identical to a reference protein if (a) the overall sequence identity is within the natural range of variation of homologous proteins (e.g, of all HIV-1 gpl20 variants, if the referent is a particular gpl20); (b)most or all of the sites of mutation are sites of high variability in that family; and (c) most or all of the substitutions, especially at low variability sites, are at least semiconservative , and more oreferablv conservative or highly conservative substitutions in general, or favored by experimental data.
- homologous proteins e.g, of all HIV-1 gpl20 variants, if the referent is a particular gpl20
- most or all of the sites of mutation are sites of high variability in that family
- most or all of the substitutions, especially at low variability sites are at least semiconservative , and more oreferablv conservative or highly conservative substitutions in general, or favored by experimental data.
- X-like protein mutant implies that the protein is not identical to X.
- X-equivalent protein includes all of the possibilities (a) - (c) above. Possibility (a) is excluded by the phrasing "X-equivalent protein mutant” . Possibilities (a) and (b) are both excluded by the phrasing "X surrogate” .
- a mutant is considered substantially similar in sequence ito a reference sequence if it is at least 50% identical with the reference sequence, with percentage identity being calculated by the default procedure set forth below.
- This invention contemplates that certain genetic elements of the lentiviral genome will be substantially inactivated to render the genome more suitable (e.g., safer) for use as a vector in the delivery of therapeutic genes to a patient.
- the inactivation may, but need not be, absolute.
- the level of inactivation is at least 50%, more preferably at least 9%, still more preferably at least 95%, most preferably at least 99%.
- a gene is substantially inactivated if it is mutated so that it substantially is no longer capable of being transcribed and translated into a polypeptide retaining a substantial amount of the objectionable biological activity of the originally encoded polypeptide.
- a gene may be inactivated by (1) completely deleting it, (2) removing its initation codon so that it is not translated, (3) inserting one or more stop codons into the coding sequence, preferably immediately after or in place of the initiation codon, so as to stop translation prior to production of a functional polypeptide, (4) inserting or deleting a number of bases, other than a multiple of three, so as to cause a frameshift, and thus the production of an erroneous polypeptide sequence downstream of the frameshift mutation, (5) inserting or deleting one or more whole codons, at either end or internally, so that a nonfunctional polypeptide (or a polypeptide of substantially different function) is encoded, (6) making one or more base substitutions (point mutations) in the gene which alter the encoded
- the gene is inactivated by completely deleting it.
- some genes overlap, so that it is not possible to inactivate one gene by complete deletion without inactivating the overlapping gene.
- some genes contain regulatory elements (like RRE in env) , and so cannot be completely deleted without loss of a regulatory function. Hence, in these circumstances, methods (2) -(6) above are appropriate. If point mutations are employed, preferably these are multiple mutations.
- This specification explains how to identify which mutations of a gene are likely to be functional. By disregarding such advice, one may obtain inactivating mutations .
- a genetic element other than a gene is inactivated if it is mutated so that it is no longer capable of performing its normal biological function. Normally, this means that it is mutated so that it is no longer recognized by a nucleic acid- binding protein.
- the protein recognizes the primary structure (nucleotide sequence) of the genetic element, in other cases, it recognizes a secondary structure arises from the folding of the strand. A single nucleic acid strand can fold upon itself to bring complementary regions into proximity; these are then held together by hydrogen bonding between the complementary G:C or A:T bases.
- the HIV major packaging signal is an example of a genetic element recognized on the basis of its secondary structure.
- the secondary structure of a nucleic acid sequence may be predicted by conventional methods, such as those of Tinoco, and one or more segments predicted to have a secondary structure (e.g., a stem loop) deleted or modified until an acceptable level of inactivation is obtained.
- a secondary structure e.g., a stem loop
- Complete deletion is the preferred method of inactivation, if a genetic element is not at all desirable .
- a genetic element A which is to be inactivated lies between two genetic elements B and C which are to be retained, and which preferably are at a particular distance from each other, or that all or part of the sequence forming the genetic element to be inactivated is also part of another genetic element which is to be retained. In these situations, complete deletion is not desirable. If so, single or multiple insertions, deletions or substitutions, whether consecutive or nonconsecutive in the primary sequence, may be used to alter the sequence sufficiently so that the objectionable genetic element is inactivated without substantial adverse consequences.
- a vector may be said to comprise an inactivated genetic element even if the inactivation is accomplished by completely deleting the element, so it is not present in the vector; the phrase then indicates that at least one of the differences between the vector and a source (or cognate) lentivirus is that the element in question, which is missing in the former, is found in the latter.
- this specification refers to "deletion" of a genetic element, but does not specify complete deletion, it should be taken to include a partial deletion (even a single nt ) if sufficient to accomplish inactivation of the element. However, in general, unless said to be impossible, complete deletion should be considered a more preferred embodiment of any preferred "deletion" .
- the deletion is at least 5%, 10%* 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. With the higher values being most preferred.
- a genetic element performs an essential function, but one nonetheless would like to modify it to reduce the risk of homologous recombination. In that case, one may replace it with an equivalent element which has the same function but which differs in nucleotide sequence.
- a replacement element which exists in nature, for example, as an element of a nonlentiviral retrovirus, a virus other than a retrovirus, or even of a cell.
- RSV is a preferred source or replacement element because it is on nonhuman and indeed nonmammalina (avian) origin, and hence less likely to do harm to human or other mammalian cells.
- MLV is another potential source of replacement elements .
- a replacement element may be substantially, but not exactly, identical to a natural element.
- Homologous recombination is the formation of a hybrid of two sequences, wherein the point of crossover between one sequence and the other lies at a region of significant length wherein the two sequences are substantially identical. This region is called a region of homology.
- the packaging vector (s) and transducing vector are chosen so that the frequency of homologous recombination between them is less than that experienced with the vector systems of Naldini, et al . , Science, 272: 263 (1996) or of Corbeau, et al . , PNAS USA 93:14070-5 (1996) .
- homologous recombination so occurs, it is not enough by itself to form a replication-competent virus.
- mutations which inactivate a gene do not necessarily prevent that gene from being a site for a crossover event, and, conversely, mutation of a gene in one vector to eliminate homology with a corresponding gene in another vector will not necessarily inactivate the gene.
- insertion of a stop codon does not prevent crossover within the untranslated downstream sequence, and silent mutations may be used to destroy homology, without affecting the nature of the encoded polypeptide.
- a gene is entirely deleted, it is both inactivated and incapable of participating in homologous recombination.
- Cell GeneSys which relates to the deliberate induction of homologous recombination of a transgene, states that homologous sequences as short as 14 bases may provide for homologous recombination, but that its preferred flanking sequences are at least about 150 bp . They cite Rubnitz and Subramani , Mol. And Cell.
- the likelihood of recombination can be decreased by minimizing the lengths of identical segments in the two vectors.
- this means that it is preferable to (1) completely delete any genes or other genetic elements which do not substantially contribute to the functionality of the vector (packaging or transducing) in question, (2) when a function of a genetic element does make such a contribution, replace it, where possible, in one of the vectors (or differently in both vectors) with a genetic element which is different in sequence but similar in function, and substitutable for the original element (e.g., the cognate element in another strain or clade, in another lentivirus, in another retrovirus, in another virus, or in a microbial cell, or a functional mutant of the original element or a cognate element of another organism) .
- the PBS and the RT, Rev and RRE, and Tat and TAR it may be necessary to make coordinated changes in two or more genetic elements .
- silent mutations may be introduced so as to reduce its sequence identity with the original gene, even though both the mutated and original genes encode the same protein.
- the third base of the codon is a wobble position in which any of 2-4 different bases can appear without alteration of the encoded amino acid. While there are a few codons (Met and Trp) which do not allow silent alteration of the third base, these are counterbalanced by the codons which allow some variation of the first base, too.
- Method and Trp codons which do not allow silent alteration of the third base, these are counterbalanced by the codons which allow some variation of the first base, too.
- the alterations being fairly evenly distributed. Care must be taken that such silent mutations do not substantially interfere with any important regulatory element of the gene, such as the slippage region in gag-pol, unless a functional substitute for that regulatory element is provided.
- paired bases may be identified and swapped, i.e., if a G at position 1 pairs to a C at position 10, forming part of a stem, position 1 may be changed to C and position G to C. Indeed, it may also be possible to replace G:C with A:T, and A:T with G:C, pairs, although such changes are less certain to be tolerated in view of the difference in strength between the two interactions.
- the percentage sequence identity between two sequences is to be determined by (1) aligning' to maximize the local similarity score (as hereafter defined) between the two sequences, and (2) expressing the number of identical aligned pairs as a percentage of (a) the total length of the overlap region, including nulls (gaps) , or (b) the original length of the shorter sequence, whichever of (a) or (b) is larger.
- the two sequences are to be aligned by a rigorous (linear programming) based local alignment algorithm in which the overall similarity score for a given alignment is obtained by summing the pairwise alignment scores, for each aligned pair of bases or amino acids, and a gap penalty for each gap introduced into either sequence in an attempt to improve the overall similarity score for the alignment.
- the pairwise alignment scores are derived from a 20x20 scoring matrix for amino acids and a 4x4 scoring matrix for nucleotides.
- the gap penalties are a linear combination of a gap initiation penalty imposed for the first null of a given gap, and a gap extension penalty for each additional null added to that gap. Only internal gaps will be penalized.
- the alignment must be statistically significant (chance expectation ⁇ 0.001 as elsewhere defined) in order to be considered.
- the scoring matrix will be the PAM250 matrix, in the form wherein the scores range from +17 to -8; the gap initiation penalty will be -12; and the gap extension penalty will be -4.
- the scoring matrix will be an identity matrix in which all identities are scored 6 and non-identities are scored zero, the gap initiation penalty will be -12, and the gap extension penalty will be -4.
- insertions or deletions are directed to the termini of the molecule, or to surface loops or interdomain boundaries.
- internal insertions and deletions are of no more than five residues, absent evidence (such as an example in a homologous protein) that a larger internal insertion or deletion could be tolerated.
- fusion protein there is no preferred maximum with respect to an insertion at a terminus, which is more aptly termed an “addition” or “fusion”. It is routine to fuse one protein to another to facilitate expression, or to provide a fusion protein which has the combined biological activities of its components.
- a fusion protein may be useful as a precursor, which can be cleaved to liberate an active protein, even if the fusion protein itself lacks activity.
- truncation With regard to deletion at a terminus, more aptly termed "truncation", the purpose of the modification is important. It is routine to extensively truncate a protein when one is interested only in its immunological properties. One may abstract from a protein an epitope as small as five amino acids, and use it by itself to elicit a T cell response, or conjugated to copies of itself or to an immunogenic carrier to elicit a B cell response. When it is a biological activity which must be preserved, the limits on truncation may be more stringent .
- the mutant is at least 50%, more preferably at least 80%, identical in sequence to the original protein.
- a protein is more likely to tolerate a mutation which (a) is a substitution rather than an insertion or deletion;
- (e) is a substitution of one amino acid for another of similar size, charge, and/or hydrophobicity
- (f) is at a site which is subject to substantial variation among a family of homologous proteins to which the protein of interest belongs.
- the preferred mutants are those which comprise an amino acid sequence which is
- the internal deletion or insertion corresponds to a difference between the first reference protein and a homologous second reference protein .
- the second reference protein is any corresponding lentiviral protein. In some instances, it may be appropriate to consider the possible second reference proteins as including corresponding nonlentiviral proteins, especially retroviral proteins, as well.
- the predicted or experimentally determined 3D structure of the modified protein has a main chain (C ⁇ -carbon) conformation whose root -mean-square deviation from the predicted or experimentally determined 3D structure of the original protein is preferably less than 5A, more preferably less than 3A, still more preferably less than 2A, most preferably less than 1A.
- Constant modifications are defined as (a) conservative substitutions of amino acids as hereafter defined; and (b) single or multiple insertions or deletions of amino acids at the termini, at interdomain boundaries, in loops or in other segments of relatively high mobility.
- no more than about five amino acids are inserted or deleted at a particular locus, and the modifications are outside regions known to contain binding sites important to activity.
- Surface residues may be identified experimentally by various labeling techniques, or by 3-D structure mapping techniques like X-ray diffraction and NMR. A 3-D model of a homologous protein can be helpful .
- Residues forming the binding site may be identified by (1) comparing the effects of labeling the surface residues before and after complexing the protein to its target, (-2) labeling the binding site directly with affinity ligands, (3) fragmenting the protein and testing the fragments for binding activity, and (4) systematic mutagenesis (e.g., alanine- scanning mutagenesis) to determine which mutants destroy binding. If the binding site of a homologous protein is known, the binding site may be postulated by analogy.
- Protein libraries may be constructed and screened that a large family (e.g., 10 8 ) of related mutants may be evaluated simultaneously.
- 3-D Structure Determination The determination of the 3-D structure of a protein can provide considerable guidance to one seeking to modify that protein for a useful purpose, or at least to avoid inactivating modifications. If the full 3-D structure is known, the practitioner knows which residues are on the surface and which are on the interior, which residues have side chains pointing toward the outside, which residues are packed closely together and which are not, where the chain is flexible and where it is constrained, which residues are in secondary structures, which residues are brought into proximity as a result of chain folding, and which may be interacting in the form of H-bonding and salt bridges.
- a protein may be modified at an interior residue, a surface residue distant from the binding site of interest, or at a surface residue which is part of, or close enough to affect, the binding site of interest.
- Mutations at surface residues are more likely to be tolerated than at internal residues. Mutations at the latter positions have greater potential to destabilize the protein, thereby, by denaturing the protein, affecting all of its binding activities. Mutation at a surface residue may have no effect on binding activity at all, or it may affect some activities but not others. In any event, they are unlikely to denature the protein.
- the principal methods of determining the complete 3-D structure of a protein are X-ray crystallography and NMR spectroscopy .
- Amino acid-specific chemical affinity labels may be used to ferret out which residues are in fact exposed.
- the most useful labels are likely to be those which react with charged residues, as those are most likely to appear on the surface.
- Sample labels include the following: Amino Acid Affinity Label
- Labeled and unlabeled protein are then separately subjected to a fragmentation reagent such as cyanogen bromide, pepsin, papain, chymotrypsin, trypsin or iodosobenzoic acid.
- a fragmentation reagent such as cyanogen bromide, pepsin, papain, chymotrypsin, trypsin or iodosobenzoic acid.
- the peptides resulting from cleavage of the labeled protein are compared to those derived from the native protein, using two- dimensional electrophoresis. Peptides that have altered mobility are sequenced, and modified amino acids are determined. Surface residues may also be identified by means of photoaffinity labels which, upon exposure to light, form highly reactive intermediates, e.g. nitrenes and carbenes . These species are capable of insertion into C-H bonds, and therefore can react with any accessible amino acid.
- a nonspecific labeling reagent is tritium.
- a folded protein may be tritiated (by hydrogen exchange with tritiated water) , denatured, and fragmented, and the fragments sequenced and tested for the presence of tritium (which is radioactive) .
- All of these labeling methods may also be used to determine whether residues, besides being on the surface, are part of a binding site.
- the distribution of label obtained when free protein is labeled is compared with that obtained when the complexed protein is labeled. Since in the complex, the binding partner occludes the binding site residues of the binding protein, binding site residues should be labeled in the free protein and not in the complexed protein.
- the basic approach is to (1) identify related sequences and structures; (2) identify structurally equivalent residues;
- model structurally conserved regions SCRs
- SVRs model structurally variable regions
- the model of the SCRs acts, to a greater or lesser degree, as a constraint in the modeling of the SVRs.
- core residues are usually more structurally conserved than surface residues, they are usually modeled first.
- helices and strands are usually modeled before loops.
- the main chain (C ⁇ atom) conformation is determined first, and then the side chain conformations. Modeling steps may be iterated to arrive at successively improved approximations of the true structure.
- the predicted structures are more accurate for protein cores than for protein loops.
- the 3-D structures are "weighted" to reflect teh relatedness of the homologous protein to the protein of interest.
- One popular scheme is to weight by the square of the percentage sequence identity.
