WO2002083902A2 - Adenovirus de serotype 30 (ad30) - Google Patents

Adenovirus de serotype 30 (ad30) Download PDF

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WO2002083902A2
WO2002083902A2 PCT/US2002/000362 US0200362W WO02083902A2 WO 2002083902 A2 WO2002083902 A2 WO 2002083902A2 US 0200362 W US0200362 W US 0200362W WO 02083902 A2 WO02083902 A2 WO 02083902A2
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region
cell
polypeptide
polynucleotide
fiber
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WO2002083902A3 (fr
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Beverly L. Davidson
Lane K. Law
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University of Iowa Research Foundation UIRF
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University of Iowa Research Foundation UIRF
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention provides adenovirus serotype 30 (Ad30) fiber proteins, such as the polypeptide encoded by SEQ ID NO: 1.
  • Ad30 fiber proteins such as the polypeptide encoded by SEQ ID NO: 1.
  • the present invention also provides a polynucleotide encoding such Ad30 fiber protein, such as the polynucleotide encoded by SEQ ID NO: 12.
  • the term "fiber protein” includes variants or biologically active or inactive fragments of this polypeptide.
  • a "variant" of the polypeptide is a fiber protein that is not completely identical to a native fiber protein.
  • a variant fiber protein can be obtained by altering the amino acid sequence by insertion, deletion or substitution of one or more amino acid.
  • the amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide.
  • the substitution may be a conserved substitution.
  • a "conserved substitution” is a substitution of an amino acid with another amino acid having a similar side chain.
  • a conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide retains its spacial conformation but has altered biological activity.
  • Glu is commonly used to substitute for other amino acids.
  • the 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains. Stryer, L. Biochemistry (2d edition) W. H. Freeman and Co. San Francisco (1981), p. 14-15; Lehninger, A. Biochemistry (2d ed., 1975), p. 73-75.
  • variant polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve biological activity. For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide that result in increased bioactivity.
  • amino acid substitutions in certain polypeptides may be used to provide residues that may then be linked to other molecules to provide peptide- molecule conjugates that retain sufficient properties of the starting polypeptide to be useful for other purposes.
  • hydropathic index of amino acids in conferring interactive biological function on a polypeptide, wherein it is found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity.
  • substitution of like amino acids may be made on the basis of hydrophihcity, particularly where the biological function desired in the polypeptide to be generated in intended for use in immunological embodiments.
  • the greatest local average hydrophihcity of a protein, as governed by the hydrophihcity of its adjacent amino acids correlates with its immunogenicity.
  • U.S. Patent 4,554,101 Accordingly, it is noted that substitutions can be made based on the hydrophihcity assigned to each amino acid. In using either the hydrophihcity index or hydropathic index, which assigns values to each amino acid, it is preferred to conduct substitutions of amino acids where these values are ⁇ 2, with ⁇ 1 being particularly preferred, and those with in ⁇ 0.5 being the most preferred substitutions.
  • the variant amino acid molecule of the present invention has at least 50%, at least about 80%, or even at least about 90% but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of a corresponding native nucleic acid molecule or polypeptide.
  • the amino acid sequence of the variant fiber protein corresponds essentially to the native fiber protein's amino acid sequence.
  • “corresponds essentially to” refers to a polypeptide sequence that will elicit a biological response substantially the same as the response generated by native fiber protein. Such a response may be at least 60% of the level generated by native fiber protein, and may even be at least 80%> of the level generated by native fiber protein.
  • a variant of the invention may include amino acid residues not present in the corresponding native fiber protein, or may include deletions relative to the corresponding native fiber protein.
  • a variant may also be a truncated "fragment" as compared to the corresponding native fiber protein, i.e., only a portion of a full-length protein.
  • the polypeptide of the present invention may contain one or more of the three regions of an Ad30 fiber, i.e., a tail region (such as amino acids 1-46 of SEQ ID NO:l), a shaft region (such as amino acids 47- 400 of SEQ ID NO:l) or a knob region (such as amino acids 401-582 of SEQ ID NO:l).
  • Fiber protein variants also include peptides having at least one D-amino acid.
  • the variant fiber protein of the present invention may be expressed from an isolated DNA sequence encoding the variant fiber protein.
  • the amino acid changes from the native to the variant fiber protein are achieved by changing the codons of the corresponding nucleic acid sequence.
  • "Recombinant” is defined as a peptide or nucleic acid produced by the processes of genetic engineering. It should be noted that it is well-known in the art that, due to the redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon, and still result in an identical amino acid sequence.
  • the terms “protein,” “peptide” and “polypeptide” are used interchangeably herein.
  • the Ad30 fiber protein as described above may be operably linked to an amino acid sequence for a therapeutic agent.
  • amino acid or nucleic acid is "operably linked" when it is placed into a functional relationship with another amino acid or nucleic acid sequence.
  • DNA a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked means that the amino acid or nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader in DNA, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • therapeutic agent refers to any agent or material that has a beneficial effect on the mammalian recipient.
  • therapeutic agent embraces both therapeutic and prophylactic molecules having nucleic acid or protein components.
  • the mammalian recipient may have a condition that is amenable to gene replacement therapy.
  • gene replacement therapy refers to administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ.
  • condition amenable to gene replacement therapy embraces conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (i.e., a pathological condition that is not attributable to an inborn defect), cancers and prophylactic processes (i.e., prevention of a disease or of an undesired medical condition) .
  • the mammalian recipient has a genetic disease and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the disease.
  • the mammalian recipient has an acquired pathology and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the pathology.
  • the patient has a cancer and the exogenous genetic material comprises a heterologous gene encoding an anti-neoplastic agent, hi yet another embodiment the patient has an undesired medical condition and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the condition.
