EP1027451A1 - Systeme de vecteurs recombinants bases sur des parvovirus chimeres et specifiquement diriges sur le lignage erythrocitaire - Google Patents

Systeme de vecteurs recombinants bases sur des parvovirus chimeres et specifiquement diriges sur le lignage erythrocitaire

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Publication number
EP1027451A1
EP1027451A1 EP98956097A EP98956097A EP1027451A1 EP 1027451 A1 EP1027451 A1 EP 1027451A1 EP 98956097 A EP98956097 A EP 98956097A EP 98956097 A EP98956097 A EP 98956097A EP 1027451 A1 EP1027451 A1 EP 1027451A1
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Prior art keywords
gene
aav
promoter
vector
cells
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German (de)
English (en)
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Arun Srivastava
Selvarangan Ponnazhagan
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14211—Erythrovirus, e.g. B19 virus
    • C12N2750/14241—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14211—Erythrovirus, e.g. B19 virus
    • C12N2750/14241—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14245—Special targeting system for viral vectors

Definitions

  • the present invention relates generally to the fields of virology and molecular biology. More particularly, it concerns the engineering, propagation and use of viral vectors in the delivery of exogenous genes to cells.
  • AAV adeno-associated virus
  • AAV possesses several unique features that makes it more desirable than the other vectors. Unlike retroviruses, AAV can infect non-dividing cells; wild-type AAV has been characterized by integration, in a site-specific manner, into chromosome 19 of human cells (Kotin and Berns, 1989; Kotin et al, 1990; Kotin et al, 1991; Samulski et al, 1991); and AAV also possesses anti-oncogenic properties (Ostrove et al, 1981; Berns and Giraud, 1996). Recombinant AAV genomes are constructed by molecularly cloning DNA sequences of interest between the AAV ITRs, eliminating the entire coding sequences of the wild-type AAV genome.
  • the AAV vectors thus produced lack any of the coding sequences of wild-type AAV, yet retain the property of stable chromosomal integration and expression of the recombinant genes upon transduction both in vitro and in vivo (Berns, 1990; Berns and Bohensky, 1987; Bertran et al, 1996; Kearns et al, 1996; Ponnazhagan et al, 1997a).
  • AAV infection is receptor-mediated (Ponnazhagan et al, 1996; Summerford and Samulski, 1998).
  • the B19 parvovirus is a pathogenic virus and is an etiologic agent for a variety of human diseases (Anderson et al, 1983; Brown et al, 1984; Saarinen et al, 1986; Serjeant et al, 1981).
  • B19 is known to infect only cells of erythroid lineage (Ozawa et al, 1986; Ozawa et al, 1987; Srivastava and Lu, 1988; Srivastava et al, 1990). It recently has been determined that erythrocyte P antigen functions as the receptor for B19 infection in target cells.
  • the present invention provides an expression vector comprising two adeno-associated virus (AAV) inverted terminal repeats and an expression cassette comprising a selected DNA sequence and a promoter active in eukaryotic cells, wherein the cassette is located between the inverted terminal repeats, the selected DNA sequence is operably linked to the promoter, and the vector lacks any AAV structural genes.
  • the inverted terminal repeats comprises nucleotides 1 to 125 of SEQ ID NO:l.
  • the promoter may be selected from the group consisting of CMV IE, LTR, SV40 IE, HSV tk, ⁇ - actin, bl9p6, human globin ⁇ , human globin ⁇ and human globin ⁇ promoter.
  • the selected DNA sequence encodes a polypeptide.
  • the polypeptide may be selected from the group consisting of a gene encoding alpha-globin, beta-globin, gamma -globin, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, neomycin resistance, luciferase, adenine phosphoribosyl transferase (APRT), retinoblastoma, insulin, mast cell growth factor, p53, p21, pi 6 and adenosine deaminase.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • TNF tumor necrosis factor
  • IL-1 IL-2, IL-3, IL-4, IL-5, IL-6, IL
  • the selected DNA encodes an antisense RNA. More particularly, the antisense RNA is complementary to a segment of an oncogene.
  • the oncogene may be selected from the group consisting o myb, myc, ras, raf, erb, src.
  • the present invention provides a B19 viral particle comprising an expression vector comprising two adeno-associated virus (AAV) inverted terminal repeats and an expression cassette comprising a selected DNA sequence and a promoter active in eukaryotic cells, wherein the cassette is located between the inverted terminal repeats, the selected DNA sequence is operably linked to the promoter, and the vector lacks any AAV structural genes.
  • AAV adeno-associated virus
  • a helper virus construct comprising two adenovirus inverted terminal repeats, an AAV rep gene and a B 19 VP2 gene, wherein the rep and cap genes are under the control of at least one promoter and are located between the inverted terminal repeats.
  • the VP2 gene is under the control of the CMV IE promoter.
  • the rep gene is under the control of the B 19 p5 promoter.
  • the helper virus further may comprise a B19 VP1 gene.
  • Also contemplated by the present invention is a method for packaging an AAV expression vector comprising the steps of providing an expression vector comprising two adeno-associated virus (AAV) inverted terminal repeats and an expression cassette comprising a selected DNA sequence and a promoter active in eukaryotic cells, wherein the cassette is located between the inverted terminal repeats, wherein the selected DNA sequence is operably linked to the promoter, and the vector lacks any AAV structural genes; providing a helper virus construct comprising two adenovirus inverted terminal repeats, an AAV rep gene and a B19 VP2 gene, wherein the rep and cap genes are under the control of at least one promoter and are located between the inverted terminal repeats; contacting the expression vector and the helper virus construct with the host cell under conditions permitting the uptake of the expression vector and the helper virus construct by the cell; infecting the transfected host cell with adenovirus; and harvesting B19 particles from the host cell.
  • the helper virus further may
  • the multiplicity of infection of the adenovirus is about 10 pfu.
  • this is an exemplary number, it is understood that values greater or less than this value also will be useful.
  • MOIs of 5 pfu, 6 pfu, 7 pfu, 8 pfu, 9 pfu, 10 pfu, 11 pfu, 12 pfu, 13 pfu, 14 pfu, 15 pfu, 16 pfu, 17 pfu, 18 pfu, 19 pfu, 20 pfu, 25 pfu, 30 pfu per cell or more may also be employed.
  • the host cells are infected about 8 hours after transfer of the expression vector and the helper virus construct.
  • the contacting conditions may comprise calcium phosphate precipitation, electroporation, microprojectile bombardment or lipofection.
  • the harvesting comprises host cell disruption, virus isolation and heat inactivation.
  • the host cell disruption comprises freeze-thawing.
  • the harvesting is performed between about 65 and about 72 hours after infection of the adenovirus.
  • the virus isolation may comprise a technique selected from the group consisting of centrifugation, filtration and ion exchange chromatography.