- homologous substructures of nonhomologous proteins may be used in addition to, or even in lieu of, a 3-D structure of a homologous protein. See Jones and Thirup, Curr. Comm. Molec . Biol., :75-76 (1986); Unger, et al . , Proteins, 5:335 (1989); Claesseus, et al . , Protein Eng., 4:335 (1989); Levitt, et al . , J. Mol. Biol., 226:507 (1992) for the building of models by combining "spare parts" from different proteins. It is not necessary for a molecular biologist to be an expert in protein modeling, as several programs exist which automate the modeling process. These include COMPOSER (Tripos Associates) .
- molecular modeling software may be used to predict potential binding sites, or to predict the effect of a proposed mutation on a binding site, by attempting to "dock" the binding partner to the site. See, e.g., Guruprasad, et al . , Protein Eng., 9:849- 56 (1996); Constantino and Pelliccian, J. Med. Chem. 39:3998- 4006 (1996) .
- Some surface residues are directly involved in the binding surface by which a protein exercises a particular binding activity. Mutation of such residues is likely to affect binding; however, it is not necessarily undesirable to make such mutations. For example, mutation of the binding site of a serine protease can alter what is bound, as opposed to simply rendering the protein inactive altogether.
- the ability of a protein to tolerate mutations which alter the volume of core residues is dependent more on the net change in the total core residue volume, then on the magnitude of the individual residue volume changes. In other words, an increase in the volume of one core position can compensate for a decrease in the volume of another core position.
- the net change in the total core residue volume is not more than 10%, more preferably, not more than 5%. See Lim and Sauer, Nature, 339:31-36 (1989); Lim et al . , Biochemistry, 31:4324-33 (1992) .
- Amino acids differ in terms of their propensity to be buried residues.
- the following table shows, for each residue, the percentage which were in buried positions, based on a study of the 3D structures of a collection of unrelated proteins: Amino Acid % Buried
- the makeup of the buried core of a protein is dependent, not only on the propensity of each amino acid, if present, to be buried, but also on the overall frequency of occurrence of that amino acid in the protein.
- the most commonly buried residues are, in descending order, Val, Gly, Leu, Ala, He and Ser.
- Buried Cys, (-S-S- form) , Asp, Gly, His, Pro, and Trp are more than 70% likely to be unchanged in a homologous protein. Therefore, if these residues occur in a buried position in the protein of interest it is preferable to leave them unchanged. Their conservation is probably explainable as follows: Cys (disulfide bonding) , Asp (charged but rigid side chain) , Gly (chain flexibility) , His (charged and uncharged states both available at physiological pH) , Pro (unique chain geometry) , and Trp (largest side chain) .
- the other residues, with the exception of Met are 40-60% likely to left unchanged when buried (Met is unchanged only 26% of the time, but it is 25% likely to be replaced by Leu) .
- the most useful information for determining which residues are safely mutatable is knowledge of the sequence of proteins of similar sequence which have similar activity.
- the sequences of these homologous proteins may then be aligned, and residues which are not conserved are more likely to be safely mutatable.
- the degree of confidence which one has as to the tolerance of a residue to mutation is a function of the degree of variation of amino acid type at that site among the protein family, as well as the extent to which all of the proteins in the family, despite their differences, retain the desired activity. While studies of homologous proteins are useful in identifying sites which, by virtue of their variability, are likely to be tolerant of mutation, it is less certain that sites which are strongly conserved are necessarily invariant. According to Shenkin, et al . , (1991), random mutagenesis studies indicate that "proteins are able to accommodate, both structurally and functionally, a far greater variety of mutations than occur naturally" .
- Homologous proteins are those which are similar in structure to the protein of interest, to a statistically significant degree, and which perform the same or an analogous biological function. Examples are human growth hormone and human prolactin, and human alpha globin and human myoglobin. When homologous proteins occur in nature, the similarities may imply that they have a common evolutionary origin, and the time of origin may be estimated by calculating the number of mutations which would give use to the observed sequence divergence and dividing by the mutation rate.
- Cognate proteins are homologous proteins, expressed in a different species of organism, which perform the same biological function as that performed by the protein of interest, although they may differ in activity, specificity, timing of expression, etc.
- cognate proteins are human and fish growth hormones, or human and other vertebrate alpha (or beta) globins .
- the possession of the cDNA or genomic DNA (the “starting DNA”) encoding the protein of interest (the “starting protein”) greatly facilitates the isolation of homologous proteins.
- starting DNA the cDNA or genomic DNA
- starting protein greatly facilitates the isolation of homologous proteins.
- probes to identify homologous genes in other species see, e.g., Schwinn, et al . , J. Biol. Chem., 265:8183-
- genetic elements may be defined, in addition to or in place of definition by percentage identity, by their ability to hybridize.
- a genetic element When a genetic element is desired to be similar to a reference element, it preferably will specifically hybridize under the relaxed conditions set forth above, and more preferably under stringent conditions (wash temperature which is not more than 15°C, more preferably not more than 10°C, below the Tm of a perfect duplex of the reference element) .
- wash temperature which is not more than 15°C, more preferably not more than 10°C, below the Tm of a perfect duplex of the reference element.
- homologous proteins In order to derive guidance from the sequences of homologous proteins, it is necessary to identify which proteins are homologous and to align the sequence of the protein of interest with that of the homologous proteins. Such alignment is guided by calculating a homology or alignment score for each possible alignment, and determining the highest such score for each pair of potentially homologous proteins. Homologous proteins are distinguished from nonhomologous proteins by having an alignment score which is significantly higher.
- Global alignment algorithms e.g., Pearson's ALIGN and ALIGN0 programs
- ALIGN and ALIGN0 programs consider both complete sequences in generating similarity scores for a given alignment, and, in general, allow "gapping" . They are most appropriate when the sequences are known or expected to be similar over their entire length. The most popular form are those which do not penalize end-gaps.
- Local alignment algorithms e.g., Pearson's LALIGN search for similar fragments of two sequences, and, in general, do not allow gaps. They are useful in locating common subdomains between long sequences that otherwise share little similarity.
- a mismatch occurs when the amino acid residues at the same site in two different aligned amino acid sequences are different.
- Several systems are used in the art for scoring matches (identities) and mismatches.
- GCM Genetic Code Matrix
- a Mutation Data Matrix scores amino acid similarity on the basis of the frequency of the exchange of the two amino acids in question between two members of a family of homologous proteins, or of a member and the inferred ancestor of that protein. It is customary for an MDM to take into account the apparent evolutionary distance, too. Thus, it calculates the probability that one residue will be mutated into another residue in a specified unit of a evolutionary time. To calculate this matrix, proteins of known sequences are clustered into families of homologous proteins, a phylogenetic type is constructed for each protein, and an ancestral sequence inferred the amino acid exchanges which apparently occurred between each modem sequence and the ancestral sequence are tailied, and the minimum number of base changes which could explain those exchanges are calculated.
- MDM molecular evolution
- the Structural Similarity Matrix weighs pairings according to the similarity of the amino acids in size, hydrophilicity, and/or other structural measures. Hybrid matrixes have also been devised.
- the standard protein matrix is the Dayhoff PAM 250 matrix.
- the default matrix is the PAM120 matrix, which is more selective.
- overhangs are not counted.
- the simplest method of scoring for internal gaps is to count each deleted residue as a simple mismatch.
- Another approach is to impose an initiating gap penalty and an extension gap penalty.
- the cost of initiating a gap is at least R and more usually at least 2*R.
- Another approach is to set the initiation cost at 1.5 times the most negative aa pair in the matrix, which is -8 in pam 250.
- the extension cost is typically 1/3 to 1/20 the initiation cost .
- the default penalties associated with the pam250 matrix in FASTA are -12 for the first deletion and -4 for each additional consecutive deletion.
- Two random amino acid sequences (of equimolar amino acid composition) would have, an average, an identity of 5% if gapping is not allowed. If gapping is permitted, two random sequences can be 10-20% identical. In general, if two sequences are longer than 100 residues, and are more than 25% identical after suitable gapping, it is likely that they are genuinely related, i.e., that the similarity is not due to chance.
- the "twilight zone" is 15-25% identity.
- the statistical significance of an alignment may be determined by comparing the alignment score obtained when the two authentic sequences are aligned, with the mean and standard deviation of the alignment scores obtained when both sequences are repeatedly randomized, and each "jumbled" sequence in one set is aligned with the jumbled sequences in the other set.
- the score of the authentic sequence is then expressed as so many standard deviations above the mean of the jumbled group. See, e.g., Doolittle, Science, 214:149 (1981); Lipman and Pearson, Science, 277:1435 (1985). However, since similarity scores are distributed according to the extreme value distribution, not the normal distribution, the extreme value statistical form of the z-value should be used. See Altschul, et al . , Nature Genetics, 6:119-129 (1994).
- the program PRSS which is part of the FASTA package, may be used to evaluate the statistical significance of a local alignment, e.g., with LALIGN.
- Lipman and Pearson are of the opinion that the z . value of the optimized alignment similarity score obtained with FASTP should be evaluated as follows: >3 , possibly significant; >6, probably significant; >10, significant. Doolittle used a more stringent identity matrix scoring system and considered a score of 3.0 S.D. or more to be significant.
- a second approach to determining significance of a particular alignment is to compare the alignment score for that alignment with the mean and standard deviation of the alignment scores for the alignments of the query sequence with all sequences in a sequence library. Once again, a z . value is calculated. See Wilbar and Lipman Proc. Nat. Acad. Sci. USA 80:726-30 (1983). Most of these sequences will be unrelated to the query sequence. Of course, the choice of sequences in the library will reflect the interests of the scientific community, e.g., it will tend to favor the sequences of those organisms which are most closely studied, e.g., humans, fruit flies, S. cerevisiae, and E. coli.
- V k The variability index (V k ) is a simple method of quantifying the degree of variation of amino acid residues at a particular aligned site. It is the ratio of the number of different amino acid types which appear at the position, to the fraction of the time which the site is occupied by the most common of these types.
- V k ranges from 1 to 400 for proteins.
- mutations are directed to sites having a variability index which is within the upper 50%, more preferably the upper 20%, of all sites of the protein.
- the informational entropy is less likely to "jump" than the variability index when a new sequence is added.
- mutations are directed to sites which have an informational entropy S greater than 1.0, more preferably greater than 2.0.
- V s is defined as 6 x 2 s ; it ranges from 6 to 120 for proteins. The factor 6 was chosen empirically to make the V k and V s scales roughly comparable .
- V k ranged from 1 to 96, S from 0 to 3.4792, and Vs from 6 to 66.91.
- Binding sites may also be identified by mutagenesis strategies designed to locally perturb the protein.
- One such strategy is alanine scanning mutagenesis. In this technique, all non-alanine residues of the protein (or of a region of the protein suspected to contain the binding site are replaced, one-by-one, with alanine, yielding a collection of single substitution mutants.
- Alanine is used because (1) it is the most common amino acid residue in proteins, (2) it has a small side chain, and therefore is not likely to sterically hinder other residues, and (3) its side chain (-CH 3 ) does not form H- bonds, but is not especially hydrophobic.
- Ala-scanning mutagenesis may be applied to all residues of a protein, or to residues selected on some rational basis, such as amino acid type (e.g., charged and aromatic residues) , degree of variability in a homologous protein family, or relevance to function as shown by homologue- scanning mutagenesis.
- mutations are made at sites where an alanine substitution does not worsen the activity of interest by more than 20-fold, more preferably, by more than 10-fold, even more preferably, by more than 5-fold, still more preferably, by more than 2-fold.
- mutations are made at sites at which an alanine substitutions improves activity.
- the expected (additive) effect of the mutations is one which does not worsen the activity more than 10-fold, more preferably, by more than 5 fold, still more preferably, by more than two fold. Most preferably, the expected effect is to improve activity.
- the expected effect of a conservative substitution is the effect of that mutation as a single substitution if known, or otherwise neutral .
- the expected effect of a nonconservative substitution is the effect of that mutation as a single substitution if known, or otherwise the effect of a single substitution of a different residue of the same exchange group as the actual replacement residue, if known, or otherwise the effect of a single Ala substitution.
- Another approach is homologue-scanning mutagenesis. This involves identifying a homologue which can be distinguished in an activity assay from the protein of interest, and screening mutants in which a segment of the protein of interest is replaced by corresponding segments of the homologue (or vice versa) .
- the segment in question presumably contributes to the observed difference in activity between the protein of interest and the homologous protein, and comparison of the interchanged segments helps to explain the character of the binding site involved in that activity.
- segments of prolactin which does not bind the GH receptor
- a substitution disrupts GH binding it implies that the replaced segment was part of the GH receptor binding site, and one may then focus on how the replaced and replacing segments differ. See WO90/04788.
- the most common reason for combining mutations is to benefit from their additive or synergistic effect in combination. For example, if a mutation has both favorable and unfavorable activities, it may be possible to combine it with a second mutation that neutralizes the unfavorable activity of the first mutation.
- One use of multiple mutation is to achieve, by combining mutations which individually have a small but favorable effect on activity, a mutant with a more substantial improvement in activity. It is not necessary that the mutations be strictly additive; it is sufficient that they be at least partially additive for the combination to be advantageous. See Blacklow, et al . , Biochemistry, 30:8470-6 (1991) (improved catalytic effectiveness of triosephosphate isomerase) ,- Akasako, et al . , Biochemistry, 34:8115-22 (1995) (multiple thermostabilizing mutations in ribonuclease HI); Lowman et al . , J. Biol.
- the protein is too large for all sites of interest to be sampled by binomial Ala-scanning mutagenesis in a single experiment, one may divide the protein into segments and subject each segment in turn to such mutagenesis, and then, as a cross-check, similarly mutate one residue from each segment.
- chimera implies a protein which is a hybrid of two or more different parental proteins which are associated with two or more different organisms.
- Functional chimeras may be identified by a systematic synthesize-and-test strategy. It is not necessary that all theoretically conceivable chimeras be evaluated directly.
- One strategy is described schematically below.
- the units correspond to functional domains or are demarcated so as to correspond to special features of the sequence, e.g., regions of unusually high divergence or similarity, conserved or unconserved regions in the relevant protein family or the presence of a sequence motif, or an area of unusual hydrophilicity or hydrophobicity.
- Let "1" represent a unit of the protein 1, and "2" a corresponding unit of protein 2. If there are five units (the choice of five instead of two, three, four, six, ten, etc. is arbitrary) , we can synthesize and test any or all of the following chimeras, which will help us rapidly localize the critical regions:
- the transgene is a gene encoding a polypeptide which is foreign to the lentivirus (es) from which the vector is primarily derived, and which has a useful biological activity into the organism which is ultimately infected with the transducing vector in its virion-packaged form.
- the transgene may be identical to a wild-type gene, or it may contain one or more mutations.
- the transgene may be derived from genomic DNA, cDNA, synthetic DNA, or a combination thereof.
- Intronless "minigenes” which are normal genes from which introns have been removed, have been especially popular. Intron-containing genes may be employed, but they may be inserted into the vector in the reverse orientation if removal of the introns is not desired.
- Silent mutations may be introduced to facilitate gene maipulation, to avoid undesirable secondary structure in the mRNA, to inhibit recombination, to control splicing, etc. Nonsilent mutations alter the encoded protein, and may be either gratuitous, or aimed at beneficially altering the biological activity of the protein.
- transgene is a remedial gene.
- the term "remedial gene” refers to a gene whose expression is desired in a cell to correct an error in cellular metabolism, to inactivate a pathogen or to kill a cancerous cell.
- the adenosine deaminase (ADA) gene is the remedial gene when carried on a retroviral vector used to correct ADA deficiency in a patient.
- the applications of transgenes include the following:
- anti-pathogen genes or anti- parasite can be introduced into a host infested, or especially vulnerable to infestation, by the pathogen or parasite in question.
- an inherited genetic defect may be ameliorated by supplying a functional gene.
- Suitable approaches include providing genes encoding the enzyme adenosine deaminase (ADA) , especially to hematopoietic stem cells so as to provide longterm treatment of ADA deficiency; and correcting familial hypercholesterolemia with a vector encoding the low density lipoprotein (LDL) receptor.
- ADA adenosine deaminase
- LDL low density lipoprotein
- adenosine deaminase gene has been introduced into peripheral blood lymphocytes and cord blood stem cells via retroviral vectors in order to treat patients with severe combined immunodeficiency due to a lack of functional adenosine deaminase (K.W. Culver et al . , Human Gene Ther., 2:107 [1991]) .
- Partial correction of familial hypercholesterolemia has been achieved using existing retroviral vectors to transfer the receptor for low density lipoproteins (LDL) into hepatocytes.
- LDL low density lipoproteins
- hemophilia (lack of Factor VIII or Factor IX)
- cystic fibrosis (lack of cystic fibrosis transmembrane regulator)
- emphysema defective ⁇ -1-antitrypsin
- thaiassemia and sickle cell anemia defective synthesis of 3-globin
- phenylketonuria deficient phenylalanine hydroxylase
- muscular dystrophy defective dystrophin
- CF cystic fibrosis
- F508 single amino acid deletion
- CFTR CF transmembrane regulator
- Other forms of genetic mutations in the CFTR genes have also been identified. This rich genetic information makes CF an ideal gene therapy candidate.
- the target cells for CF patients are undifferentiated, proliferating and differentiated, non-proliferating lung epithelial cells. It is hoped that both of the dividing and non-dividing lung epithelial cell types can be efficiently targeted by VSV-G pseudotyped lentiviral vectors carrying a wild type CFTR cDNA.
- CF patients have CFTR mutations which leads to basic chloride flux defect in the respiratory ciliated epithelial cells. This CFTR dysfunction causes chronic infection and inflammation of the respiratory tract and leads to high morbidity and mortality in CF patients.
- the CFTR cDNA gene transfer by adenoviral vectors or liposomes has demonstrated partial correction of the defective CFTR channel activity in the nasal epithelium of CF patients.
- An important indication that CFTR dysfunction in CF patients could be treated by gene therapy protocols came from the study of Johnson et al . who demonstrated that overexpression of CFTR which numerically corrected 6-10% of a mutant CF epithelial monolayer resulted in a bioelectric phenotype similar to sheets of 100% corrected cells.
- cancers may be treated with vectors carrying genes which express cancer antigens, or immunomodulatory proteins, and thereby stimulate an immune response against the cancer cells, or which express a normal tumor suppressor gene to replace the function of a mutated, tumor-prone gene, such as a p53 mutant.
- Gene therapy originally developed for treating inherited and acquired diseases by introducing therapeutic genes to somatic cells, has great potential for cancer treatment. With the rapid advances in molecular medicine and gene delivery technology during the past decade, gene therapy approaches have brought excitement and new hopes to fighting cancers. Currently, more than 70% of approved clinical trial gene therapy protocols worldwide are designed for treating cancers. The list is growing rapidly because of the ineffectiveness of conventional cancer treatments, especially to those late stage, metastatic cancers. There are three major components to be considered in the design and development of a gene therapy regimen: the therapeutic genes, the mode of gene delivery (ex vivo or in vivo) , and an appropriate preclinical study model for the assessment of the therapeutic efficacy. Various therapeutic genes have been utilized in cancer treatments.
- the common examples include: (1) genes that are capable of changing the cellular sensitivity to chemo- or radiation therapy in cancer patients either to sensitize tumor cells, or to minimize the damage of chemotherapy to normal cells such as the hematopoietic stem cells, (2) genes that interfere with proliferating tumor cell cycle by either replacing the mutated genes (i.e. tumor suppresser genes and apoptotic genes), or inactivating the oncogenes to prevent further tumor development, and (3) genes that can augment a systemic anti-tumor immunity in cancer patients; this can be accomplished by the injection of modified tumor infiltrating lymphocytes (TIL) or immunomodulatory gene-modified tumor cells, or by the modification of antigen presenting cells (APC) .
- TIL tumor infiltrating lymphocytes
- APC antigen presenting cells
- Retroviral vectors containing genes encoding tumor necrosis factor (TNF) or interleukin-2 (IL-2) have been transferred into tumor- infiltrating lymphocytes in patients (A. Kasid et al . , Proc Natl Acad Sci USA. 87:473-477 [1990]; and S.A. Rosenberg, Human Gene Therapy 5: 140 [1994]). It is postulated that the secretion of TNF or IL-2 stimulates a tumor-specific immune response resulting in the destruction of the tumor or the recruitment of effective tumor infiltrating lymphocytes from nearby lymph nodes.
- Other proposed anti-tumor gene therapy strategies include the delivery of toxin genes to the tumor cell.
- antisense genes or antisense oligonucleotides in inhibition of oncogenes and modulation of growth factors have the potential to reduce the mortality of cancer, in particular, human leukemia (For review see, A.M. Gewirtz, Stem Cells 3:96 [1993]; and L. Neckers and L. Whitesell, Amer. J. Physiol . , 265 :L1 [1993]).
- vectors may be used to deliver transgenes which protect susceptible cells against HIV by synthesizing proteins, antisense RNAs, or ribozymes that block HIV binding and entry, reverse transcription, integration, or replication.
- transgenes must be regulated so they do not interfere with the packaging of the transducing vector.
- a vector may contain one or more selectable or screenable markers. Such markers are typically used to determine whether the vector has been successfully introduced into a host or target ceil .
- a selectable marker is a gene whose expression substantially affects whether a cell will survive under particular controllable conditions.
- a selectable marker may provide for positive selection (cells with the marker are more likely to survive) , negative selection (cells with the marker are less likely to survive) , or both (the choice of environmental condition dictating whether positive or negative selection occurs) .
- Selectable markers include those which confer antibiotic resistance (or sensitivity) , the ability to utilize a particular nutrient, and resistance (or sensitivity) to high
- Suitable selectable markers include the bacterial neomycin and hygromycin phosphotransferase resistance genes, which confers resistance to G418 and hygromycin, respectively, the bacterial gpt gene, which allows cells tog row in a medium containing mycophenolic acid, xanthine and aminopterin; the bacterial hisD gene which allows cells to grow in a medium lacking histidine but containing histidinol; the multidrug resistance gene mdr; the hprt and HSV thymidine kinase genes, which allow otherwise hprt- or tk- cells to grow in a medium containing hypoxanthine , amethopterin and thymidine, and the bacterial genes conferring resistance to puromycin or phleomycin. Positive or negative selection may require the use of a particular strain of host cell for the selection to be effective.
- Screenable markers are genes which encode a product whose presence is readily detectable, directly or indirectly, but which do not necessarily affect cell survival.
- the green fluorescent protein (GFP) is an example. Any cell surface protein not native to the host cell can be used as an immunoscreenable marker. Transformed cells may be segregated out by using a fluorescent antibody to the protein and a cell sorter.
- Many enzyme-encoding genes are useful as screenable markers, especially those encoding enzymes which can act upon a substrate to provide a colored or luminescent product.
- the luciferase and beta-galactosidase genes have been especially popular .
- a dominant marker encodes an activity which can be detected in any eukaryotic cell line.
- dominant selectable markers include the bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene
- selectable markers are not dominant in that their use must be in conjunction with a cell line that lacks the relevant activity.
- examples of non-dominant selectable markers include the thymidine kinase ( tk) gene which is used in conjunction with tk " cell lines, the CAD gene which is used in conjunction with CAD-deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is used in conjunction with hprt ' cell lines.
- transgene (s) of the transducing vector and the marker (s) and viral genes (or replacements) of the packaging and transducing vectors, are expressed under the control of regulatory elements.
- regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
- a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
- Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (defined infra) .
- a constitutive promoter is one which is always active at essentially a constant level.
- Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (T. Maniatis et al . , Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i . e . , promoters, are also found in prokaryotes) . The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest . Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types
- the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (R. Dijkema et al . , EMBO J. 4:761 [1985]) .
- Two other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor l ⁇ gene (T. Uetsuki et al .
- promoter/enhancer denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element, see above for a discussion of these functions) .
- the enhancer/promoter may be "endogenous” or “exogenous” or “heterologous.”
- An “endogenous” enhancer/promoter is one which is naturally linked with a given gene in the genome.
- An “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation ( i .
- a regulatable promoter is one whose level of activity is subject to regulation by a regulatory molecule.
- An inducible promoter is one which is normally substantially inactive, but which is activated by the binding of an inducer to an operator site of the promoter.
- a repressible promoter is one which is normally active, but which is substantially inactivated by the binding of a repressor to an operator site of the promoter. Similar terminology applies to enhancers.
- the inducer or repressor molecules are typically expressed only in particular tissues, at a particular developmental stage, or under particular environmental conditions (e.g., damage to the cell, infection, overproduction of a metabolite, absence of a nutrient, etc.) .
- an inducible promoter may be inactive or may produce a low level of The level of activity in the presence of the inducer will be higher than the basal rate.
- a tightly inducible promoter is one whose basal level of activity is very low, e.g., less than 10 % of its maximum inducible activity.
- Different promoters may have different levels of basal activity in the same or different cell types. When two different promoters are compared in a given cell type in the absence of any inducing factors, if one promoter expresses at a higher level than the other it is said to have a higher basal activity.
- the activity of a promoter and/or enhancer is measured by detecting directly or indirectly the level of transcription from the element (s) .
- Direct detection involves quantitating the level of the RNA transcripts produced from that promoter and/or enhancer.
- Indirect detection involves quantitation of. the level of a protein, often an enzyme, produced from RNA transcribed from the promoter and/or enhancer.
- a commonly employed assay for promoter or enhancer activity utilizes the chloramphenicol acetyltransferase (CAT) gene.
- CAT chloramphenicol acetyltransferase
- the level of enzymatic activity is proportional to the amount of CAT RNA transcribed by the cell line.
- This CAT assay therefore allows a comparison to be made of the relative strength of different promoters or enhancers in a given cell line.
- a promoter is said to express at "high” or “low” levels in a cell line this refers to the level of activity relative to another promoter which is used as a reference or standard of promoter activity.
- Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.
- the term "poly A site” or "poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
- the poly A signal utilized in an expression vector may be "heterologous” or "endogenous.”
- An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
- a heterologous poly A signal is one which is one which is isolated from one gene and placed 3' of another gene.
- a commonly used heterologous poly A signal is the SV40 poly A signal.
- the SV40 poly A signal is contained on a 237 bp Bam HI /Bel I restriction fragment and directs both termination and polyadenylation (J.Sambrook et al . , supra , at 16.6-16.7) .
- the cytomegalovirus immediate early promoter-enhancer (CMV- IE) is a strong enhancer/promoter. See Boshart M, Weber F, Jahn G, Dorsch-Hasler K, Fleckenstein B,
- the internal promoter for a transgene may be the promoter native to that transgene, or a promoter native to the target cell (or viruses infecting the target cell) , or another promoter functional in the target cell.
- the preferred promoters and enhancers are those exhibiting tissue or cell type sepecificity which can direct the transgene expression in the target cells at the right time(s) .
- a promoter to control human preproinsulin must be operable under control of carbohydrate in the liver.
- An example of such a promoter is the rat S-14 liver-specific promoter.
- Promoters (and enhancers) may be naturally occurring sequences, or functional mutants thereof, including chimeras of natural sequences and mutants thereof.
- a tissue-specific, development -specific, or otherwise regulatable element of one promoter may be introduced into another promoter.
- vector is used in reference to nucleic acid molecules that can be used to transfer nucleic acid (e . g. , DNA) segment (s) from one cell to another.
- nucleic acid e . g. , DNA
- vector is sometimes used interchangeably with “vector.” It is intended that any form of vehicle or vector be encompassed within this definition.
- vectors include, but are not limited to viral particles, plasmids, transposons, etc.
- transfection refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, elect roporat ion , microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
- Vectors may contain "viral replicons "or "viral origins of replication.”
- Viral replicons are viral DNA sequences which allow for the extrachromosomal replication of a vector in a host cell expressing the appropriate replication factors.
- Vectors which contain either the SV40 or polyoma virus origin of replication replicate to high copy number (up to 10 4 copies/cell) in cells that express the appropriate viral T antigen.
- Vectors containing the replicons from bovine papillomavirus or Epstein-Barr virus replicate extrachromosomally at low copy number (-100 copies/cell) .
- expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
- Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional) , and a ribosome binding site, often along with other sequences.
- Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
- "expression vectors" are used in order to permit pseudotyping of the viral envelope proteins.
- the host cell is a cell into which a vector of interest may be introduced and wherein it may be replicated, and, in the case of an expression vector, in which one or more vector-based genes may be expressed. It is not necessary that the host cell be infectable by the transducing vector virions of the present invention. Indeed, it is preferable that they not be so infectable, so the hos cells do not bind the virions and thereby reduce the vector production titer. This can be achieved by choosing (or engineering) cells which do not functionally express the receptor to the vector particle envelope protein.
- the transducing vector may be administered to a target organism by any route which will permit it to reach the target cells. Such route may be, e.g., intravenous, intramuscular, subcutaneous, or, with an enteric coating, oral. Alternatively, target cells may be removed from the organism, infected, and they (or their progeny) returned to the organism. Or the transducing vector may simply be administered to target cells in culture.
- the target cells into which the transgene is transferred may be any cell which the transducing vector, after packaging into a virion, is capable of infecting, and in which the control sequences governing expression of the transgene are functional.
- it will be a eukaryotic cell, preferably a vertebrate cell, more preferably a cell of a mammal or bird.
- the mammal will preferably belong to one of the orders Artiodactyla (e.g., cows, pigs, goats, sheep), Perissodactyla (e.g., horses), Rodenta (e.g., rats, mice), Lagomorpha (e.g., rabbits), Carnivora (e.g., dogs, cats) or Primata (e.g., humans, apes, monkeys, lemurs) .
- Artiodactyla e.g., cows, pigs, goats, sheep
- Perissodactyla e.g., horses
- Rodenta e.g., rats, mice
- Lagomorpha e.g., rabbits
- Carnivora e.g., dogs, cats
- Primata e.g., humans, apes, monkeys, lemurs
- a bird it will preferably be of the orders Anseriformes (e.g., ducks, geese, swans) or Galliformes (e.g., quails, grouse, pheasants, turkeys, chickens) . Most preferably it will be a human cell.
- Anseriformes e.g., ducks, geese, swans
- Galliformes e.g., quails, grouse, pheasants, turkeys, chickens.
- Non-dividing cells of particular interest include neuronal cells and astrocytes .
- Dividing cells of particular interest include hematopoietic stem ceils, muscle cells, white blood cells, spleen cells, liver cells, epithelial cells and eye cells .
- TE671, HepG2, HeLa, 293T, and MT4 are of particular interest for experimental studies .
- TE671 rhabdomyosarcoma cells can be induced to differentiate into muscle cells by HIV-1 Vpr.
- HepG2 hepatoma, HeLa cervical carcinoma, 293T human kidney carcnoma and MT4 lymphoma cells are all transformed by HTLV-I human T cell leukemia virus type I.
- MT4 cells are very susceptible to wild-type HIV-1 NL4-3 and hence have been used as indicator cell for RCV. Miscellaneous Defini tions
- endogenous virus is used in reference to an inactive virus which is integrated into the chromosome of its host cell (often in multiple copies) , and can thereby exhibit vertical transmission. Endogenous viruses can spontaneously express themselves and may result in malignancies .
- gene refers to a DNA sequence of a vector or genome which comprises a coding sequence and which is operably linked to one or more control sequences such that, in a suitable host cell, under suitable conditions, a biologically active gene product, or a gene product which is a precursor of a biologically active molecule, is produced which is encoded by the coding sequence.
- This gene product may be a transcriptional product, i.e., a messenger RNA, as in the case of an antisense RNA or a ribozyme .
- polypeptide may be a translational product, i.e., a polypeptide (the term "polypeptide” as used herein includes oligopeptides) , which is either biologically active in its own right, or further processed by the cell to generate one or more biologically active polypeptide products.
- a polypeptide the term “polypeptide” as used herein includes oligopeptides
- the term “gene” also refers to the RNA sequence of the retroviral genome which a suitable host cell reverse transcribes into a DNA sequence which acts as a gene in the classic sense.
- the term “gene” may refer to the DNA sequence encoding a single mRNA transcript, or only to that portion of the DNA sequence which is ultimately expressed as a single polypeptide chain.
- each gene may be constructed from genomic DNA, complementary DNA (DNA reverse transcribed from mRNA) , synthetic DNA, or a combination thereof.