  • the invention also provides a method for expressing a selected DNA sequence in a host cell comprising the steps of providing a B19 viral particle comprising an expression vector comprising two adeno-associated virus (AAV) inverted terminal repeats and an expression cassette comprising a selected DNA sequence and a promoter active in eukaryotic cells, wherein the cassette is located between the inverted terminal repeats, wherein the selected DNA sequence is operably linked to the promoter, and the vector lacks any AAV structural genes; contacting the viral particle with the host cell under conditions permitting infection of the host cell; and culturing the host cell under conditions permitting the transcription of the DNA sequence.
  • the DNA sequence encodes a polypeptide
  • the DNA sequence encodes an antisense mRNA.
  • the host cell is an erythroid cell. In more preferred embodiments, the erythroid cell is a human erythroid cell.
  • FIG. 1 Diagrammatic representation of construction of recombinant parvovirus B19- lacL.
  • pCMVp-/ ⁇ cZ contains a CMV promoter linked to the lacZ gene, flanked by AAV ITR's (SEQ ID NO:l).
  • pSP-42 contains AAV rep and B19 cap genes under the endogenous promoter (SEQ ID NO:2) and the CMV promoter, respectively, and flanked by Ad ITR's. 293 cells are transfected with both plasmids, thereby producing a B19-like particle containing an AAV-like genome, lacking all coding regions other than CM p-lacZ.
  • FIG. 2 Electron microscopy of chimeric viral particles.
  • FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E and FIG. 3F Expression of the transduced lacZ gene mediated by the recombinant AAV- and B19-/ cZ vectors in human 293 and Epo-differentiated MB-02 cells.
  • Approximately equivalent numbers of 293 (FIG. 3 A, FIG. 3B and FIG. 3C) and Epo-differentiated MB-02 cells (FIG. 3D, FIG. 3E and FIG. 3F) were either mock-infected or infected with 200 particle/cell each of AAV -lacZ (FIG. 3B and FIG. 3E) and B19-/ ⁇ cZ (FIG. 3C and FIG. 3F) recombinant vectors under identical conditions. Forty-eight hrs post-infection, the cells were fixed and stained for the analysis of expression of the lacZ gene as previously described (Ponnazhagan, 1996).
  • FIG. 4 FACS analysis of the lacZ gene expression in glycophorin-positive primary human low-density bone marrow (LDBM) cells transduced with recombinant AAV- and B19- lacZ vectors.
  • Glycophorin-positive cells from human LDBM were either mock-infected or infected with either 1x10 ⁇ particles/cell of AA N-lacZ vector, or 2x10 ⁇ particles/cell of B19- lacZ vector under identical conditions.
  • Forty-eight hrs post-infection cells were harvested and processed for analysis of the lacZ gene expression using FITC-conjugated X-Gal substrate using a Becton-Dickinson FACScanner as described in Example 1.
  • FIG. 5 FACS analysis of the lacZ gene expression in erythroid and non-erythroid populations of primary human bone marrow-derived CD34 + cells transduced with AAV- and ⁇ 9-lacZ recombinant vectors.
  • CD34 + cells isolated from human LDBM were either mock- infected or infected with either 1x10 ⁇ particles/cell of the AAV -lacZ vector or 2x10 ⁇ particles/cell of the B19-/ cZ vector under identical conditions.
  • FIG. 6 FACS analysis of the lacZ gene expression in erythroid- and myeloid- differentiated CD34 + primary human bone marrow cells transduced with AAV- and B19-/ ⁇ cZ recombinant vectors.
  • Primary human bone marrow-derived CD34+ cells were allowed to undergo differentiation into erythroid or myeloid lineages with the use of respective cytokine combinations for 10 days in vitro as described in Example 1.
  • cells were either mock-infected or infected with either 1x10 ⁇ particles/cell of the AAV-lacZ vector or 2x10 ⁇ particles/cell of the B19-/ ⁇ cZ vector under identical conditions.
  • Viral vectors are widely utilized for a variety of gene transfer endeavors.
  • retroviral vectors have been used for a number of years to transform cell lines in vitro for the purpose of expressing exogenous polypeptides.
  • various other vectors including adenoviruses and herpesviruses, along with retroviruses, have been utilized to transfer therapeutic genes into the cells of patients.
  • the inventors exploited the unique features of the AAV and B19 viruses to create a chimeric, parvovirus-based recombinant vector system.
  • This system does not suffer from toxicity, oncogenicity and immunogenicity concerns, as do many other viral vectors.
  • high titers of this vector may be generated, facilitating in vivo therapy.
  • this system is designed to specifically target primitive progenitor and differentiated cells of erythroid lineage, and can achieve stable integration and expression of transduced genes.
  • Production of the recombinant vector is achieved by creating a helper plasmids of the rep gene sequences of AAV and the cap gene sequences of B19.
  • the AAV -rep gene in one example, is expressed using its own promoter, designated p5, based on the map unit determination, and the VP2 capsid gene of B19 is expressed under the control of a strong promoter such as the cytomegalovirus immediate early (CMV IE) gene promoter.
  • CMV IE cytomegalovirus immediate early
  • This helper plasmid is cotransfected, along with a recombinant AAV plasmid containing either the bacterial ⁇ -galactosidase gene or human ⁇ -globin gene sequences, and a thymidine kinase (TK) promoter-driven bacterial neomycin resistance gene, cloned between a pair of AAV ITRs, into
  • 293 cells by calcium phosphate precipitation protocol, which cells subsequently are infected with adenovirus. Harvesting, purification and heat inactivation of the recombinant virus are then performed.
  • Adeno-Associated Virus AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.
  • the sequence of AAV is provided by Srivastava et al, (1983) and in U.S. Patent 5,252,479 (entire text of which is specifically incorporated herein by reference).
  • the three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pi 9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced.
  • the splice site derived from map units 42-46, is the same for each transcript.
  • the four non- structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
  • AAV is not associated with any pathologic state in humans.
  • AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus.
  • the best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication.
  • Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.
  • the terminal repeats of the AAV vector of the present invention can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al (1987).
  • the terminal repeats may be obtained by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV.
  • the ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration.
  • the ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.
  • B19 is known to be the etiologic agent of a variety of serious clinical disorders in humans.
  • B19 is the causative agent of transient aplastic crises associated with various hemolytic anemias, erythema infectiosum or the
  • the B19 virus is very similar to AAV, having rep and cap genes flanked by ITRs.
  • One notable difference between the two is the transcriptional pattern, which results in nine different transcripts, all initiated at a single start site at the left end of the genome.
  • Another difference is the 3' ITR, which for most parvo viruses is about 1 15 bases, but in the case of B19, is more than 300 bases in length.
  • the present invention utilizes the cap gene sequences of the B19 virus.
  • This gene encodes the structural components of the B19 virion and provides the host cell specificity of the virus.