- the gene may duplicate a gene which exists in nature, or differ from it through the omission of introns (noncoding intervening sequences) , a so-called mini-gene, silent mutations (i.e., mutations which do not alter the amino acid sequence of the encoded polypeptide), or translated mutations (i.e., mutations which do alter that sequence) .
- the mutations may be functional mutations (ones which preserve at least a substantial portion of at least one of the biological activities or functions of the encoded polypeptide) or nonfunctional (inactivating) mutations.
- transcription unit refers to the segment of DNA between the sites of initiation and termination of transcription and the regulatory elements necessary for the efficient initiation and termination.
- a segment of DNA comprising an enhancer/promoter, a coding region and a termination and polyadenylation sequence comprises a transcription unit.
- RNA especially lentiviral RNA (e.g., packaging vector RNA) ; whether recombination occurs between the packaging vector and the transducing vector, or between these two vectors and defective retroviruses endogenous to the host
- the packaging cell line packages the transducing vector into the viral particles; whether the transducing vector-containing viral particles are infectious vis-a-vis the target cells; whether the latter particles are cytotoxic to the target cells; whether the latter particles are immunogenic to the target organism; whether infected target cells themselves produce viral RNA-containing particles, infectious or otherwise; and the level and duration of expression of the transgene in the target cells.
- the successful establishment of the packaging or transducing vector in the host (or target) cell may be verified by selecting for the presence of a selectable marker, or screening for the presence of a screenable marker, carried by the vector.
- the integration of the relevant packaging or transducing vector genes may be determined by collecting genomic DNA, amplifying the gene of interest by PCR, and detecting the amplified sequence with a suitable hybridization probe.
- the production of viral proteins may be detected by an immunoassay; the sample may be a cell lysate or a cell supernatant .
- An immunoassay by itself cannot determine whether the viral proteins are produced in functional form, although there is greater assurance of this if the antibody used is directed to a conformational epitope, or is an activity- neutralizing antibody.
- the functionality of the produced Gag and Env protein may be determined by examining the cell lysate or supernatant for the presence of viral particles; these may further be examined for proper morphology by means of an electron microscope. It is also possible that antibodies could be used which bind to the formed viral particles, but not to gpl20 or gp41 by itself.
- the functionality of the Pol reverse transcriptase may be determined by assaying the viral particles for RT activity.
- the functionality of the Pol integrase is apparent only in assays which examine whether RNA from viral particles is integrated into the target cell .
- Viral particles produced by the packaging ceil line may be collected and assayed for total RNA content. If more specific information is desired as to the nature of any packaged RNA, a suitable hybridization probe may be employed.
- the vector is introduced into a first culture of susceptible cells. Then, either a second culture is layered onto the first, so that infectious particles may travel by cell-to-cell contact, or the second culture is exposed to the supernatant of the first culture.
- the cells of the first and second culture are examined for a least one of the following indicia: RT activity, p24 Gag antigen expression, production of viral particles, and cytotoxic effects.
- the stringency of the assay is dependent on the susceptible of the cells to infection and to cytotoxicity, and the time allowed for the recombination and spread of the virus in the first and second cultures.
- the infectivity of the vector or vector system will be compared with that of a wild-type, unattenuated, replication-competent lentivirus.
- the ability of a packaging vector to generate transmissible virus, as opposed to defective virus, may be measured.
- One method is described by Mann, et al . , Cell, 33: 153-9 (1983) .
- the packaging vector and its wild-type counterpart are independently transfected into suitable host cells, and reverse transcriptase activity in the culture supernatants is assayed over a period of days or weeks.
- a rapid increase in RT activity over 24-48 hrs is indicate of gene expression after transient transfection.
- a continued increase is indicative of the efficient spread of virus from the initially transfected cells to the remaining cells on the plate.
- a slow or delayed increase could be indicative of either a steady but attenuated spread of virus, or to generation of competent virus by mutation, or by recombination with a cellular sequence capable of providing the missing function.
- the packaging efficiency of a packaging cell line in the presence or absence of the packageable transducing vector may be measured in a variety of ways.
- One method is described by Mann, et al . , Cell, 33: 153-9 (1983).
- total cellular RNA is purified from the culture supernatant of the test and control cell lines, and viral RNA is extracted from purified viral particles released from the test and control cell lines.
- the two virion preparations are normalized by reference to their reverse transcriptase activity just prior to RNA extraction.
- the purified RNAs are probed with a virus-specific hybridization probe (e.g., a plasmid containing the entire viral genome) in a slot-blot assay, and the amount of viral RNA in the particles and in the cells is thereby quantified.
- a virus-specific hybridization probe e.g., a plasmid containing the entire viral genome
- packaging efficiency of a packaging cell line it is not unusual for the packaging efficiency of a packaging cell line to be less than 1% that of a host cell infected by wild-type virus.
- the packaging cell line be able to efficiently package the highly defective transducing vector into viral particles, and bud the particles into the culture supernatant (in vitro) or extracellular environment (in vivo) without also budding helper virus (the packaging vectors) .
- the transducing vector is transfected into the packaging (helper) cell line. After 24 hours, the culture supernatants are used to infect fresh potential host cells (reporter cells) . Two days later, selection pressure for the transferred gene is applied, and 8-10 days later, the transferred gene-positive colonies or cells are counted. In addition, one determines the reverse transcriptase activity of the supernatant colelcted from the packaging cell lines, and the reverse transcriptase activity of the fresh cells. A transducing vector-specific packaging cell line will produce a high transfer gene activity and a low reverse transcriptase activity in the reporter cells. In addition, the reporter cells will not produce reporter gene-positive colony-forming units (cfus) .
- the ability of a packaging vector to provide all viral functions required in trans may be assayed by cotransfecting host cells with the packaging vector (or control virus) and with a reporter vector carrying a selectable reporter gene. After 24 hours, culture supernatants of the transfected cells are used to infect a second plate of host cells. Selection pressure for the reporter gene is applied, and reporter- positive colonies are counted. If the helper activity is of wild-type magnitude, the count for the packaging vector should be of the same order of magnitude as that for the control virus, and no reporter activity should be detectable in the second plate when the reporter vector or the control wild-type virus expressing all viral functions is transfected into the host cells of the first plate by itself.
- Several sensitive assays are available for the detection of RCV in the present lentiviral vector systems. These include: (1) co-cultivation with a sensitive cell line such as MT4, AA2 or PBLs ; (2) the CD4 HeLa MAGI cell assay which relies on Tat transactivation of an integrated LTR-lacZ gene; and (3) a sensitive immunohistochemical staining method for the detection of HIV antigen expression at the individual cell level. As described in the Examples below, the latter method was modified and developed for the characterization of "Tat-minus" HIV-1 infection, although all three methods are suitable for the routine titration of infectious HIV-1.
- RC-HIV can also be studied in an in vivo model by transduction of humanized SCID/beige mice. In the latter model, a long in vivo incubation time can be performed, mimicking the situation that exists in a human clinical trial. In addition, the possibility of generating HIV/HERV recombinants may be carefully tested using an artificially constructed HIV/HERV-env recombinant.
- the stability of the packaged transducing vectors under adverse conditions, especially those which might be encountered during storage, is of interest.
- Thermostability may be ascertained by subjected them to elevated (e.g., 37 deg. C) or depressed (e.g., 4 deg. C) temperatures for various periods of time (e.g., 2, 4, 6 or 8 hrs . , or overnight), or to a number (e.g., 1-6) freeze-thaw cycles, and determining the number of infectious particles remaining as a percentage of the number of such particles prior to treatment. See Burns et al . 1993.
- a preferred method for determinining whether the contemplated vectors, or their gene products, could elicit an immune response in a subject involves evaluating cell-mediated immunity (CMI) using either an immunocompetent mouse model or a a humanized SCID/beige mouse model .
- CMI cell-mediated immunity
- mice lacking T, B and natural killer (NK) cell functions are severely immunodeficient .
- This strain of mice can be successfully reconstituted with fresh human peripheral blood lymphocytes (PBLs) , and exhibits functional human naive, memory and activated T cell markers for more than 2-3 months (See e . g. , copending U.S. Patent Appln. Ser. Nos. 08/848,760, and 08/838,702, both of which are herein incorporated by reference) .
- spleen and peripheral blood lymphocytes were harvested 38 days after reconstitution from reconstituted SCID/beige mice, and red blood cells were lysed prior to incubation with anti-mouse 2Kd, anti -human CD45, anti-human CD3 , anti-human CD4 and anti-human CD8 labeled antibodies.
- Reconstituted human lymphoid cell populations in the spleen and in the peripheral blood of the SCID/beige mice can reach up to 50-80% and 5-12%, respectively.
- mice repetitively injected with the viral vectors will be analyzed. Their sera will be assayed for Ab response to viral antigens, such as p24 Gag or the pseudotype env (e.g., VSV-G) .
- viral antigens such as p24 Gag or the pseudotype env (e.g., VSV-G)
- the mouse splenocytes will be isolated and an in vitro assay for cellular immunity will be performed as described below.
- T cell response to recall antigen is normally characterized by the production of interferon gamma (IFN ⁇ ) .
- IFN ⁇ interferon gamma
- This assay requires activation of lymphocytes with the test Ags, such as Gag p24 or Gag-Pol or VSV-G env proteins of the vector .
- the Thl lineage of T cells Upon activation, the Thl lineage of T cells produce interferon gamma (IFN-g) and the measurement of IFN-g production has been shown to be a reliable assay for CMI.
- IFN-g interferon gamma
- a sensitive ELISPOT assay for the detection of IFN-g producing cells was developed. With the computer assisted imaging system integrated into this protocol, the ELISPOT method was shown to be very convenient and more sensitive than the conventional limiting dilution assay for the determination of the effector T cell precursor frequency.
- This in vivo model and the ELISPOT assay system were developed for the evaluation of in vivo CMI after lentiviral gene transfer. (See, e.g., PCT/US98/06944 ) .
- RCR replication-competent retrovirus
- RCV replication-competent virus
- WT wild-type
- PBL peripheral blood lymphocyte
- M molar
- mM millimolar
- ⁇ M micromolar
- mol molecular weight
- base pair base pair
- kb kilo base pair
- TEM transmission electron microscope
- SEM scanning electron microscope
- TLC thin layer chromatography
- tRNA transfer RNA
- nt nucleotide
- VRC vanadyl ribonucleoside complex
- RNase ribonuclease
- DNase deoxyribonuclease
- poly A polyriboadenylic acid
- HBS HBS buffered saline
- SDS sodium dodecyl sulfate
- LB Lia-Bertani broth: 10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter, pH adjusted to 7.5 with IN
- AIDS Research and Reference Reagent Program (AIDS Research and Reference Reagent Program of the National Institutes of Health, Bethesda, MD) ; Beckman (Beckman Instruments Inc., Fullerton CA) ; BM (Boehringer Mannheim Biochemicals, Indianapolis, IN) ; Bio-101 (Bio-101, Vista, CA) ; BioRad (BioRad, Richmond, CA) ; Brinkmann (Brinkmann Instruments Inc. Wesbury, NY); BRL, Gibco BRL and Life Technologies (Bethesda Research Laboratories, Life Technologies Inc., Gaithersburg, MD) ; CRI (Collaborative Research Inc. Bedford, MA); Eastman Kodak (Eastman Kodak Co., Rochester, NY); Eppendorf (Eppendorf, Eppendorf North America, Inc., Madison,
- non-attenuated HIV strains used- include the NL4-3 HIV-1 strain, HIV-1 primary isolates covering the different HIV clades ( e . g. , 92RW008, 92HT593, etc.), the ROD strain of HIV-2, and the SIVmac239 strain of SIV, all of which are available from the AIDS Research and Reference Reagent Program.
- HIV-1 LTR and tat mutations were constructed as described previously (Chang et al.1993; Chang and Zhang, 1995) .
- Cloned HIV proviruses with heterologous enhancer/promoters were constructed by ligating three fragments from an HIV-1 molecular clone HIVNL4-3 (Adachi et al.1986), two fragments isolated from the U3-R-CAT plasmids containing inserted heterologous enhancer/promoters and the BamHI plus Pstl digested pT7T318U vector.
- the proviral segments used in the ligation were as described before (Chang et al.1993) .
- the structures of the reconstructed HIV proviral DNAs were verified by extensive restriction enzyme mapping, and the LTR regions were checked by nucleotide sequencing.
- RT assay and p24 ELISA for the detection of HIV gag and pol products.
- RT assays detect functional reverse transcriptase activity which were performed as described below. The supernatants from transfected cells were spun in a microfuge at 3000 rpm for 5 min before being added to the reaction mixture. Supernatants from virus infections were removed from cultures after the cells had settled.
- Each reaction mixture contained 10 ml of supernatant and 50 ml of RT cocktail (60 mM Tris-HCl, pH 7.8, 75 mM KCl, 5 mM MgCl2, 0.1% Nonidet P-40, ImM ' EDTA, 5 mg/ml poly rA and 0.16 mg/ml oligo-dT) and was incubated at 37oC for 1 h.
- the radioactive products generated in the CAT and RT assays were quantitated by using a Fuji phosphoimager . The results obtained were comparable to those derived by scintillation counting.
- p24 antigen is derived from p55 gag precursor. The p24 antigen expression was quantified using a commercial ELISA kit from Coulter (Coulter Corp., Hialeah, FL) .
- Cell-free particles present in the supernatants of vector producing cells, were harvested (100 microl) , centrifuged at top speed for 5 min in a microcentrifuge at room temperature, and filtered through a 0.45 mm-pore-size Eppendorf spin filter. The particles present in the filtrate was dissociated by vortexing in the presence of an equal volume of 8 M LiCl, placed on dry ice for 20 min, transferred to a -20oC freezer for at least 2 h, and centrifuged at top speed in a microcentrifuge at 4o C for 20 min.
- RNA pellet was then rinsed with 70% ethanol, dried briefly under vacuum, resuspended in water and reverse transcribed by using an appropriate primer and the RiboClone cDNA Synthesis System (Promega) for the synthesis of the first DNA strand.
- a control reaction excluding the reverse transcriptase was performed in parallel.
- the cDNA was amplified by PCR using the polymerase and reagents obtained from Perkin Elmer Cetus ; 5' and 3' primers (0.1 micromole each) were added to a reaction mixture containing the cDNA (1/20 of the RT product) and amplified for 30 cycles under the following conditions: 94oC for 1 min, 58oC for 1 min and 72oC for 3 min.
- the product obtained was then subjected to asymmetric PCR amplification (i.e., two primers at 10:1 molar ratio) to generate single stranded DNA for sequencing as described by Meltzer et al . (39) .
- Excess primers were removed with a centricon 100 filtration device (Amicon) after each amplification step. Nucleotide sequencing was performed using Sequenase and protocols supplied by USB.
- non-adherent ceils were attached to the surface of a microscope cover glass (12 mm circle, Fisher Scientific, Pittsburgh, PA) which had been pretreated with poly-D-lysine (1 mg/ml, Sigma) at room temperature for 10 min.
- the attached cells were washed with phosphate buffered saline (PBS) three times, fixed in cold acetone and methanol (1:1) for 5 min, washed three times in PBS, and incubated in blocking (20% FBS, 0.1% TritonXlOO in PBS) solution for 30 min.
- PBS phosphate buffered saline
- the primary antibody was diluted at 1:2000 in blocking solution, and the cells were incubated at room temperature for 1 h or at 4oC overnight with constant shaking. After washing in PBS 4 times for 5 min each, the cells were incubated with normal goat or sheep antisera (1:200 dilution) at room temperature for 30 min to block non-specific binding.
- the secondary antibody was FITC-labeled goat anti-human IgG (Fab specific, Sigma Chemical Company, St. Louis, MO) . After staining, the cover glass was washed four times in PBS and examined using a fluorescent microscope. For direct immunohistochemical staining, a peroxidase-linked sheep anti-human Ig (Amersham) was used as the secondary antibody.
- Northern analysis was performed as previously described (Robinson et al.1995) .
- cells were lysed in a buffer containing 50 mM Tris pH 7.4, 300 mM NaCl , 0.5% Triton X100, 1% (v/v) aprotinin and 1 mM PMSF at 4oC for 10 min and freeze-thawed once.
- Virus particles were collected by centrifugation in a refrigerated micro centrifuge in a small volume (200 microliters) at 23,000 g for 1 hr .
- SDS sample buffer final 2% SDS, 5% glycerol, 0/001% BPB, 0.5% NP-40
- PAGE polyacrylamide gel electrophoresis
- the protein was transferred to a 0.2 micron nitrocellulose filter, stained with Ponceau S to identify the molecular weight marker, and blocked with 10% dried milk in TBS-T (Tris-buffered saline with 0.3% Tween 20) at room temperature for 30 min to 1 hr .
- the blot was placed into a "seal-a-meal" bag and incubated with an AIDS patient's serum (diluted at 1:2,000, or a rabbit polyclonal anti-Vpr antibody at 1:1,000, or a monoclonal anti-Nef antibody at 1:1000) in TBS-T containing 2% dry milk at 4oC overnight .
- TBS-T normal goat sera (the same species as the secondary Ab) at 1:200 dilution in a shallow tray or in a bag at room temperature for 30 min.
- the blot was then transferred to a second bag containing a horse radish peroxidase (HRP) conjugated goat anti-human (or goat anti-rabbit, or goat anti-mouse) antibody and incubated at room temperature for 1 hr .
- HRP horse radish peroxidase
- the blot was washed four times in TBS-T and developed using the chemiluminescence ECL immunodetection reagents from Amersham.
- the blot was then exposed to a hyperfilm (Amersham) normally for 1 min and developed.
- HeLa (human cervical carcinoma) and HepG2 (human hepatoma) cells were obtained from ATCC, Rockville, Maryland.
- TE671 (human rhabdomyosarcoma) and 293T (transformed human primary embryonal kidney) cells were kindly provided by Dr. Takeuchi (Chester Beatty Laboratories, obtained from ECACC, England) and Dr. H. Goldstein (Albert Einstein College of Medicine, N. Y. ) , respectively.
- H9, CEM, MT4 , C8166 and AA2 were obtained from NIH AIDS Research and Reference Reagent Program.
- HeLa, HepG2 , TE671 and 293T cells were propagated in Dulbecco' s modified Eagle' s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco Canada) , penicillin and streptomycin.
- DMEM Dulbecco' s modified Eagle' s medium
- FBS fetal bovine serum
- the macrophage culture was prepared from HIV-sero-negative donors by adherence of PBLs to plastic flasks as described previously with minor modifications (Hassan, et al . 1986) .
- PBLs was prepared using lymphocyte separation medium (Organon Teknika Corp. , Durham, NC) by density gradient. The PBLs were resuspended in RPMI 1640 medium supplemented with 20% heat-inactivated human serum. Approximately 5 x 107 PBLs were attached to a T-75 flask and incubated overnight at 37oC. The next day cells were washed three times with phosphate buffered saline and the attached cells were incubated with 0.02% EDTA in PBS for 5-10 min.
- the cells were collected with a cell scraper and plated onto a 48-well plate at 5 x 10 4 cells per well. The viability approached 100% as determined by trypan blue staining.
- the initial monocytes were characterized by Wright's staining and the mature macrophages by both Wright's staining and microscopic examination.
- Sprague Dawley rats (180-200 gram of body weight) were purchased from the Health Science and Laboratory Animal Service (HSLAS) at the University of Alberta.
- tat-A, tat-B and tat-C site-directed mutations were generated by the "Megaprimer” method of Sarkar and Sommer (Sarkar and Crissman, 1990) using the following mutagenic oligos : 5' -GAATTGGGTGTCGACATAGCGGCCGCTTGTACCAATTGCTATTG-3' , 5 ' -GGTACAAGCAGTTTAAGGCTAACTTCCTGGATGCTTCC-3 ' , and
- a fragment containing the tat mutation [EcoRI-SacI (260 nt) ] generated by PCR mutagenesis was used to construct the full-length two LTR HIV plasmids. Construction of the tat-A and tat-C mutations have been described elsewhere (Dimitrov, et al . 1993; Amendt, et al . 1994) .
- the dl.Spl/CMV tat-B macrophage-tropic virus was made by replacing the EcoRI to BamHI fragment in a T-cell tropic construct (pNL4-3, Adachi, et al . 1986) with the same fragment from a macrophage-tropic construct (pNLAD8, kindly provided by Eric Freed) . Sequences of the PCR fragment and its flanking region in the final constructs were verified by DNA sequencing.
- HeLa cells were split into 6-well plates 20 h prior to transfection.
- the plasmid DNA was in 90 ml of ddH 2 0 and mixed with 10 ml of 2.5 M CaCl 2 (Mallinckrodt) in a polycarbonate tube.
- a 100 ml of BES-buffered solution 50 mM
- transfection was done using 10 mg of DNA of different HIV-1 constructs.
- HeLa cells were transfected with 10 mg of cloned HIV-1 plasmids and virus was harvested, filtered through a 0.45 m filter (MILLEX-HV, Millipore Products Division, Bedford, MA) and frozen at -80oC for later use. All transfections were performed in the presence of a control human growth hormone plasmid pXGH5 (Nichols Institute Diagnostics) .
- Northern analysis of viral RNA was done as described previously (Chang et al.1993) and analyzed using a phosphoimager (Fuji, BAS1000) .
- Adherent cells were washed with phosphate buffered saline (PBS) three times, fixed in cold acetone and methanol (1:1) for 2 min, washed three times in PBS, and incubated in blocking solution (20% FBS, 0.1% TritonXlOO in PBS) for 30 min.
- Non-adherent cells were attached to the surface of a 24 -well plate which had been pretreated with poly-D-lysine (1 mg/ml, Sigma) at room temperature for 10 min.
- the cells were washed four times in PBS-Tween 20 (0.3%) and incubated in the ultra-sensitive ABC staining solution (containing avidin and biotinylated horseradish peroxidase, Pierce Chemical Co.) at room temperature for 30 min. After four more washes in PBS-Tween 20, the cells were incubated in 3, 3 ' -Diaminobenzidine tetrahydrochloride (DAB) solution (Sigma) containing 0.3%NiCl 2 for 2-3 min. The reaction was stopped by washing cells with tap water for 1-2 min. Cell staining was scored under an inverted microscope and photographed. To reduce background staining, both the primary and the secondary antisera were preabsorbed with fixed human PBLs. Pretreatment of fixed cells with 0.01% H202 at room temperature for 5 min essentially eliminated all nonspecific background signals. The percentages of positive cells were determined by taking the average of more than three representative counts of 1,000 or 10,000 cells.
- Genomic and Hirt DNA preparation and Southern and PCR analyses were used which allowed simultaneous preparation of genomic and Hirt DNA. Motmans, et al . , BioTechniques, 23:1044-6 (1997). Briefly, cells were washed three times with PBS and resuspended in 250 ⁇ l 25 mM Tris-HCl pH 8.0 buffer containing 50 mM glucose, and 10 mM EDTA. The resuspended cells were incubated at room temperature for 5 min, and then lysed in 200 ⁇ l lysis buffer containing 200 mM NaOH and 1% SDS on ice for 5 min. The lysate was neutralized by adding 150 ⁇ l potassium acetate (5 M, pH 4.8) .
- Cell debris and chromosomal DNA were removed by centrifugation at 10,000 x g for 5 min.
- the supernatant containing the Hirt DNA was loaded onto a QIAprep Spin Columns and centrifuged for 1 min. Columns were washed to remove residual endonucleases and salts, and the DNA was eluted with 100 ⁇ l distilled water (75°C) by centrifugation at 10,000 x g for 1 min.
- the pellet containing the genomic DNA was processed using a Qiagene genomic DNA harvesting kit starting from the proteinase digestion step according to the manufacturer ⁇ s instructions. Southern analysis was performed using standard protocols as described by Maniatis et al .
- PCR analysis of unintegrated lentiviral proviral DNA was performed using the following two sets of nested primers flanking the LTR of the circular lentiviral proviral DNA: 5 ' -ACG ACT CCT GGA GCC CG- (3' end of the lacZ gene) and 5'-ACA AGG CAG CTG TAG ATC TTA GCC- (5' end of poly- purine tract (PPT) of HIV-1); 5 ' -ACT TTC GCT TTC AAG TCC C- (upstream of primer binding site) and 5 ' -ACT GAC GCT CTC GCA CCC AT- (downstream of gag AUG) .
- the amplified products from the one-LTR lentiviral proviral circular DNA will be 715 bp and from the two-LTR proviral circular DNA will be 1351 bp.
- the amplified products from the one-LTR MLV proviral DNA will be 733 bp or 1195 bp .
- Two-LTR circles were infrequently amplified from both HIV and MLV vector transduced cells.
- lentiviruses to infect non-dividing cells such as macrophages and neurons makes them good candidates for use as gene transfer tools.
- HIV-1 Gag-Pol requires the activation of LTR by Tat and the interaction of Rev-RRE to mediate nuclear export of mRNA, whereas the accessory gene functions of vif, vpr, vpu and nef have been shown to be dispensable for viral replication, as well as for vector function in tissue culture. Both tat and rev genes are functional in the gag-pol packaging construct pHP .
- pHP pHP
- several cis-elements essential to viral replication have been deleted, including both the 5' and the 3' LTRs, the 3' PPT and the entire 5' leader sequences except for TAR.
- a 59 bp artificial RSV splice donor sequence has been inserted into pHP which supports tat and rev mRNA splicing.
- the RSV gag AUG is located in the 59 bp artificial leader sequence 5' to the RSV SD site.
- the RSV gag AUG was mutated in pHP .
- the expression of functional tat and rev genes by pHP indicates that the 5' RSV gag AUG mutation does not interfere with the RSV SD function.
- Naldini et al have compared transduction efficiencies of an HIV-1-based vector versus an MLV-based vector using both proliferating and growth-arrested HeLa cells and 208F rat fibroblasts.
- the results of their studies show that the HIV- based vectors infect Gl/S and G2 arrested HeLa cells and GO arrested 208F cells more efficiently than the MLV-based vector.
- the HP/TV vector transduced growth-arrested or terminally differentiated cells efficiently in tissue culture including primary rat and human neuronal cells.
- pTV contains a CMV- IE enhancer/promoter which has been shown to frequently be inactivated after integration. Promoter inactivation could therefore have contributed to loss of HP/TV gene expression.
- HIV-1 LTR Mutants Investigation of virus attenuation was essential to the understanding of viral pathogenesis, the development of preventive vaccines, and development of a safe lentiviral vector system.
- a safe HIV vector For production of a safe HIV vector, attenuated mutant molecular constructs of HIV-1 were viewed as better starting materials than wild-type constructs.
- One approach to developing these attenuated constructs was establishing mutations in the LTRs of HIV-1.
- the function of HIV-1 LTR enhancer/promoter elements has been studied using recombinant LTRs containing heterologous enhancer/promoters ( See, Figure 1) .
- NF-kB and Spl binding sites in the HIV-1 LTR are not absolutely required for viral replication and pathogenicity in vivo, a correlation of LTR mutations with low viral load and prolonged asymptomatic state has been observed for isolates of long term survivors of HIV-1 infection.
- LTR deletion mutants containing a cytomegalovirus enhancer element were capable of attenuating HIV-1 (i.e., the mutants were capable of infecting human lymphocytes with reduced cytopathic effects when the tat gene also was deleted) . Instead of killing the entire culture, infection with these LTR- and tat mutants led to rapid cell recovery and establishment of persistent infection. The replication efficiency was not markedly affected by these mutations.
- CMV-IE-HIV-LTR recombinant LTRs
- the tat gene was also a target, as it is a gene that is essential for efficient HIV-1 replication.
- HIV-1 Tat has been implicated in the induction of Kaposi's sarcoma, repression of MHC Class I gene promoter, induction of functional unresponsiveness of T cells, modulation of monocyte function, induction of IL-10 expression, potentiating TNF-induced NF-kB activation and cytotoxicity, and sensitizing T cells to Fas-mediated apoptosis (L.-J. Chang et al . , J Virol., 67:743-752; N. Chirmule e al., J Virol . 69:492-498 [1995]; B. Ensoli et al .
- LTR/tat double mutants were established. These LTR/tat double mutants were generated using the LTR mutant constructs which exhibited enhanced transcriptional activity after inserting heterologous enhancer elements.
- the recombinant LTR (CMV-IE-HIV-LTR) , which has been shown to exhibit increased basal level of promoter activity, can support HIV-1 replication without Tat (L.-J. Chang and C. Zhang, Virol., 211:157-169 [1995]; D. Robinson et al . , Gene Therap., 2:269-278 [1995]).
- the tat-C mutant is more defective than the tat-A and -B mutants
- the dl.Spl/CMV tat-B double mutant is more defective than the dl.Spl/CMV LTR mutant or the dl.Spl/CMV tat-A double mutant reported previously (L.-J. Chang and C. Zhang, Virol., 211:157-169 [1995]).
- the dl.Spl/CMV tat-B double mutant infects human lymphoid cell lines with delayed kinetics and exhibited reduced cytopathic effects .
- the first column lists the cell line used and the virus used to infect the cells.
- MT4/mock means that MT4 cells were tested without infection with HIV-1 virus (i.e., it was a control) .
- WT refers to wild-type virus.
- HIV-1 LTR/tat/nef Triple Mutants Prolonged asymptomatic survival of macaques infected with a nef-deleted
- SIV strain SIVmac239 suggested that the nef gene is a pathogenesis factor (H.W. Kestler et al . , Cell 65:651-662
- nef-A mutant an additional stop codon was inserted in the nef ORF upstream of the polypurine tract (PPT) in the nef-A mutant, to generate a more defective nef-minus mutant (nef-B mutant, see below) .
- the nucleotide sequence of pNL4-3 (HIV-1) from 9001 to 9031 (WT) was 5'- CTCAGGTACCTTTAAGACCAATGACTTACAA-3' (SEQ ID NO : 2 )
- the nef-B mutant sequence generated by site-specific mutagenesis was 5' -CTCAGGTACCTTTAAGACTCTAGATCTAGAA-3' (SEQ ID NO : 3 ) .
- Figure 13B provides a schematic showing a portion of the wild- type HIV-1 sequence, as well as the nef-B mutations (Figure 13B; wild- ype sequence provided in SEQ ID NOS : 5 and 6) .
- the nef-A mutations are also shown in this Figure 13B.
- the nef-A and nef-B mutations contain the same mutations in the sequence shown starting at base 8781 (i.e., SEQ ID NO : 5 corresponds the the nef-A sequence and nef-B sequence for this stretch of bases) .
- the nef-A sequence is the same as the wild-type sequence for the sequence shown starting at base 9001 (i.e., SEQ ID NO : 6 represents the sequences for both wild-type and nef-A) .
- the T cell-tropic env gene of the LTR/tat/nef mutant was also substituted with a macrophage-tropic env (HIVADA) .
- HIVADA macrophage-tropic env
- FIG 3. A schematic diagram of these HIV-1 mutants is shown in Figure 3.
- These infectious molecular clones are further modified and attenuated by mutating other accessory genes including vpr, vif and vpu , as well as the U3 transcriptional regulatory elements NF-AT, NRT-1, USF and TCF-la.
- a safe HIV-1 vector construct is developed from these attenuated HIV-1 LTR/tat/nef mutant constructs with a total deletion of U3 except for the att site.
- HIV-2 and SIV vectors will be constructed using two molecular clones, HIV-2ROD and SIVmac .
- Example IB Replication-Competent HIV-1 Vectors Carrying Heterologous Foreign Genes
- the nef gene has been shown to play an important role in viral pathogenesis (Z. Du et al . , Cell 82:665-674 [1995]; B.D. Jamieson et al . , J. Virol., 68:3478-3485 [1994]). Thus, it was considered to be safer to delete the nef allele from the lentiviral vector system to produce useful vectors. Since the nef gene of HIV is dispensable for viral replication in tissue culture, and since the nef ORF does not overlap with other genes, a foreign gene can be inserted into the nef ORF without inactivating the virus.
- Figure 4 shows a comparison of the replication efficiencies of recombinant HIV-1 constructs carrying heterologous foreign genes.
- TE671 cells were transfected with plasmid DNA; 48 hours later, culture supernatants were used for the in vi tro RT (reverse transcriptase) assay.
- Virus titer i.e., transduction efficiency
- the MAGI cells carry an integrated LTR-lacZ gene which can be transactivated by transduced HIV-1 Tat (J. Kimpton and M. Emerman, J Virol., 66:2232-2239.30 [1992] ) .