  • Relevant sequences from B19 may be obtained from B19 DNA or cloned B19 DNA. See, for example, Cotmore et al. (1984); Shade et al. (1986). Standard techniques for the construction of such B 19 vectors are well-known to those of ordinary skill in the art and can be found in references such as Sambrook et al. (1989), or any of the myriad of laboratory manuals on recombinant DNA technology that are widely available. A variety of strategies are available for restricting and ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by the skilled artisan.
  • the present invention provides a unique chimeric vector system in which heterologous
  • DNA sequences are cloned within the inverted terminal repeats (ITR) of AAV, and subsequently packaged inside the capsid structure of B19.
  • ITR inverted terminal repeats
  • Production of such a recombinant chimeric vector is achieved by creating a helper plasmid consisting the rep gene sequences of AAV and the cap gene sequences of B19.
  • the AAV -rep was expressed using its own promoter, termed p5, based on the map unit determination and the VP2 capsid gene of B19 was expressed under the control of cytomegalovirus early gene promoter.
  • the B 19 for the purposes of the present invention, needs only to have a cap gene that is capable of facilitate packaging.
  • the cap gene may be a VP2 gene alone or in combination with the B19 VP1 gene.
  • These cap genes may be wild-type or a recombinant genes. Additional disclosure regarding B19 parvovirus capsids and genes thereof may be found in U.S. Patent 5,508,186 (specifically incorporated herein by reference) which describes recombinant baculoviruses encoding parvovirus structural proteins, method of infecting host cells therewith, and methods of packaging and delivering genetic information utilizing the noninfectious capsids.
  • U.S. Patent 5,508,186 specifically incorporated herein by reference
  • Adenovirus is a linear, double-stranded DNA virus with a genome of about 36 kB. Adenovirus can infect a wide range of host cells in a non-integrative fashion. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans. Both ends of the viral genome contain 100-200 base pair inverted terminal repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • E2A and E2B results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990).
  • the products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
  • MLP major late promoter
  • the MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.
  • TPL 5'-tripartite leader
  • Racher et al (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100- 200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
  • cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • adenovirus is relatively easy to grow and manipulate and exhibits broad host range in vitro and in vivo.
  • This group of viruses can be obtained in high titers, e.g., 10 -10 plaque-forming units per ml, and they are highly infectious.
  • the life cycle of adenovirus does not require integration into the host cell genome.
  • the foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • two vectors are provided that, when introduced into the same cell, produce an apathogenic, recombinant, chimeric parvovirus capable of targeting cells of erythroid origin.
  • the first vector is derived primarily from AAV and carries a recombinant DNA construct comprising a heterologous gene to be delivered to a target cell.
  • the second vector is derived from adenovirus, containing ITRs from this virus, but contains AAV and B19 sequences as well. This second vector serves to provide structural and replicative functions that facilitate the packaging of the first vector.
  • the AAV-derived vectors comprise a first and second AAV ITR, which flank at least a first promoter operably linked to a heterologous gene.
  • the terminal repeats can comprise all or an active portion of the ITRs of AAV. By active, it is meant that sufficient portions of the ITR exists to permit replication and packaging of the vector.
  • the ITRs mediate stable integration of the DNA sequence into a specific site in a particular chromosome, specifically, on human chromosome 19. The entire DNA sequence, including the ITRs, the promoter, and the heterologous gene, is integrated into the genome. Therefore, in preferred embodiments, the ITRs or portions thereof also permit integration.
  • the hybrid Ad-AAV-B19-Ad vectors of the present invention comprise a first and a second adenovirus ITR, which flank an AAV rep gene and a B19 cap gene.
  • promoters are provided for each of the rep and cap genes.
  • the terminal repeats may comprise all or an active portion of the ITRs of adenovirus, with active defined, in this instance, as permitting the replication of the vector.
  • Packaging of the vector into a viral capsid is not necessary as the DNA can be transfected into cells along with the AAV -derived vector, although in some embodiments, it may be desired that this hybrid vector be packaged in an adenoviral particle. In this situation, an active adenoviral packaging signal is necessary. This signal is located at approximately 290-390 bases pairs from the left end of the genome.
  • Crude cell lysates are prepared 65-72 hrs post-infection by three cycles of freezing and thawing followed by heat-inactivation of Ad2 at 56°C for 30 min.
  • Clarified supernatants are digested with DNase I (100 U/ml) for 1 fir at 37°C and adjusted to a density of 1.40 g/ml by addition of CsCl and centrifuged at 35,000 rpm for 40 hrs at 20°C.
  • Equilibrium density gradients are fractionated by collecting drops through a puncture in the bottom of the centrifuge tube. The densities of all fractions are determined from refractive index measurements.
  • Viral titers of rAAV generally ranges from 10 n to 10 13 particles/ml, and for rB19 from 10 8 to 10 9 particles/ml.
  • Virions can be produced by cotransfer of the helper plasmid and the AAV plasmid, followed by infection with a helper virus such as adenovirus, herpes virus or vaccinia virus. Transfer may be accomplished any standard gene transfer mechanism: calcium phosphate precipitation, lipofection, electroporation, microprojectile bombardment or other suitable means.
  • helper virus Following transfer, host cells are infected with a helper virus, virions are isolated and the helper virus is inactivated (e.g., heated at 56°C for one hour). The resulting helper free stocks of virions are used to infect appropriate target cells. Mature virions may further be isolated by standard methods, e.g., cesium chloride centrifugation, and to inactivate any contaminating adenovirus.
  • Bone Marrow Cells are an example of host cells that may be transformed conditions for transformation of host cells.
  • Equivalent numbers of primary human low-density bone marrow (LDBM) mononuclear cells are either mock-infected or infected with 1,000 - 10,000 particles/cell of rAAV at 37°C for 1 hr, washed with sterile phosphate-buffered saline (PBS), and incubated in IMDM containing 20% FBS and antibiotics at 37°C in a 5% CO 2 incubator.
  • LDBM low-density bone marrow
  • the LDBM and differentiated and undifferentiated CD34+ cells are either mock-infected or infected with the recombinant B19-lacZ or recombinant AAV-lacZ vectors at a particle to cell ratio of 200:1 and 100,000:1, respectively, at 37°C for 1 hr following which the cells are grown in the presence of cytokines (interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF)) for a period of 48 hrs.
  • cytokines interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF)
  • Analysis of expression of the transduced lacZ gene is performed using the FITC-conjugated substrate for beta-Gal and the PE-conjugated substrates for CD34 and CD33 antigens or glycophorin.
  • cells are incubated with 300 ⁇ M chloroquine for 30 min. at 30°C following which chloroquine is removed by centrifugation and cells are incubated further with 33 ⁇ M Imagreen C12FDG ⁇ -gal substrate for 30 min. at 37°C. Following centrifugation, the cells are resuspended in fresh culture medium and analyzed using a Beckton-Dickinson FACScanner.