- the two scales in this Figure are numerically identical.
- reporter genes including human T cell receptor CD8 , T cell costimulator B7-2 (B70) , the bacterial hygromycin-B-phosphotransferase ( h y g ) , neomycin-phosphotransferase ( neo) , xanthine-guanine phosphoribosyltransferase ( gpt) , puromycin-resistant gene, and histidinol dehydrogenase (hisD) with or without an internal promoter (SV40) were inserted into the nef ORF at the new Hindlll site or a downstream Xhol site in the nef-A mutant.
- SV40 histidinol dehydrogenase
- heterologous HIV-1 vector constructs were assessed by transfecting human TE671 cells, and quantitatively measuring viral RT expression and transduction efficiencies on a human CD4 cell line. Transduction efficiency was determined by counting the blue nucleated cell foci after X-gal staining. Two independent transfections were done. Representative results are shown in Figure 4 (the standard deviation is not shown) . An insertion of up to 1.5 kb of nucleotide sequences, such as B70 and SV-his, seemed to have no effect on RT production. Furthermore, the infectivity of HIV-SVhis is as high as wild-type HIV-1.
- HIV-1 can sustain extensive changes in the enhancer and promoter region. Indeed, the replacement of the entire U3 , except for att, can be tolerated. Partial substitution of the intron region for the regulatory genes (tat and rev) in the env ORF with foreign sequences can affect the splicing efficiency of the singly-spliced messages, although the nearest splice acceptor site is almost 1 kb away (See e . g. , B.A. Amendt et al . , Mol. Cell. Biol., 14:3960-3970 [1994]).
- HIV-1 packaging and transducing vectors were constructed.
- Figure 7 is a structural diagram of seven different pHP vector constructs, including pHP-1 and pHP-VSVG.
- Attenuated HIV-1 constructs were modified to produce the "pHP-1" expression vector capable of synthesizing all viral structural proteins, but lacking the packaging signal function.
- This vector included a strong promoter (in preferred embodiments, it is preferably not a native HIV-1 LTR), the gag-pol gene, the RRE element, the tat, and the rev gene.
- the RRE-Rev interaction is of great importance to the efficient synthesis of the Gag-Pol protein. This dependency may be compensated for if the INS's are deleted and RRE is replaced by a surrogate regulatory element such as the CTE of the Mason-Pfizer monkey virus.
- pHP-1 which contained a modified 5' HIV-1 LTR, a novel major splice donor site derived from RSV, the entire gag-pol -env, vif , vpr, vpu, tat, and rev genes, a selectable gpt marker gene, and an SV40 polyadenylation signal as shown in Figure 5 was developed.
- the wild type HIV-1 genome contains genetic elements in the 5' to 3 ' order:
- pHP construct contains from 5' to 3 ' :
- pHP-1 lacks the native HIV-1 U3 TATA box, the primer binding site (PBS), polypurine tract (PPT), 3' LTR and most of the untranslated 5' leader sequences including the conventional retroviral packaging signal ( ⁇ ) and the major HIV-1 splice donor (SD) site.
- pHP-1 contains all HIV structural and accessory genes except for the nef gene and thus is capable of expressing the vast majority of the viral proteins, and also contains the bacterial gpt gene. pHP-1 provides a provirus capable of mimicking HIV-1 infection in terms of the viral proteins expressed yet this virus cannot be packaged into viral particles.
- pHP-1 and its derivatives provide excellent HIV packaging vectors.
- Examples of th pHP-derived packaging vectors include: pHP-dl.Vpr, pHP- Vpr/ala/leu. PHP-dl . env/Vpu I, and pHP-dl . env/Vpu II. pHP-1 was constructed as follows. First, the
- Tat -responsive enhancer promoter CMV-TATA-TAR fragment (approximately 400 bp) was isolated from dl . kB/Spl -CMV-TATA-TAR
- This linker was formed by annealing the following oligonucleotides :
- gag coding sequence for the pHP-1 construct was obtained by PCR from pNL4-3 (a full-length HIV-1 plasmid) using a 5' primer (5' -CGG GAT CCA CCA TGG GTG CGA GAG CGT C-3' [SEQ ID NO:ll])
- the PCR product was digested with SamHI-Sphl (-660 bp) and this fragment was ligated with BamHI-Sp l digested chimeric pSP-CMV-TAR-SD to obtain pSP-CMV-TAR-SD-dl . gag.
- a poly-A minus subclone of pHP-dl.pA was constructed by ligating the following three fragments: a 1112 bp Hpal -Sphl fragment isolated from pSP-CMV-TAR-SD-dl . gag
- pNLgpt was generated by cloning the E. coli xanthine-guanine phosphoribosyltransferase (gpt) gene from pMSG
- pHP-1 was made by the following ligation: No I -Xhol (9059 bp) of pHP-dl.pA containing dl .CMV-TATA-TAR-SD-gag-pol-env-gpt, a 422 bp poly-A site from Xhol-Pstl digested pREP9 (Invitrogen) , and Notl -Pstl digested pBS-KS(-).
- pHP-1 (12,479 kb) is provided in SEQ ID NO : 13 ; this sequence begins at the promoter of the half- BbrPI site from pNL4-3 (an HIV clone available from the AIDS Research and Reference Reagent Program; the sequence of this recombinant clone is shown in Genbank Accession No. M19921) . Additional mutations of pHP-1 to generate pHP-ldl2 and pHPl- dl.28 are described above ( See also, Figure 5) .
- pHP-VSVG was derived from pHP-1, with the HIV-1 env gene being replaced by the VSV-G gene, and with wild- type vpr and tat, or the vpr and tat genes mutated by site-specific mutagenesis.
- pHP-CMV was derived from pHP-1 with the promoter being replaced by the cytomegalovirus immediate early promoter (CMV-IE) and the tat, rev, env, vpr and pu deleted.
- CMV-IE cytomegalovirus immediate early promoter
- PHP-CMV ⁇ TAR/SD was derived from pHP-CMV, with the TAR and RSV SD deleted. In other words, this construct lacks any major SD site.
- pHP-CMV-EFl ⁇ -intron was derived from pHP-CMV ⁇ TAR/SD, with an insertion of the EFl ⁇ - intron between the promoter and the Gag AUG.
- pHP-EF was derived from pHP-CMV ⁇ TAR/SD by replacing the CMV-IE promoter-enhancer and the synthetic SD site with the human elongation factor l ⁇ (EFlce )'s promoter and enhancer- containing intron (the latter being of course proceeded by a splice donor site) .
- the intron-containing EFla has been shown to be a stronger promoter than the CMV-IE promoter.
- the TAR sequence was also deleted. It also contains a poliovirus- derived internal ribosomal entry site (IRES) and the vpr gene. The expression of Vpr may increase the vector transduction efficiency in non-dividing cultures.
- Both packaging constructs i.e., pHP-1 and pHP-VSVG
- a recombinant CMV/HIV-TAR as promoter and a synthetic major splice donor site.
- No sequence homology was observed with the HIV-1 genome between TAR (in the 5' end of the RNA) and the gag AUG in these two constructs .
- a BamHI site was generated near the gag AUG for the purpose of inserting recombinant HIV-2 and SIV gag-pol sequences in subsequent experiments.
- pHP-VSVG construct with vpr and tat mutations lacks vpr and tat genes, and the VSV-G gene is substituted for the env gene exactly at the env AUG by PCR mutagenesis. These two constructs were the first two packaging plasmids tested. The construction of these pHP-1 derivatives is described in greater detail below. The three pHP-CMV derivatives were tested, and found to be inefficient in synthesizing HIV proteins, indicating that the pHP-1, pHP-VSVG and pHP-1 dl derivatives are the preferred embodiments of the efficient HIV vector system of the present invention. pHP-VSVG.
- This clone was made to delete the HIV-1 env gene and replace it with the VSV-G gene, as well as delete the HIV-1 vpr and tat genes. It was constructed by combining the following four pieces of DNA fragments: 1) the recombinant LTR (dl.kB/Spl-CMV-TATA-HIV-TAR) gag-pol from Notl to EcoRI fragment of pHP-1; 2) a fragment from HIV-1 with deletion in the C-terminal of Vpr and the ⁇ -terminal of Tat by PCR using the following two primers:
- pHP-CMV This clone was derived from pHP-1, with the 5' recombinant LTR replaced by a CMV-IE enhancer-promoter and the entire env, tat, vpu, rev, vpr, nef deleted, but with the vif gene remaining intact.
- This clone was constructed by ligation of the following 3 pieces of D ⁇ A: 1) the vector pcD ⁇ A3. lZeo (+) from Invitrogen cut with Nhel -Xhol; 2) the TAR/ SO- gag-pol from pHP-1 digested with Xbal -EcoRI; and 3) the RRE element from pBS-RRE digested with EcoRI-XhoI.
- pBS-RRE was constructed by ligating Bglll (nt. 7611) to Hindlll (nt . 8131) of p ⁇ L4-3 of HIV-1 with Bglll-Hindlll digested pBS-EF.
- pHP-CMV- ⁇ TAR/SD This clone is the same as pHP-CMV except that the 5' TAR and splice donor site are deleted. This construction was made by ligating the following two fragments: 1) a 702 bp fragment of MiuI-BamHI digested pcDNA3. lZeo (+) containing the CMV enhancer; and 2) the vector containing MluI -BamRI digested pHP-CMV which has deleted TAR and contains the RSV splice donor site. pHP-CMV-EFl ⁇ -intron.
- This clone is similar to pHP-CMV- ⁇ .TAR/SD but with an intron from human EF-la gene inserted between the CMV promoter and the gag AUG. It was made by ligating the following three DNA fragments: 1) pHP-1 BamHI -EcoRI fragment containing gag-pol and vif; 2) the MluI-EcoRI of pcDNAZeonlacZ-RRE containing the vector backbone of pcDNA3.lZeo(+) , HIV-1 RRE and part of the CMV promoter; and 3) the rest of the CMV enhancer promoter was obtained from BamHI-MluI digested pcDNAZeoHGHP2EF, a pcDNAZeo3.1 (+) vector containing EFl ⁇ intron and the human growth hormone gene.
- pHP-1 dl2 and pHP-1 dl28 To further mutate pHP-1 for safety reasons (as discussed below) , portions of the env gene were deleted by Bal31 excision. To generate HIV-1 env deletions, pHP-1 was digested at the unique restriction enzyme site Nhel in the env gene, and treated with Bal31 exonuclease for 1, 2 or 5 minutes. The digested product was self-ligated after T4 D ⁇ A polymerase treatment.
- the self-ligated plasmid D ⁇ A was then transformed into competent E. coli DH5 ⁇ and from a pool of more than 48 deletion mutants, two clones (pHP-ldl.2 and pHP-ldl.28) , were selected, sequenced and used in this study.
- pHP-ldl.2 and pHP-ldl.28 have 2 and 28 nucleotide deletions in the env gene respectively ( See, Figure 5) .
- the vpr gene was mutated by site-specific mutagenesis so it retains the nuclear localization function but loses its cell cycle arrest function. Specifically, a mutation was made at amino acid 30, changing from ala to leu, as described in Mahalingam S, Ayyavoo V, Patel M, Kieber-Emmons T, Weiner DB. Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J Virol 1997; 71:6339-47.
- the env/vpu was also mutated by site-specific mutagenesis to delete the env initiation AUG codon and part of the vpu reading frame . These mutations were first made individually and then combined.
- the mutant I has a long deletion from nt 6195 at the vpu amino acid codon 45 to nt . 6330 at the 38th amino acid codon of env gene and a stop codon TAA inserted in the sequence .
- the primer used was a forward primer: -GTTAATTGATAGACTAGTCTAATATGGGGTACCTG- .
- the mutant II has a small deletion from nt . 6216 at vpu a. a. codon 52 to nt . 6237 at vpu a. a. codon 59 and env a. a. codon 6 which also has a stop codon mutation TAA at the vpu a. al . codon 50. Note that although these mutations are Gl mutations, they are made into the G2 vector pHP-dl.28 backbone.
- pHP-dl.vpr is a frame-shift mutant which was made by EcoRI digestion at nt . 5745, near vpr amino acid codon #62, and resulted in a 4 bp insertion which caused a frameshift.
- pHP-vpr/ala/leu this is a point mutation which has changed alanine to leucine at a. a. #30.
- This mutation deleted the cell cycle arrest function but not the nuclear localization function of vpr as reported before [Zhang, 1997 #3492; Mahalingam, 1997 #3791] .
- the primer used for mutagenesis is: -CCTAGGAAAATGTCTAACTAGTTCACTCTTAAGTTCCTC- . Note that although these mutations are Gl mutations, they were made with the G2 vector pHP-dl.28 backbone.
- Example ID Construction of HIV-1 Transducing Vectors (TV) Two families of transducing vectors were constructed. In the pTV ⁇ vectors, the major packaging signal was modified relative to the source HIV-1 signal. In the pTV ⁇ vectors, the source major packaging signal was used.
- Figure 8 provides a diagram of six HIV-1 transducing vectors, in which the vector backbone is derived from pNL4-3 and different LTRs.
- the IRES element shown in this Figure was derived from poliovirus, which could allow bicistronic gene expression.
- HIV-1 transducing vectors TV
- an artificial HIV-li/ sequence using four synthetic oligonucleotides was synthesized, which comprised sequences between the PBS and the gag AUG (.referred to as “i'lOO” or “PAKIOO”) and sequences extending into the gag ORF (referred to as " ⁇ 140” or “PAK140”).
- dl.kB-CMV/HIV-TAR (“dl.kB-CMV/HIV-TAR”) , the PBS and 5' leader sequences, an SV40 -driven neo resistance gene, and the 3' PPT.
- the titer of the wild-type HIV-1 was determined by a sensitive immunohistochemical staining method using anti -Gag p24 mAb as described by Chang and Zhang (L.-J. Chang and C. Zhang, Virol., 211:157-169
- site-specific mutagenesis was performed to change 1-2 nucleotides in the splice donor site, and the Gag AUG in pTV ⁇ s using primers :
- the major SD site, the gag AUG and the env sequences must be restored, because they are deleted from the modified pHP and pTV constructs.
- the wild-type genome was gradually deleted (pTV ⁇ ) .
- the two replication-competent HIV-1 vectors “HIV-1-SVneo” and “HIV-1-SVhyg” (See, Figure 4) were used as a starting point. These two constructs are nef-minus, and exhibited up to 50-70% of the wild-type HIV-1 replication efficiency.
- a deletion was made starting from the middle of the gag ORF to the middle of the env ORF. This did not delete the RRE element.
- pTV ⁇ SVneo was created by digesting pNL-SVneo with Nhel (with a site located in the middle of the env gene) , and Spel (with a site in the middle of the gag gene) , and then self-ligating it to delete the gag-pol -env, and vif , vpu, vpr, tat , and rev genes.
- p ⁇ LSV neo was created by inserting SVneo (an SV40 promoter-driven neomycin-resistant gene) at a Hindlll site generated by site-specific mutagenesis, inbetween the nef
- pTV ⁇ CMVnlacZ was made by digesting pTV ⁇ SVneo with Xhol and Kpnl , which deleted SVneo and part of the nef sequences near the 5' end of the PPT of HIV-1. The product was then ligated with a Sail - Kpnl fragment containing CMV-nlacZ sequence from pcDNAzeo-nlacZ. pcDNAzeo-nlacZ was generated by inserting nlacZ of pSP72nlacZ into pcDNA3. lzeo (+) .
- the nuclear lacZ gene was generated by fusing a nuclear localization signal of SV40 large T antigen to the N-terminus of the bacterial lacZ gene that was obtained from the pBlueBacHisA vector (Invitrogen) using PCR mutagenesis.
- CMV-GFP green fluorescent protein
- CMV-nlacZ pTV ⁇ CMV-nlacZ
- CMV-nlacZ-hyg pTV ⁇ CMV-nlacZ-hyg
- PTV ⁇ EFGFP was generated by replacing the CMVnlacZ gene of pTV ⁇ CMVnlacZ withthe human elongation factor l ⁇ promoter of pHEF kindly provided by D Denny plus a GFP reporter gene. pTV ⁇ EFGFP with nlacZ gene.