  • the present invention contemplates the use of helper vectors packaged in viral particles.
  • the Ad-AAV-B19-Ad vectors can be engineered to contain sufficient cw-acting signals for their packaging in adenoviral capsids.
  • the requirements for adenoviral packaging are well-defined by the field and largely reside in the adenoviral left end ITR. The only other major concerns relate to the compatibility of inserted sequences and the overall size of the vector, which is up to about 40 kB.
  • This system would require provision of adenoviral replicative and packaging functions in trans, either by a cell line or by a helper virus.
  • Essential functions from adenovirus, which would need to be provided, include El A, E1B, E2A, E2B, E4 and L1-L5. 293 cells are well known in the art and can provide El A and E1B functions.
  • Function of the hybrid vectors of the present invention i.e., the ability to mediate transfer and expression of the heterologous gene in hematopoietic stem or progenitor cells, can be evaluated by monitoring the expression of the heterologous gene in transduced cells.
  • the assay for expression depends upon the nature of the heterologous gene. Expression can be monitored by a variety of methods including immunological, histochemical or activity assays. For example, Northern analysis can be used to assess transcription using appropriate DNA or RNA probes. If antibodies to the polypeptide encoded by the heterologous gene are available. Western blot analysis, immunohistochemistry or other immunological techniques can be used to assess the production of the polypeptide.
  • heterologous gene is an enzyme.
  • the heterologous gene encodes antibiotic resistance
  • a determination of the resistance of infected cells to the antibiotic can be used to evaluate expression of the antibiotic resistance gene.
  • Site-specific integration can be assessed, for example, by Southern blot analysis.
  • DNA is isolated from cells transduced by the vectors of the present invention, digested with a variety of restriction enzymes, and analyzed on Southern blots with an AAV-specific probe. A single band of hybridization evidences site-specific integration.
  • Other methods known to the skilled artisan, such as polymerase chain reaction (PCRTM) analysis of chromosomal DNA can be used to assess stable integration.
  • the nucleic acid encoding a gene product is placed under the transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the phrases "under transcriptional control” or “operably linked” mean that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of genes.
  • CMV cytomegalovirus
  • the SV40 early promoter the Rous sarcoma virus long terminal repeat
  • rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
  • Tables 2 and 3 list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is. they are composed of many individual elements, each of which binds to one or more transcriptional proteins. Thus, in certain situations, it will be desirable to further include an enhancer in the regulatory cassette.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • NC AM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • Transgene expression will be driven by a selected promoter. The promoter selection will depend on the polypeptide to be expressed, the target tissue and the purpose for expression.
  • a high level promoter will be utilized. If the protein is toxic to the cells, it may be desirable to regulate the expression of the protein such that cell proliferation is maximized prior to polypeptide expression. If the protein's processing or secretion is dependent upon a particular stage in the host celfs cycle, it may be desirable to employ a promoter that is regulated in an appropriate, cell cycle dependent fashion.
  • the B19 p6 promoter provides for expression specific to erythroid progenitor cells.
  • a consensus promoter-like sequence TATATATA is located at nucleotide 320 in B19 and. thus, transcription is likely to originate about 30 nucleotides downstream. It is known that B19 fragments containing these sequences direct expression that is specific for erythroid progenitor cells, and that deletion of B19 coding sequences downstream from the promoter prevents replication of B19.
  • the promoter sequence can be derived by restriction endonuclease digestion of B19 or a cloned B19 plasmid such as pYT103 and pYT107 (Cotmore et al. (1984)) or by any other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis based upon the published sequence of B19.
  • the vectors of the present invention include a "helper" vector that supplies the replication (rep) function of AAV and the capsid (cap) function of B19. Each of these genes may be contained in a single expression cassette.
  • the exemplified construct utilizes a CMV IE promoter operably linked to the cap gene, and a the p5 promoter of AAV operably linked to the rep gene. Where multigene constructs are utilized, internal ribosome entry sites (IRES) may increase the level and fidelity of expression of the downstream gene (discussed below).
  • any high level expression promoter will suffice to drive these genes.
  • certain advantages may accrue through the use of the homologous (normal) promoter for these genes and/or viruses.
  • position effects in the 5' untranslated portion of a gene may be difficult to duplicate in synthetic constructs and may be responsible for dramatic variations in expression levels.
  • it may prove advantageous to utilize the homologous promoter.
  • the B 19 p6 promoter is very useful in the context of the present invention, and is a homologous promoter with respect to the B19 cap gene.
  • Another homologous promoters include the AAV p5 promoter for the AAV rep gene.
  • a cell may be identified in vitro or in vivo by including a marker in the vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the vector and, hence, the gene of interest.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase or chloramphenicol acetyltransferase (CAT) may be employed. Further examples of selectable markers are well known to one of skill in the art.
  • IRES internal ribosome binding sites
  • IRES elements are able to bypass the ribosome scanning model of 5'-methylated Cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis
  • IRES elements from two members of the picanovirus family Polio and encephalomyocarditis
  • IRES elements from a mammalian message Macejak and Sarnow, 1991.
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • the heterologous gene encodes a biologically functional protein, i.e., a polypeptide or protein which affects the cellular mechanism of a cell in which the biologically functional protein is expressed.
  • the biologically functional protein can be a protein which is essential for normal growth of the cell or for maintaining the health of a mammal.
  • the biologically functional protein can also be a protein which improves the health of a mammal by either supplying a missing protein, by providing increased quantities of a protein which is underproduced in the mammal or by providing a protein which inhibits or counteracts an undesired molecule which may be present in the mammal.
  • the biologically functional protein can also be a protein which is a useful protein for investigative studies for developing new gene therapies or for studying cellular mechanisms.
  • (i) Secreted Proteins The cDNA's encoding a number of useful human proteins are available for insertion into vectors of the present invention. Examples would include soluble CD4, Factor VIII, Factor IX, von Willebrand Factor, TPA, urokinase, hirudin, interferons and ⁇ , TNF, GM-CSF, antibodies, albumin, transferin and nerve growth factors. Cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 also are contemplated.
  • Peptide hormones are grouped into three classes with specific examples given for each.
  • Class I is represented by Growth Hormone, Prolactin and Parathyroid hormone.
  • Class II is represented by Insulin and Glucagon.
  • Class III is represented by Amylin, Glucagon- like Peptide I and Calcitonin.