- the VSV-G envelope expression plasmid pHEF-VSVG was constructed by inserting a PCR amplified VSV-G fragment containing a favored translational initiation codon into the EcoRI site of pHEF (kindly provided by D. Denny) . The mutagenesis site and flanking sequences were confirmed by DNA sequencing.
- estern Blot analysis Western analyses of HIV-1 proteins in HeLa cells transfected with various vector constructs were tested.
- cell lysates were prepared and analyzed by Western blotting and compared with a wild-type HIV-1 construct (pNL4-3), in order to determine the level of viral proteins synthesized by pHP-1 and pHP-VSVG (with and without Tat), in comparison with wild-type HIV-1.
- the Western blots were performed using serum obtained from an HIV- infected individual, and methods known in the art (See e . g. , Ausubel et al . (eds.) Short Protocols in Molecular Biology, 2d ed.
- RT and vector titer analyses of HP/TV vs. wt HIV-1 To examine the efficiency of gene expression, we first compared viral reverse transcriptase (RT) production. HeLa cells were transfected with either wt HIVNL4-3 , pHP-1, pHP-ldl.2, or pHP- ldl.28 and the culture supernatant was harvested for RT assay 48 h later. Figure 6 shows that the levels of RT synthesis by the pHP-1 derived constructs were comparable to those observed for wt HIV-1; however, the vpr/tat -deleted pHP-VSVG produced only minute amounts of RT even when co-transfected with tat and vpr.
- RT viral reverse transcriptase
- Transfected HeLa cell lysates were also analyzed to see if the supernatant RT activity correlated with intraceliuiar Gag synthesis. The results showed that the amount of Gag protein produced by pHP-1 was similar to that produced by wt HIV-1 infected cells or transfected cells.
- Gag-Pol function indicates that tat and rev are functional.
- the artificially engineered splice donor (SD) site in the pHP-1 construct which is unrelated to HIV sequences, works like the wild-type SD site (i.e., allowing partition of spliced and unspliced mRNAs into the cytoplasm) .
- the packaging vector pHP-dl.28 expressed RT at 50-90% of the wild type level, indicating that the mutations in pHP-dl. did not substantially affect the synthesis and function of Gag-Pol.
- PHEF-VSVG human elongation factor 1 alpha promoter driven VSV-G envelope and pHEF-A-env: EFla promoter driven amphotropic MLV envelope, were also constructed in our laboratory and shown to express high levels of envelope proteins; better than the CMV-IE promoter driven construct.
- overexpression of Gag-Pol may not increase the vector titer because earlier studies have shown that overexpression of Gag-Pol induces protease activation and prevents virus assembly and budding (V. Karacostas et al . , Virol., 193:661-671 [1993]; J. Park and C. D. Morrow, J. Virol., 65:5111-5117 [1991]).
- the present examples describe vectors that produce measurable amounts of Gag-Pol ( e . g. , pHP-1, pHP-ldel, and pHP-VSVG) , as well as vectors that do not express detectable amounts of Gag-Pol
- pHP-CMV pHP-CMV and its derivatives
- Virus pellets (“P") and cell lysates (“L”) were prepared from Tat + (tat WT) and Tat " (tat- B and tat-C) virus-infected cells, and the protein contents were separated by a 10% SDS protein gel, and detected in Western analysis using AIDS patient's serum. The signals were amplified using the Amersham ECL chemiluminescence kit.
- the Western analysis showed that the two tat-minus mutant HIV constructs (tat-B and tat-C) derived from chronically infected cell lines can replicate at high efficiency and synthesize viral proteins at wild type efficiency.
- WT or tat-minus HIV-1 chronically infected cultures were metabolically labeled with 3 H-leucine overnight, immunoprecipitated with pooled HIV patient sera, and analyzed by SDA-PAGE (10%) .
- the relative ratio of Gag p55:p24 is shown at the bottom. Processing of the envelope gpl60 to gpl20 was not significantly different between different samples.
- the 3 H- labeled protein bands were quantified using a phosphoimager (BAS1000) .
- HIV polyprotein processing requires Tat function.
- Tat In the absence of Tat, there is a high GaG p55/p24 ratio compared with wild type.
- HeLa cells were transfected with plasmid DNA encoding HIV-1 Gag, Rev, Tat, HTLV Tax/Rex, SIV Tat, or HIV Tat exon 1.
- the results clearly demonstrate that Tat enhances p55 to p24 Gag processing.
- the effect of Tat on Gag processing is TAR- independent as GagTAR- construct which has TAR deletion is also sensitive to this Tat effect. This function of Tat resides in the exon 1 which can be partially restored by SIVTat and HTLV Tax/Rex.
- TE671 cells were transfected with plasmids as described above. Cell lysates were harvested 24 hours after DNA removal and analyzed by SDS-PAGE and Western blotting as described using anti-p24 MAb. The result indicated that Gag processing is enhanced by the presence of Tat .
- pHP-VSVG pHP-VSVG was linearized and transfected into human TE671 cells by electroporation, together with a selective marker. After selection, individual cell clones were tested for Gag-Pol expression by direct extracellular RT assay in the presence or absence of a transfected tat plasmid. The expression of VSV-G protein was detected by immunohistochemical staining. Briefly, the pHP-VSVG linearized by digestion with Notl , and transfected into the TE671 cells along with pSV2-neo (i.e., with G-418 as the selectable marker) .
- Transfection was accomplished by electroporation, using methods known in the art. Transfected cells were grown in 1 mg/ml of G418 culture in DMEM containing 10% FBS. The induced gag-pol Gag-Pol expression was then determined by direct extracellular RT assays with and without transfected tat plasmid. HIV-1 Gag and RT expression were detected by p24 antigen ELISA or RT (See, co-pending U.S. Patent Appln. Ser. ⁇ os . 08/791,994 and 08/838,702; See also, L.J. Chang and C. Zhang, Virol., 211:157-169 [1995]; and L . J . Chang etal., J Virol . , 67:743-752 [1993] ) .
- Gag-Pol in this inducible cell line still requires Tat function.
- vpr and tat genes can also be expressed by an inducible promoter.
- the vpr gene is included because of its function in promoting transduction of nondividing cells.
- Vpr is a virion-associated protein, and the vpr gene is therefore assigned to the packaging vector so that equivalents of Vpr, like those of Gag, Pol and Env, are produced only in the packaging cell line.
- tetOP-tat-P2-vpr-SVpA- inverted tk-zeo-pA
- tetOP-tat-P2-vpr-P2-tetR-VP16-SVpA- inverted tk-zeo-pA
- tetOP-tat-P2-vpr-P2-tetR-VP16-SVpA- inverted tk-zeo-pA
- IRES HIV-1 Vpr
- IRES P2 HIV-1 Vpr
- tetR-VP16 HIV-1 Vpr
- tetR-VP16 HIV-1 Vpr
- tetR-VP16 HIV-1 Vpr
- tetR-VP16 HIV-1 Vpr
- tetR-VP16 HIV-1 Vpr
- tetR-VP16 HIV-1 Vpr
- tetR-VP16 tetR-VP16
- pREP9 vector pREP9 with EBNA1 gene sequence deleted.
- the two tTA plasmids were obtainable from Display Systems Biotechnology, Inc. (now distributed by Clontech) . This clone is auto- inducible by the removal of tetracycline or doxycycline (2-10 microgram/ml) from the culture media (a Tet-OFF system) ( See, M. Gossen and H. Bujard, Proc. Natl. Acad. Sci
- TE671 cells were transfected with 5 ⁇ g of pcDNA3-nlacZ or pTV ⁇ CMVnlacZ, as described above. Following transfection and growth, cells were fixed and stained for 3-galactosidase actvity, as described below.
- the beta-galactosidase activity was detected by the following protocol as published by Kimpton and Emerman (J. Kimpton and M. Emerman, J Virol., 66:2232-2239 [1992]).
- the cells were then washed three times with PBS, and incubated with 500 ⁇ l ddH 2 0 containing 4 mM potassium ferrocyanide (100 ⁇ l of 0.4 M for final 10 ml) , 4 mM potassium ferricyanide (100 ⁇ l of 0.4 M) , 2 mM MgCl 2 (20 ⁇ l of 1 M) , 0.4 mg/ml X-Gal (200 ⁇ l of 20mg/ml) at 37°C for 50 minutes to several hours.
- the blue-staining (i.e., 3-galactosidase positive) cells were counted under an inverted microscope.
- the resulting vector titer was determined by titration on TE671 cells using transfected culture supernatants.
- the reporter genes, neo, hyg, and lacZ, were assayed by selecting resistant cell colonies using either G418, hygromycin B, or by colorimetric staining for -galactosidase activity, respectively.
- the transfected TE671 cells were co-cultured with MT4 cells, one of the most susceptible cell lines to HIV-1 NL4 . 3 infection. After co- cultivation, MT4 cells were monitored for syncytium formation and culture supernatant was subjected to HIV-1 reverse transcriptase (RT) assay.
- RT HIV-1 reverse transcriptase
- Table 2A HP/TV vector transduction and RCV assays. pHP pTV Additional Genes Reporter* RCV* pHP-1 pTV ⁇ SVneo VSV-G + + pTV ⁇ SVhyg VSV-G + + pTV ⁇ CMV-nlacZ VSV-G + + pHP-VSVG pTV ⁇ CMV-nlacz VSV-G, tat +
- neo r or hyg r of pTV ⁇ _ were assayed by infecting HeLa cells with 50 ul and 100 ul of culture supernatant followed with G418 or hygromycin B selection 24 hr after transduction, or for nlacZ reporter gene, by infecting HeLa cells with 20, 50 and 100 ul of culture supernatant and stained with X-gal 48 hr later as described in the Materials and Methods.
- pHP-l/pTV co-transfection produced >10 3 /ml vector and pHP-VSVG/pTV produced ⁇ 10 2 /ml vector; however, the titer was not precisely determined due to the generation of RCV.
- *Replication- competent virus (RCV) assayed by co-culturing with MT4 cells for up to 30 days; RCV was detected by monitoring cytopathic effects on MT4 cells, RT and MAGI assays.
- Example 6A Production Efficiency of VSV-G and pHEF- VSVG Pseudotyped TV ⁇ and TV ⁇ Vectors Ex. 6A.1
- VSV-G pseudotyped vectors were produced and the target cells were CD4 -minus human cell lines.
- pHP-VSVG which is both a packaging and a pseudotyping vector
- pCEP4tat tat plasmid
- Culture supernatant was harvested 48 hours later.
- Tat was included to transactivate both pHP-VSVG and pTV ⁇ .
- the production of virus was confirmed by RT assay, and expression of HIV-1 p24 and VSV-G was confirmed by immunohistochemical staining. Virus produced from the transfected cells were harvested without further concentration, and used to infect TE671 cells.
- pHEF-VSVG a different (pure) pseudotyping vector, pHEF-VSVG.
- This is just a eukaryotic expression vector, with the VSVG gene under human EFl-alpha promoter control. It is not derived from a lentivirus.
- TE671 or HeLa cells were co-transfected with a packaging vector (pHP-1, pHP-ldl.2 or wt pNL4-3), a transducing vector pTV ⁇ CMVnlacZ, and the envelope construct pHEF-VSVG.
- the results are summarized in Table 3.
- the pHP packaging vectors produced RT at 30-40% of the wt level in TE671 cells but produced near wt level in HeLa cells.
- the vector titer assay indicated that HP/TV co-transfections using either pHP-1 or pHP-ldl.2 produced 2-3 times more transducing vector than the wt HIVNL4-3/TV co-transfection.
- Table 3 also showns results for experiments with transducing vector pTWCMV-nlacZ-hyg-dl . Smal . They showed very low titers. Table 3. Production of High-Titer HIV-1 Derived Vectors
- Table 3A Efficacy and RCV assays of HP/TV vs. wt HIV-1 /TV vectors.
- RT cpm/ ⁇ l Titer (ti ⁇ /ml) a RCV construct vector gene pNL4-3 pTV ⁇ CMV- 19,124 1.1 x 10 J + nlacZ pHP-1 pTV ⁇ CMV- tat 6,434 7.2 x 10 5 + nlacZ pHP-ldl.2 pTV ⁇ CMV- tat 5,130 3.0 x 10 3 - nlacZ pHP-ldl.28 pTV ⁇ CMV- tat 4,744 5.0 x 10 5 - nlacZ
- TE671 cells were co-transfected with plasmid DNA as indicated and vector titer and RCV were assayed as described in Materials and Methods.
- RCV was assay by MT4 co-cultivation for 60 days and the survival MT4 cells were stained for HIV- 1 antigens by immunohistochemica! staining using an
- pTV construct (8 microgram per well in a 6-well plate), pTV construct (8 microgram per well) , pHEFVSV-G (5 microgram per well as envelope pseudotype) with a tat expression plasmid pCEP-tat (0.5-1 microgram) and a rev expression plasmid pCMV-rev
- the tat and rev expression plasmids were included because we have shown that they could enhance the vector titers for most of the pHP constructs and they were necessary for pHP-dl.Ndel which has a tat and rev deletion and for pHP-VSVG which has a tat deletion.
- pHP-dl.28 (a Gl construct) expressed RT at 50-90% of the wild type level indicating that the mutations in pHP-dl. did not affect the synthesis and function of Gag-Pol.
- the preliminary relative titers of different pHP mutants are shown below: (all included a co-transfected pTV reporter transgene)
- pHP-dl.28 (env, nef deletion, relative titer: 1.00); pNL4-3 (wild type HIV-1 control which in fact produce less vector than pHP-dl.28, relative titer: 0.40); pHP-VSVG (vpr, tat, env and nef deletion, relative titer : 0.014); pHP-dl .
- env/vpu I vpu, env, nef deletion, relative titer: 0.43
- pHP-dl pHP-dl .
- env/vpu II (vpu, env, nef deletion, relative titer: 0.38); pHP-dl.vpr (vpr, env, nef deletion, relative titer: 0.85); pHP-vpr/ala/leu (vpr funtional mutation, env, nef deletion, relative titer: 0.85); pHP-vpr/env/vpu I (vpr functional mutation and vpu, env, nef deletion, relative titer: 0.24); pHP-vpr/env/vpu II (vpr functional mutation and vpu, env, nef deletion, relative titer: 0.50) pHP-dl.Ndel (vif, vpr, tat, rev, vpu, env, and nef deletion, relative titer: 0.006).
- a G2 pHP construct should contain only gag-pol open reading frames and the RRE regulatory sequences such as the pHP-CMV, pHP-CMVdel . TAR/SD, pHP-CMVEFla- intron, or pHP-EF constructs (although the vif gene is still present in all of them) . However, these constructs exhibited reduced levels of gag-pol activity as shown by the following summary table:
- TE671 cells were transfected with 5 microgram of each test HP plasmid and 0.5 microgram of pCEPtat (except for one construct, pHP-CMVEFla-intron, we tested both with and without Tat) and 1 microgram of pCMVrev.
- the culture supernatant was harvested and p24 level was determined by ELISA as described before.
- pHP-1 (1.00) pHP-dl.28 (1.00) pHP-VSVG (0.008) pHP-dl.vpr (0.34) pHP-dl .env/vpu I (0.43) pHP-dl. env/vpu II (1.41) pHP-dl.Ndel (0.007) pHP-CMV (0.05) pHP-CMVdel. TAR/SD (0.03) pHP-CMVEFla-intron (0.21, with Tat)) pHP-CMVEFla- intron (0.04, without Tat) pHP-EF (0.27)
- the env gene was deleted in pHP and two deletion constructs were generated, pHP-ldl.2 and pHP-ldl.28.
- the transfected human rhabdomyosarcoma TE671 cells (ATCC CRL 8805) were co- cultured with the human lymphoma cell line MT4.
- MT4 cells are an HTLV-1 transformed human CD4+ lymphoma ceil line, that are very sensitive to HIV-1 infection. ⁇ These cells are available from the National Institutes of Health AIDS Reagents and Reference Program. Uninfected MT4 cells were added into the co-culture every week during these experiments. The wt HIV-1 plasmid pNL4-3 was included for comparison.
- pHPl packaging construct but not the env-deleted constructs pHP-ldl.2 (2 nt deletion) and pHP-ldl.28 28 nt deletion), produced replication-competent HIV-1 (RCV) after co-transfection with pTV plasmid.