  • Adrenocorticotropin (ACTH) Gastric Inhibitory Peptide (GIP) Angiotensin I and II Glucagon ⁇ -endorphin Insulin
  • PTH-rP Parathyroid Hormone-related protein
  • VIP Vasoactive Intestinal Peptide
  • Vasopressin Vasotocin
  • Alpha Melanocyte Stimulating Hormone alpha-MSH
  • Atrial Natriuretic Factor (5-28) (ANF)
  • SAP-1 Corticotropin Releasing Hormone (CRH)
  • GHRH Growth Hormone Releasing Factor
  • LHRH Luteinizing Hormone-Releasing Hormone
  • Non-Secreted Proteins Two general classes of non-secreted proteins can be defined. The first are proteins that, once expressed in cells, stay associated with the cells in a variety of destinations. These destinations include the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, golgi, membrane of secretory granules and plasma membrane. Non-secreted proteins are both soluble and membrane associated. The second class of proteins are ones that are normally associated with the cell, but have been modified such that they are now secreted by the cell. Modifications would include site-directed mutagenesis or expression of truncations of engineered proteins resulting in their secretion as well as creating novel fusion proteins that result in secretion of a normally non-secreted protein.
  • the cDNA's encoding a number of therapeutically useful human proteins are available. These include cell surface receptors, transporters and channels such as GLUT2, CFTR, leptin receptor, sulfonylurea receptor, ⁇ -cell inward rectifying channels, etc. Other proteins include protein processing enzymes such as PC2 and PC3. and PAM. transcription factors such as IPF1, and metabolic enzymes such as adenosine deaminase, phenylalanine hydroxylase, glucocerebrosidase.
  • proteins that fall into various classes that would be suitable for producing transgenic constructs in the present invention.
  • Such classes include tumor suppressors, inducers of apoptosis, enzymes, cytokines, enzymes and toxins, the following section describes examples of each of these classes. This list is not intended to be exhaustive, rather it is intended to provide examples of the types of proteins that may be encoded by transgenes using the vectors of the present invention.
  • Tumor Suppressors p53 currently is recognized as a tumor suppressor gene. High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses.
  • the p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently-mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al, 1991) and in a wide spectrum of other tumors.
  • the p53 gene encodes a 393 -amino acid phosphoprotein that can form complexes with host proteins such as SV40 large-T antigen and adenoviral E1B.
  • the protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue.
  • wild-type p53 appears to be important in regulating cell growth and division. Overexpression of wild-type p53 has been shown in some cases to be anti- proliferative in human tumor cell lines. Thus, p53 can act as a negative regulator of cell growth (Weinberg, 1991) and may directly suppress uncontrolled cell growth or indirectly activate genes that suppress this growth. Thus, absence or inactivation of wild-type p53 may contribute to transformation. However, some studies indicate that the presence of mutant p53 may be necessary for full expression of the transforming potential of the gene.
  • Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene. A single genetic change prompted by point mutations can create carcinogenic p53, in as much as mutations in p53 are known to abrogate the tumor suppressor capability of wild-type p53. Unlike other oncogenes. however, p53 point mutations are known to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity. Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line. Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991).
  • CDK cyclin-dependent kinases
  • CDK4 cyclin-dependent kinase 4
  • the activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit pl ⁇ ' .
  • the pi 6 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al. 1995). Since the pl6 1NR4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4. resulting in hyperphosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6.
  • pl6 ' belongs to a newly described class of CDK- inhibitory proteins that also includes pi 5 , p21 , and p27 ⁇ ,P1 .
  • the pl6 I K gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pi 6 gene are frequent in human tumor cell lines. This evidence suggests that the pi 6 gene is a tumor suppressor gene.
  • pi 6 promoter hypermethylation is one of these mechanisms (Merlo et al, 1995; Herman, 1995; Gonzalez-Zulueta, 1995). Restoration of wild-type pl ⁇ 11 ⁇ 4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995). Delivery of pi 6 with adenovirus vectors inhibits production of some human cancer lines and reduces the growth of human tumor xenografts C-C AM is expressed in virtually all epithelial cells (Odin and Obrink, 1987).
  • C-CAM with an apparent molecular weight of 105 kD, was originally isolated from the plasma membrane of the rat hepatocyte by its reaction with specific antibodies that neutralize cell aggregation (Obrink, 1991). Recent studies indicate that, structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily and its sequence is highly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti, 1989). Using a baculovirus expression system, Cheung et al. (1993) demonstrated that the first Ig domain of C-CAM is critical for cell adhesive activity.
  • Ig immunoglobulin
  • CEA carcinoembryonic antigen
  • CAM's are known to be involved in a complex network of molecular interactions that regulate organ development and cell differentiation (Edelman, 1985). Recent data indicate that aberrant expression of CAM's maybe involved in the tumorigenesis of several neoplasms; for example, decreased expression of E-cadherin. which is predominantly expressed in epithelial cells, is associated with the progression of several kinds of neoplasms (Edelman and Crossin, 1991 ; Frixen et al, 1991 ; Bussemakers et al, 1992; Matsura et al. 1992; Umbas et al, 1992).
  • C-CAM now has been shown to suppress tumor growth in vitro and in vivo.
  • tumor suppressors that may be employed according to the present invention include p21. pl5, BRCA1, BRCA2, IRF-1, PTEN, RB, APC, DCC, NF-1, NF-2, WT-1, MEN- I, MEN-II, zacl, p73, VHL, FCC and MCC.
  • Inducers of ⁇ poptosis Inducers of apoptosis. Inducers of apoptosis, such as Bax, Bak, Bcl-X s , Bad, Bim, Bik, Bid, Harakiri, Ad ElB, Bad and ICE-CED3 proteases, similarly could find use according to the present invention.
  • Inducers of apoptosis such as Bax, Bak, Bcl-X s , Bad, Bim, Bik, Bid, Harakiri, Ad ElB, Bad and ICE-CED3 proteases, similarly could find use according to the present invention.
  • Enzymes Various enzyme genes are of interest according to the present invention.
  • Such enzymes include cytosine deaminase, adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-1 -phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase, -L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase and human thymidine kinase and extracellular proteins such as collagenase and matrix metalloprotease.
  • Cytokines Another class of genes that is contemplated to be inserted into the adenoviral vectors of the present invention include interleukins and cytokines. Interleukin 1,
  • IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13.
  • METH-2 GM-CSF, G-CSF, M-CSF and tumor necrosis factor.
  • Toxins Various toxins are also contemplated to be useful as part of the expression vectors of the present invention, these toxins include bacterial toxins such as ricin A-chain (Burbage, 1997), diphtheria toxin A (Massuda et al. 1997; Lidor, 1997). pertussis toxin A subunit, E. coli enterotoxin toxin A subunit, cholera toxin A subunit and pseudomonas toxin c- terminal. Recently, it was demonstrated that transfection of a plasmid containing the fusion protein regulatable diphtheria toxin A chain gene was cytotoxic for cancer cells. Thus, gene transfer of regulated toxin genes might also be applied to the treatment of cancers (Massuda et al., 1997).
  • bacterial toxins such as ricin A-chain (Burbage, 1997), diphtheria toxin A (Massuda et al. 1997; Lidor, 1997). pert
  • Antisense constructs are one way of addressing this situation.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C cytosine
  • A:T thymine
  • A:U uracil
  • Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • ds double-stranded
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • ribozyme e.g., ribozyme; see below
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozyme Constructs Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech. 1987; Gerlach et al, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
  • the present invention contemplates the use of chimeric parvovirus vectors to transform cells for the production of mammalian cell cultures.