- Infectious virus was detected from pHP+pTV ⁇ CMVnlacZ MT4 co-culture in 8 days.
- no infectious virus was detected from pHP . dl .2 or pHP.dl.28+pTV ⁇ CMVnlacZ MT4 co-culture in 60 days (See, Table 4 , below) .
- TE671 cells were co-transfected with pHP+pTV+pHEF-VSV- G as shown in the Table below (Table 4) , and the culture supernatants were harvested 48 hours after DNA removal for RT assay and vector titer was determined as described before .
- MT4 lymphoblastoid cells were added to the HP/TV transfected cells 48 h after transfection. Fresh medium was added to the co-cultures every 3 days at which time two thirds of the culture media was discarded. Fresh MT4 cells were added to the cultures once a week. The transfected cells were co-cultured with the human MT-4 lymphoblastoid cell line, which is very sensitive to HIV-1 infection, for up to 2 months. The culture supernatants were harvested at different time points after co-culture.
- the supernatant from the co-culture was assayed for HIV-1 RT activity and for infectious RCV by passage onto CD4+HeLa cells or uninfected MT4 culture. Infection of CD4+HeLa cells was examined by anti-p24 immunohistochemical staining using pooled AIDS patients' sera. At different time points, MT4 cells in the co-culture were collected and immunostained using a monoclonal anti-p24 antibody for the detection of HIV-1 Gag antigen as described previously, Chang, Virol., 211:157-169 (1995). The assay also looked for infection of MT4 cells by cytopathic effects of RCV. Syncytium formation was observed under an inverted microscope.
- a very sensitive assay which would detect cell-cell transmission of poor replicative virus was also used. After four months of co-culture, the MT4 cells were removed and added to fresh MT4 cells and further cultured for 4 days. The co-cultured MT4 cells were fixed and immunostained with HIV patients' sera.
- Figure 12 illustrates the possible cross-over between pHP-dl.28 and pTV-dl . CMVnlacZ, to generate RCV during cotransfection.
- Example 8A In Vi tro Transduction of Mi tomycin- C- Trea ted Human Cells
- TE671 or HeLa cells were treated with mitomycin C to arrest the cell division cycle and were then transduced with pTV ⁇ CMV-nlacZ viral vector.
- two cell cultures were transduced with HP-TV and observed for its transduction efficiency.
- TE671 or HeLa cells were treated with the DNA synthesis inhibitor, mitomycin C, at 10 ⁇ g/ml for 4 hours, trypsinized and plated into a 6 -well culture plate. Cell cycle arrest was monitored by propidium iodide staining and FACS analysis.
- the cells were transduced with HP-TV HIV vector carrying a nlacZ marker gene in the presence of 4-8 ⁇ g/ml polybrene in a total volume of 0.5 ml for 2-3 hours and fed with growth media (DMEM containing 10% FBS) . After 48 hours, the expression of the transduced lacZ gene was detected by X-gal staining as described above. The results indicated that the HP-TV vector was capable of efficiently transducing mitomycin-C-treated, non-dividing human cells . This was further confirmed in numerous assays; we rountinely use mitomycin C-treated cells and normal dividing cells for vector stock titration.
- HP/TV lentiviral vectors transduce cells with different efficiencies depending on the cell cycle stage at the time of transduction.
- TE671 was treated with 5 microgram/ml of mitomycin C in DMEM growth media for 2.5 hr and the treated cells were transduced with the pTVdeltaEFnlacZ vector and 48 h later, the transduction efficiency was determined by x-gal staini assay.
- the transduction efficiency became reduced. This result suggests that although HP/TV lentiiral vector transduces post-mitotic cells, the efficiency of gene transduction is still dependent on the cell cycle stage.
- EXAMPLE 8B In Vi tro Transduction of Primary Neuronal Cells
- TE671 or HeLa cells were treated with mitomycin C to block cell division and then transduced with pTV ⁇ CMV-nlacZ viral vector.
- rat neuronal cells were isolated from the brains of Fisher rats according to the method of Ure et al . (Ure et al . , Develop. Biol., 154:388-395 [1992]).
- the cells were grown in culture medium containing L15C0 2 (GIBCO, Grand Island, New York), containing 200 ng/ml 2.5 S nerve growth factor (NGF) , 2.55 rat serum, 1 mg/ml ascorbic acid, and 10 ⁇ M cytosine arabinose (Sigma) , to inhibit divisions of non-neuronal cells.
- L15C0 2 Gib Island, New York
- NGF nerve growth factor
- rat serum 1 mg/ml ascorbic acid
- 10 cytosine arabinose Sigma
- human neurons and astrocytes were obtained from differentiated embryonal neural stem cells provided by Neurospheres, Ltd (Calgary, Alberta, Canada) . These cells were infected with the HP-TV vectors carrying the nlacZ reporter gene as described above. Briefly, cells were incubated in culture media containing the HP-TV vector. After two hours of incubation, conditioned media (i.e., supernatant medium harvested from cultured neuronal cells after 24 hours of culture) were added, and the culture continued to incubate for five days. The cells were then fixed with formaldehyde and glutaraldehyde, and incubated with X-gal substrate as described in the -galactosidase assay described above.
- conditioned media i.e., supernatant medium harvested from cultured neuronal cells after 24 hours of culture
- pTV ⁇ CMVnlacZ vector 2-5 x 10 5 tu/ml, was prepared from transfected TE671 and used for intramuscular injection. Each injection site received 200 ⁇ l of unconcentrated vector or 20 ⁇ l of 30-50x concentrated vector .
- HP-TV vector efficiently transduces primary neuronal cells obtained from rat brains, and human neuronal stem cells (neurons and astrocytes) ; both were post -mitotic, terminally differentiated cells.
- EXAMPLE 8C In Vivo Transduction of Muscle Cells
- the HP-TV HIV vector was used to transduce muscle cell in vivo .
- the hind-legs of mice CB-17 SCID/beige mice (Taconic) were intramuscularly injected with 50-100 ⁇ l of vectors carrying the nlacZ reporter gene as unconcentrated (10 5 /ml) or microcentrifuge concentrated
- Example 8D Miscellaneous In vi tro Transduction 1.
- Human TE671 cells were transduced with lentiviral vector pTV ⁇ EFGFP three times (3-5 MOI) and incubated for 4- 5 days.
- the GFP gene expression was detected directly under a Zeiss Axiovert 25 inverted fluorescent microscope with xlO Fluar objective lens. In contrast, injections with retroviral (MLV) vectors did not produce reproducible positive results.
- K562 a human chronic myelogenous leukemic cell line, can be efficiently transduced (>95%) as measured by flow cytometry analysis of GFP expression following tranduction with VSV-G pseudotyped HP/TV vectors, and transgene expression remained stable for over three months.
- KG1 an acute myelogenous leukemic cell line
- PCR analysis of integrated proviral DNA suggests that the reduced level of transduction in KG1 correlates with the low level of proviral DNA, which can be caused either by steps limiting viral entry, proviral synthesis and/or integration in KG2 cells .
- HIV Vectors are More Efficent than MLV Vectors
- the efficacy of gene therapy vectors is often judged by their transduction efficiency as wel as long-term stability.
- HIV vectors were compared with the standard MLV vectors commonly in use. The results obtained in these experiments indicated that HIV vector is more efficient than the MLV vector.
- the MLV-derived vectors were generated by transfecting the packaging cell line PA317 with pMFGnlacZ which contains the same recombinant nlacZ gene as the one cloned into pTV ⁇ CMVnlacZ.
- the HP/TV vectors were generated by co- transfecting HeLa or TE671 cells with the pHP, pTV, and pHEF-VSVG plasmids.
- a modified calcium phosphate DNA transfection protocol was performed as previously described 10 .
- the transfection efficiency normally ranging from 50- 90%, was determined by X-gal staining or by a radioimmunoassay for human growth hormone when the XGH5 plasmid was included in the transfection procedure (Nichols Institute Diagnostics) .
- the transfected cells normally produce retro- or lenti-viral vectors with titers ranging from 10--10 5 transducing units (tu) per mi.
- the VSV-G pseudotyped vectors were routinely concentrated 30-50 times by centrifugation in a table-top microfuge (21,000 g) for 2.5-3 h at room temperature.
- Virus supernatants were harvested 24, 48, and 72 hr following addition of DNA by low speed centrifugation (1200g for 5 min) or by filtration using a 0.45 ⁇ m low-protein binding filter to remove cell debris from transfected culture. No reduction in titer was observed between 24-48 h, but a one-log reduction in titer was frequently observed in harvests carried out at 72 h. The supernatants were aliquoted and stored at -80°C until use. Retroviral vector was titered on HeLa or TE671 cells and lentiviral vector was titered on mitomycin C-treated TE671 cells (5 ⁇ g/ml for 2.5 h) .
- cells were infected with diluted virus stocks at low multiplicity of infection (MOI) in a small volume of growth medium containing polybrene (8 ⁇ g/ml) . Cultures were grown for 3-4 hr, supplemented with additional growth medium, and then incubated for a final period of 48 hr before staining. A minimum of two different dilutions were examined for each titration sample. To detect the transduced cells, the cultures were washed twice with phosphate buffered saline (PBS) , and fixed at room temperature with 1% formaldehyde and 0.2% glutaraldehyde (Sigma) in PBS for 5 min.
- PBS phosphate buffered saline
- the cells were incubated at 37°C in PBS or distilled water containing 4 mM K- ferrocyanide, 4 mM K-ferricyanide , 2 mM MgCl 2 and 0.4 mg/ml X-Gal overnight.
- the transduced cells stained with X-Gal were examined with an inverted microscope the next day.
- the high background beta-galactosidase activity in HepG2 cells, primary cultures and tissues can be eliminated by increasing the pH of the incubation buffer.
- the efficacy of gene therapy vectors is often judged by their transduction efficiency as well as long-term stability.
- lentiviral and retroviral vectors To directly compare lentiviral and retroviral vectors, a combined short-term/long-term in vi tro study was carried out. Three different human cell types, TE671, 293T and HepG2 cells, were transduced with either retroviral vector MFGnlacZ, that was produced from PA317 packaging cells and contained a MLV LTR-driven nlacZ gene, or lentiviral vector pTV ⁇ CMVnlacZ, that was generated from co-transfection with pHP-ldl.28 and contained a CMV-IE promoter-driven nlacZ gene. About 10 5 transducing units of vector (approximately 1 moi) were used for each transduction in a total of three rounds of transduction.
- retroviral vector MFGnlacZ that was produced from PA317 packaging cells and contained a MLV LTR-driven nlacZ gene
- lentiviral vector pTV ⁇ CMVnlacZ that was generated from co-transfection with pHP-ld
- Transduced cultures were grown until confluent (3-5 days) , trypsinized, counted, and plated into 6 -well culture plates. Twenty four hours after plating, the cells were sampled for lacZ assay and the percentage of cells transduced was determined. The results of this short-term study showed that the lentiviral vectors transduced all three types of human cells 3 to 10 times more efficiently than did the MLV vectors (Table 5) .
- the transduced cells were continuously propagated without selection. At different passage times, the percentage of nlacZ-expressing cells and the expression kinetics were determined (Fig. 11) .
- Table 5 shows a direct comparison of the transduction efficiences observed at 48 hours and 48 days.
- TE671 are rhabdomyosarcoma cells
- 293T are kidney cells
- HepG2 are hepatoma cells.
- the numbers indicate the percent of cells transduced after one passage or multiple passages (for the 48 hour samples, the cells were transduced three times and propagated once, before staining for -galactosidase activity as previously described in Example 6.
- Example 8F Gene transduction into CD34+ human hematopoietic procursor cells Human CD34 hematopoietic progenitor cells are a slow- dividing cell population which is known to be difficult to transduce with conventional retroviral vectors.
- HSCs human hematopoietic stem ceils
- Amphotropic MLV vectors transduce mouse HSCs quite efficiently but human HSCs poorly due to the low level of cell surface MLV-env receptor expression; see Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline AP, Bodine DM. , The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc. Natl. Acad. Sci. USA 1996; 93:11097-11102; Sabatino DE, Do BQ, Pyle LC, et al .
- Amphotropic or gibbon ape leukemia virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines, Blood Cells Mol Dis 1997; 23:422-33; and possible cis -repressive elements in the MLV LTRs.Challita PM, Skelton D, El-Khoueiry A, Yu XJ, Weinberg K, Kohn DB, Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells. J Virol 1995; 69:748-755. In particular, transduction of HSCs in clinical trials has been very difficult. See Dunbar CE .
- VSV-G (VSV-G) and shown to infect CD34 cells quite efficiently.
- the HP/TV vector efficiently transduces actively dividing human cell lines including TE671 (rhabdomyosarcoma) , 293T (kidney carcinoma) HepG2 (hepatoma), and HeLa (cervical carcinoma) cells.
- TE671 rhabdomyosarcoma
- 293T kidney carcinoma
- HepG2 hepatoma
- HeLa cervical carcinoma
- Non-dividing and terminally differentiated cells such as mitomycin C-treated TE671 or HeLa cells, neruons, monocyte-derived macrophages and muscles can also be efficiently transduced by the HP/TV vectors.
- transduction of metabolically quiescent human peripheral blood lymphocytes or bone marrow mobilized blood CD34 stem cells with lentiviral vectors have not been reported, and in our experience, transduction of these cells with viral vectors including AAV, retroviral vectors or lentiviral vectors is extremely inefficient, probably because in the absence of growth factor activation these cells have very low metabolic enzyme and transcriptional activities, and accordingly, viral integration and gene expression do not proceed efficiently.
- human peripheral blood lymphocytes were collected from patients treated with G-CSF (granulocyte-colony stimulating factor) to mobilize bone marrow stem cells and purified through an anti-CD34 antibody affinity column (CellPro, Bothel, WA, USA) .
- G-CSF granulocyte-colony stimulating factor
- the collected C34+ cells were washed 2-3 times with RPMI medium containing 10% fetal bovine serum (FBS) without growth factor supplements, centrifuged at 800 g for 5 min, and resuspended in the same growth medium supplemented with 50 ng/ml human flt3 ligand, 50 ng/ml human c-kit ligand and 50 ng/ml human IL-3 at 1 x 10 5 cells/100 microliter.
- FBS fetal bovine serum
- TE671 cells were transfected with pHP-ldl.28 (8 microgram/well) , one of the lentiviral reporter vectors pTVdl . EFnlacZ or pTVdl.EFGFP (8 microgram/well) , pHEF-VSVG (5 microgram/well) and pCEP-tat
- the human CD34 cells were transduced 2-3 times with TV vectors at a multiplicity of infection (MOI) of 5-10, i.e., at MOI 10, approximately 10E5 cells were transduced with 10E6 infectious units (iu) of pTV vectors in a final volume of 100 ul in DMEM or RPMI growth medium supplemented with 8 microgram/ml of polybrene for 3-4 h each time.
- MOI multiplicity of infection
- the 10E6 iu of pTV vectors were prepared from two ml of vector stocks containing 5x105 iu/ml which can be concentrated 30-40 fold in a microfuge spun at 20,800 g at room temperature for 90-120 min.
- the transduced CD34 cells could be maintained in RPMI supplemented with growth factors for 1-4 days before they were plated into semi-solid methylcellulose colony assay media.
- the plated hematopoietic precursor cells grew and formed colonies in 2-4 weeks and the expression of transduced nlacZ and GFP genes were assayed by X-gal colorimetric staining and observed under an inverted fluorescent microscope.
- the reaction substrate was prepared in phosphate buffered saline adjusted to pH 8.5 using 150 mM Tris containing 4 mM K-ferrocyanide, 4 mM K-ferricyanide, 2 mM MgCl2 , 0.8 mg/ml X-Gal.
- One ml of the x-gal substrate was added to each 30 mm dish containing HSC-derived colonies and the dish was incubated at 37 deg.
- the colonies formed in methylcellulose agar were individually picked up and the genomic DNA extracted and subjected to polymerase chain reaction (PCR) using primers specific to the pTV vector.
- PCR polymerase chain reaction
- CD34 cells can be efficiently transduced by the VSV-G pseudotyped HP/TV vectors, at least at MOI of 5-10, but gene expression is delayed and the level of expression is very low.
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