  • chimeric parvovirus vectors to transform cells for the production of mammalian cell cultures.
  • the cells In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells are maintained with the correct ratio of oxygen and carbon dioxide and nutrients, but are protected from microbial contamination.
  • Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).
  • the construct encoding the protein of interest may be transferred by the viral vector, as described above, into appropriate host cells followed by culture of cells under the appropriate conditions.
  • the gene for virtually any polypeptide may be employed in this manner.
  • useful mammalian cell lines are those that express the appropriate receptor for B19 virus. These include bone marrow cells, peripheral blood cells, endothelial cells and myocardial cells (Rouger et al, 1987; van dem Borne et al, 1986)
  • Bone marrow cells are isolated and enriched for hematopoietic stem cells (HSC), e.g., by fluorescence activated cell sorting as described in Srivastava et al (1988).
  • HSC hematopoietic stem cells
  • HSC are capable of self-renewal as well as initiating long-term hematopoiesis and differentiation into multiple hematopoietic lineages in vitro.
  • HSC are transfected with the vector of the present invention or infected with varying concentrations of virions containing a subject hybrid vector and then assessed for the expression of the heterologous gene.
  • the assay for expression depends upon the nature of the heterologous gene. Expression can be monitored by a variety of methods including immunological, histochemical or activity assays.
  • Northern analysis can be used to assess transcription using appropriate DNA or RNA probes. If antibodies to the polypeptide encoded by the heterologous gene are available, Western blot analysis, immunohistochemistry or other immunological techniques can be used to assess the production of the polypeptide. Appropriate biochemical assays can also be used if the heterologous gene is an enzyme. For example, if the heterologous gene encodes antibiotic resistance, a determination of the resistance of infected cells to the antibiotic can be used to evaluate expression of the antibiotic resistance gene. An important consideration is the appropriate modification needed for a particular polypeptide. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the protein expressed.
  • a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
  • anti- metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
  • Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation ( . e. , a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent cells.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
  • the gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor.
  • the main advantage of this design is the simplicity and lack of need for mechanical mixing.
  • the height-to-diameter ratio is 10: 1.
  • the airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • purified protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. of homology between the two sequences suggest that human GDF-9 plays an important role during embryonic development and/or in the adult ovary.
  • FIGURE 7 shows in situ hybridization to adult ovary sections using a GDF-9 RNA probe.
  • [ 35 S] -labeled anti-sense (FIGURE 7a and 7c) or sense (FIGURE 7b and 7d) GDF-9 RNA probes were hybridized to adjacent paraffin-embedded sections of ovarians fixed in 4% paraformaldehyde. Sections were dipped in photographic emulsion, exposed, developed, and then stained with hematoxylin and eosin. Two representative fields are shown.
  • GDF-9 mRNA was detected primarily in oocytes in adult ovaries. Every oocyte (regardless of the stage of follicular development) examined showed GDF-9 expression, and no expression was detected in any other cell types. No hybridization was seen using a control GDF-9 sense RNA probe (FIGURE 7b and 7d). Hence, GDF-9 expression appears to be oocyte-specific in adult ovaries.
  • FIGURE 8 shows in situ hybridization to a postnatal day 4 ovary section using an antisense GDF-9 RNA probe. Sections were prepared as described for FIGURE 7. Following autoradiography and staining, the section was photographed under bright-field (FIGURE 8a) or dark-field (FIGURE 8b) illumination.
  • FIGURE 9 shows in situ hybridization to postnatal day 8 ovary sections using an antisense (FIGURE 9a) or sense (FIGURE 9b) GDF-9 RNA probe. Sections were prepared as described for FIGURE 7. specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N- acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • the hybrid vectors of the present invention are useful for gene therapy.
  • the vectors of the present invention can direct erythroid cell-specific expression of a desired gene, and thus are useful in the treatment of hemoglobinopathies.
  • maladies include thalassemia, sickle-cell anemia, diabetes, and cancer.
  • the heterologous gene in this context, can be the normal counte ⁇ art of one that is abnormally produced or unde ⁇ roduced in the disease state, for example ⁇ -globin for the treatment of sickle-cell anemia, and -globin, ⁇ -globin or ⁇ -globin in the treatment of thalassemia.
  • the heterologous gene can encode antisense RNA as described hereinabove.
  • alpha -globin is produced in excess over ⁇ -globin in ⁇ -thalassemia.
  • ⁇ -thalassemia can be treated in accordance with the present invention by gene therapy with a vector in which the heterologous gene encodes an antisense RNA.
  • the antisense RNA is selected such that it binds to a target sequence of the ⁇ -globin mRNA to prevent translation of ⁇ -globin, or to a target sequence of the ⁇ -globin DNA such that binding prevents transcription of ⁇ -globin DNA.
  • the heterologous gene can be a gene associated with tumor suppression, such as retinoblastoma gene, p53, pl6, p21 or the gene encoding tumor necrosis factor.
  • the use of the hybrid vectors of the present invention for the treatment of disease involves, in one embodiment, the transduction of hematopoeitic stems cells or progenitor cells with the claimed vectors. Transduction is accomplished, following preparation of mature virions containing the AAV vectors, by infection of HSC or progenitor cells therewith. Transduced cells may be located in patients or transduced ex vivo and introduced or reintroduced into patients, e.g., by intravenous transfusion. Rosenberg, 1990).
  • HSC or progenitor cells are provided by obtaining bone marrow cells from patients and optionally enriching the bone marrow cell population for HSC.
  • HSC can be transduced by standard methods of transfection or infected with mature virions for about one to two hours at about 37°C.
  • Stable integration of the viral genome is accomplished by incubation of HSC at about 37°C for about one week to about one month. The stable, site- specific integration and erythroid cell-specific expression is assessed as described above.
  • the presence of the heterologous gene product can be monitored or assessed by an appropriate assay for the gene product in the patient, for example in peripheral red blood cells or bone marrow of the patient when expression is erythroid cell-specific.
  • an appropriate assay for the gene product in the patient for example in peripheral red blood cells or bone marrow of the patient when expression is erythroid cell-specific.
  • the specific assay is dependent upon the nature of the heterologous gene product and can readily be determined by one skilled in the art.
  • ⁇ -thalassemia represents a heterologous group of clinical syndromes that are inherited as mutated alleles of genes that encode the human ⁇ -globin chain. These mutations affect all aspects of ⁇ -globin gene expression including transcription, splicing, polyadenylation, translation, and protein stability.
  • the hallmark of ⁇ -thalassemia is the marked reduction or total absence of synthesis of normal adult hemoglobin (HbA; ⁇ 2 ⁇ 2 ).
  • HbA normal adult hemoglobin
  • Treatment is limited to regular red blood cell transfusions and iron-chelation therapy. Treatment by bone marrow transplantation has also been attempted (Thomas et al. 1982), but an effective cure has not been found.
  • the vectors of the present invention are useful in the treatment of ⁇ - thalassemia.
  • An AAV-B 19 vector is constructed in which the heterologous gene is the normal human ⁇ -globin gene, with the resulting AAV-B 19- ⁇ -globin vector allowing parvovirus- mediated transfer, site-specific integration and erythroid cell-specific expression of the normal human beta -globin gene in human hematopoietic cells.
  • Abnormal beta-globin expression in beta-thalassemia may result in the overabundance of alpha-globin mRNA relative to beta-globin mRNA.
  • the present invention can not only provide a normal beta-globin gene, as described hereinabove, but can further be utilized to down-regulate the production of excess alpha-globin by providing a vector with an antisense
  • RNA as the heterologous gene As the heterologous gene.
  • the present invention contemplates gene therapy for ⁇ -thalassemia comprising transduction of hematopoietic stem or progenitor cells with a hybrid vector encoding normal ⁇ - globin chains, or simultaneous transduction with a vector encoding a normal ⁇ -globin chain and a vector encoding an RNA antisense to alpha -globin mRNA or DNA.
  • a construction with more than one B19 p6 promoter, as described hereinabove permits coincident expression of ⁇ -globin and antisense ⁇ -globin. Accordingly, transduction with a single vector effects both the provision of a normal ⁇ -globin gene and the down-regulation of excess ⁇ -chains.
  • bone marrow cells are transfected with the subject vectors, and transduced cells are introduced, by intravenous transfusion, into a patient.
  • the stable integration of the vector can be assessed by PCRTM or Southern blot analysis and the expression of the heterologous gene can be evaluated by assaying for the heterologous gene product in the patient's peripheral blood cells or bone marrow cells. As described previously, the particular assay depends upon the nature of the heterologous gene product.
  • Yet another aspect of the present invention provides a method for delivery of a pharmaceutical product, a protein or an antisense RNA in a mammal. Since the normal differentiation of these stem cells results in production of mature erythrocytes, the transduction of stem cells with the subject vector ultimately yields a population of circulating, enucleate vesicles containing the gene product.
  • This method comprises transducing hematopoietic stem or progenitor cells with the hybrid vector of the present invention and introducing, by intravenous transfusion or injection, the transduced cells into a mammal. Transduction can be accomplished by transfecting cells with the hybrid vector by standard methods or infecting cells with mature AAV virions containing the hybrid vector at about 37°C. for about one to two hours.
  • the pharmaceutical product is encoded by the heterologous gene of the hybrid vector, and can be any pharmaceutical product capable of being expressed by the hybrid vector.
  • Such products include alpha, beta and gamma -globin, insulin, GM-CSF, M-CSF, G-CSF, EPO, TNF, MGF, interleukins, the gene product of the retinoblastoma gene, p53 or adenosine deaminase.
  • the coding sequences of the respective genes are known (Lee et al. (1985: GM-CSF); Broderick et al, (1987; APRT); Tratschin et al. (1985; Neo r ); Huang et al. (1988; RB-1); Liebhaber et al. (1980; ⁇ -globin); Lawn et al. (1980; ⁇ -globin); Enver et al. (1989; ⁇ -globin)) and thus can be easily provided as described hereinabove. Therefore, the present invention can provide production of constitutive levels of heterologous gene products inside membrane vesicles, specifically red blood cells, for in situ treatment of disease.
  • the hybrid vector can further comprise a sequence which encodes a signal peptide or other moiety which facilitates the secretion of the gene product from the erythroid cell.
  • sequences are well-known to one of ordinary skill in the art (Michaelis et al. 1982) and can be inserted into the subject vectors between the promoter and coding region by methods described herein above. This method can be used to treat a variety of diseases and disorders and is not limited to the treatment of hemoglobinopathies, since the heterologous gene is constitutively expressed and can be released from the red blood cell by virtue of a secretory sequence, or released when red blood cells are lysed in the liver and spleen.
  • compositions containing the viral vectors of the present invention in a form appropriate for the intended application.
  • this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be inco ⁇ orated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the active compounds also may be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • EXAMPLE 1 Materials And Methods Cells, plasmids and viruses:
  • the recombinant AAV-helper plasmid, pAAV/Ad, and the recombinant B19 plasmid, pYT-103 were provided by R.J. Samulski (University of North Carolina, Chapel Hill. NC) and P. Tattersall (Yale University School of Medicine, New Haven, CT), respectively.
  • the details of construction of the recombinant AAV plasmid containing the bacterial b-galactosidase (lacZ) gene under the control of the cytomegalovirus immediate-early promoter (pCMVp-lacZ) has been described previously (Ponnazhagan et al, 1997a).
  • the human adenovirus type 2 (Ad2) was provided by K.H. Fife (Indiana University School of Medicine, Indianapolis) and was propagated as previously described (Ponnazhagan et al, 1994; Ponnazhagan et al, 1997a ).
  • the recombinant helper plasmids containing the B 19-cap genes under the control of the CMV promoter were constructed by first cloning either the VP2 gene alone or VP1+VP2 genes isolated from the plasmid pYT-103 to yield plasmids pSP-37 and pSP-38, respectively. Subsequently, the portion of the B 19-cap genes with the CMV promoter was cloned in the plasmid pAAV/Ad replacing the AAV-cap genes to yield plasmids pSP-42 and pSP-46, respectively.
  • the recombinant helper plasmids pSP-42 and pSP-46 were used to package rescued and replicated recombinant AAV genomes containing the CMVp-lacZ gene sequences in 293 cells by the calcium phosphate transfection protocol as previously described (Ponnazhagan et al, 1997a; Ponnazhagan et al, 1997b). Harvesting and CsCl density gradient purification of the virus were done as described previously (Ponnazhagan et al, 1997a; Ponnazhagan et al, 1997b; Wang et al, 1995).
  • Bone marrow aspirates were immediately diluted with an equal volume of Iscove's- modified Dulbecco's Medium (IMDM) containing 20 U/ml heparin.
  • IMDM Iscove's- modified Dulbecco's Medium
  • LDBM Low-density bone marrow cells
  • CD34+ cells the mononuclear cells were labeled with anti- CD34 antibodies that were conjugated with magnetic particles.
  • the labeled cells were passed through a magnetic separation column (Miltenyi Biotech. Sunnyvale, CA) and CD34- cells were allowed to flow through the column.
  • CD34+ cells were subsequently eluted using MACS buffer (0.5% BSA and 5 mM EDTA in IxPBS). The purity of the isolated CD34+ cells were determined by fluorescence-activated cell sorting (FACS) and ranged between 90-95%.
  • FACS fluorescence-activated cell sorting
  • CD34+ cells into erythroid and myeloid lineages in vitro was achieved by the addition of 5 U/ml erythropoietin (Epo) and 10 ng/ml of granulocyte-colony stimulating factor (G-CSF), respectively, to the cells in liquid cultures supplemented with interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) (Stem Cell Technologies, Vancouver, BC) as previously described.
  • Epo erythropoietin
  • G-CSF granulocyte-colony stimulating factor
  • the LDBM and differentiated and undifferentiated CD34+ cells were either mock-infected or infected with the recombinant B19-lacZ or recombinant AAV-lacZ vectors at a particle to cell ratio of 200:1 and 100,000:1, respectively, at 37°C for 1 hr following which the cells were grown in the presence of respective cytokines mentioned above for a period of 48 hrs.
  • Analysis of expression of the transduced gene was performed using the FITC-conjugated substrate for ⁇ - Gal and the PE-conjugated substrates for CD34 and CD33 antigens or glycophorin. Briefly, cells were incubated with 300 mM chloroquine for 30 min.
  • the production of the recombinant B 19 vectors was achieved by generating two helper plasmids, designated pSP-42 and pSP-46, by substituting the AAW-cap gene sequences in the AAV helper-plasmid pAAV/Ad (Samulski et al. 1987) with the B ⁇ 9-cap gene sequences isolated from plasmid pYT103c (Srivastava et al, 1989) leaving the rep gene sequences of AAV intact.
  • the B 19 sequences were cloned under the control of the CMV promoter.
  • helper-plasmid pSP-42 contains only the VP2 gene
  • the plasmid pSP-46 contains both VP1+VP2 genes of the B19-c p sequences.
  • Both helper-plasmids were initially tested for their ability to mediate efficient rescue and replication of the AAV genome from a recombinant AAV plasmid indicating functional trans-complementation by the AAV-rep proteins.
  • the efficiency of rescue/replication of the recombinant AAV CMVp-/ cZ genome by plasmids pSP-42 and pSP-46 was nearly the same as that by pAAV/Ad.
  • the packaging of the recombinant B19-/ ⁇ cZ vectors was carried out by co-transfecting the pCMVp-/ ⁇ cZ plasmid with either pSP-42 or pSP-46 in 293 cells as described in Example 1.
  • Recombinant AAV vector stocks containing the same CMVp-/ ⁇ cZ transgene were also prepared as previously described (Ponnazhagan et al, 1996; Ponnazhagan et al, 1997a). Based on quantitative DNA slot-blot analyses (Kube et al, 1997), the recombinant B19 viral titers were determined to be approximately lxlO 8 particles/ml when pSP-42 was used as a helper-plasmid.
  • the recombinant B ⁇ 9-lacZ vector was tested for its ability to infect both 293 cells, known to be permissive for AAV infection, and undifferentiated or eiythroid-differentiated MB-02 cells, known to be permissive for B19 infection and replication upon differentiation with Epo (Munshi et al, 1993), but non-permissive for AAV infection (Ponnazhagan et al., 1996). Both recombinant vector stocks were used at a virus particle-to-cell ratio of 200:1. Forty-eight hrs post infection, cells were analyzed for the transduced lacZ gene expression. These results are depicted in FIG. 3A-FIG. 3F.
  • Recombinant B19 virions mediate high-efficiency, selective transduction of primary human hematopoietic progenitor cells in the erythroid lineage
  • the functional studies to determine erythroid cell targeting of the recombinant viruses were first performed using the recombinant virus containing the ⁇ -gal sequences. In each of the studies, the recombinant AA V-lacZ vector was also included as an appropriate control.
  • LDBM Human low-density bone marrow
  • the recombinant B 19 vector-mediated delivery of the transduced gene was significantly higher than that by the recombinant AAV vector in the erythroid fraction of primary human bone marrow cells which strongly suggested, but did not prove, the erythroid cell-specific targeting by the recombinant B 19 vector.
  • CD34 + cells from human LDBM were isolated and either mock-infected or infected with the recombinant AA N-lacZ or the B ⁇ 9-lacZ vectors at a particle to cell ratios of 100,000:1 and 200:1, respectively as described above.
  • the transgene expression was analyzed 48 hrs post-infection using the FITC-X-Gal substrate and the PE- glycophorin marker as previously described.
  • the results shown in FIG. 5 indicated specific targeting of the erythroid population of primitive progenitor cells by the B 19 vector.
  • the AAV vector showed higher expression in the non-erythroid population of cells, an observation consistent with the inventors previously published studies (Wang et al, 1995).
  • the low transduction efficiency of the AAV vector in CD34 + cells is also consistent with the inventors recent studies documenting a wide variation in the level of transduction by these vectors.
  • CD34+ cells were first differentiated into erythroid and myeloid lineages by treatments with Epo and G-CSF, respectively, for 10 days followed by either mock-infection or infection with the recombinant AAV-lacZ or the B19-lacZ vectors precisely as described above. Forty-eight hrs post-infection, cells were analyzed for the transgene expression using the FITC-X-Gal substrate and PE-glycophorin for the erythroid population, and with the FITC-X-Gal substrate and PE-CD33 for the myeloid population, respectively. These results shown in FIG. 6, once again indicated that the recombinant B19- lacZ vector was highly efficient in selectively transducing the erythroid-differentiated population of CD34+ cells.
  • Recombinant parvoviral vectors were constructed. These recombinant vectors that contained the bacterial ⁇ -galactosidase (lacZ) gene under the control of the human cytomegalovirus (CMV) immediate-early promoter within AAV inverted terminal repeat sequences encapsidated either in AAV capsids (v AAV -lacZ) or in B19 capsids (vB19-/ ⁇ cZ). Both recombinant vectors were purified by cesium chloride density centrifugation and used to transduce HUVEC under identical conditions.
  • lacZ bacterial ⁇ -galactosidase
  • CMV human cytomegalovirus
  • Transduction of HUVEC with recombinant revealed a 5- to 9- fold higher transduction efficiency compared with recombinant vAAV-lacZ after 2 and 6 days, respectively.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention se rapporte au génie, à la propagation et à l'utilisation de vecteurs de parvovirus chimères. On peut utiliser ces vecteurs, qui utilisent des séquences provenant de virus associés aux adénovirus (AAV) et du B19, pour transporter des gènes à destination de diverses cellules cibles, y compris de celles faisant partie du lignage érythrocitaire.
EP98956097A 1997-10-08 1998-10-08 Systeme de vecteurs recombinants bases sur des parvovirus chimeres et specifiquement diriges sur le lignage erythrocitaire Withdrawn EP1027451A1 (fr)

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US7368546B2 (en) 2003-01-21 2008-05-06 The Board Of Regents Of The University Of Nebraska Human SAA3 nucleic acid molecule, protein, and methods of use for same
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