  • the present invention also provides expression vectors containing an Ad backbone nucleic acid sequence and polynucleotide encoding a chimeric Ad fiber polypeptide comprising a tail region, a shaft region and a knob region, wherein at least one of these regions comprises an Ad30 tail region, an Ad30 shaft region or an Ad30 knob region.
  • the expression vector may also contain a nucleotide sequence encoding a therapeutic agent.
  • the present invention also provides viral particles and mammalian cells containing the expression vector described above.
  • the cell may be human, and may be from prostate, brain, breast, lung, spleen, kidney, heart, or liver. Alternatively, the cell may be a neuroprgenitor or stem cell.
  • the present invention also provides a method of transducing cells lacking CAR comprising contacting the cells with an expression vector or virus particle containing Ad backbone nucleic acid sequence and polynucleotide encoding a chimeric Ad fiber polypeptide comprising a tail region, a shaft region and a knob region, wherein at least one of these regions comprises an Ad30 tail region, an Ad30 shaft region or an Ad30 knob region.
  • the cell may be a neuronal or epithelial cell, such as a human umbilical vein epithelial cell (HUNEC), or may be a tumor cell.
  • HUNEC human umbilical vein epithelial cell
  • the present invention further provides a method of treating a genetic disease or cancer in a mammal by administering a polynucleotide, polypeptide, expression vector, or cell described above.
  • the genetic disease or cancer may be one of the diseases listed in Tables 1-3 below.
  • the invention relates to polypeptides that can be used as a therapeutic agent, and polynucleotides, expression vectors, virus particles and genetically engineered cells, and the use of them, for expressing the therapeutic agent.
  • the invention may be used as a method for gene therapy that is capable of both localized and systemic delivery of a therapeutically effective dose of the therapeutic agent.
  • a cell expression system for expressing a therapeutic agent in a mammalian recipient comprises a cell and an expression vector for expressing the therapeutic agent.
  • the expression vector further includes a promoter for controlling transcription of the heterologous gene.
  • the promoter may be an inducible promoter.
  • the expression system is suitable for administration to the mammalian recipient.
  • the expression system may comprises a plurality of non-immortalized genetically modified cells, each cell containing at least one recombinant gene encoding at least one therapeutic agent.
  • the cell expression system can be formed ex vivo or in vivo.
  • one or more isolated cells are transduced with a virus or transfected with a nucleic acid or plasmid in vitro.
  • the transduced or transfected cells are thereafter expanded in culture and thereafter administered to the mammalian recipient for delivery of the therapeutic agent in situ.
  • the genetically modified cell may be an autologous cell, i.e., the cell is isolated from the mammalian recipient.
  • the genetically modified cell(s) are administered to the recipient by, for example, implanting the cell(s) or a graft (or capsule) including a plurality of the cells into a cell-compatible site of the recipient.
  • a method for treating a mammalian recipient in vivo includes introducing an expression vector for expressing a heterologous gene product into a cell of the patient in situ.
  • an expression vector for expressing the therapeutic agent is introduced in vivo into target location of the mammalian recipient by, for example, intraperitoneal injection.
  • the expression vector for expressing the heterologous gene may include an inducible promoter for controlling transcription of the heterologous gene product. Accordingly, delivery of the therapeutic agent in situ is controlled by exposing the cell in situ to conditions that induce transcription of the heterologous gene.
  • a pharmaceutical composition comprising a plurality of the above- described genetically modified cells or polypeptides and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be for treating a condition amenable to gene replacement therapy and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the condition.
  • the pharmaceutical composition may contain an amount of genetically modified cells or polypeptides sufficient to deliver a therapeutically effective dose of the therapeutic agent to the patient. Exemplary conditions amenable to gene replacement therapy are described below.
  • a method for forming the above-described pharmaceutical composition includes introducing an expression vector for expressing a heterologous gene product into a cell to form a genetically modified cell and placing the genetically modified cell in a pharmaceutically acceptable carrier.
  • a cell graft is disclosed.
  • the graft comprises a plurality of genetically modified cells attached to a support that is suitable for implantation into the mammalian recipient.
  • the support may be formed of a natural or synthetic material.
  • an encapsulated cell expression system is disclosed.
  • the encapsulated expression system comprises a plurality of genetically modified cells contained within a capsule that is suitable for implantation into the mammalian recipient.
  • the capsule may be formed of a natural or synthetic material.
  • FIG. 3 Generation of chimeric virus, Ad5RSVeGFPf30.
  • a chimeric fiber sequence was generated containing the Ad5 tail and the Ad30 shaft and knob. This chimeric fiber was then cloned into the Ad5 backbone in place of the endogenous Ad5 fiber.
  • FIG. 4 Western blot analysis comparing viruses containing the endogenous Ad5 fiber to viruses containing the chimeric Ad5f30 fiber indicated that Ad5f30 indeed had a shorter fiber as compared to the endogenous fiber.
  • FIG. 5a FACS analysis indicating that Ad5RSVeGFP infected 93% and 95% > of A549 and HeLa cells respectively, whereas Ad5RSVeGFPf30 infected only 33% and 29% of A549 and HeLa cells respectively.
  • FIG. 5b Graph depicting that after A549 and HeLa cells were incubated with Ad5:CaPi and Ad5f30:CaPi coprecipitates, Ad5 infected 95% of A549 and HeLa cells, and Ad5f30 infected 85% and 88% of A549 and HeLa cells respectively.
  • Ad5 has been shown to infect cells via CAR.
  • 3T3 and CHO cells were used, as both cell types have been shown to express little if any CAR.
  • the graph of Figure 6 shows that Ad5 infected 3% and 0% of 3T3 and CHO cells respectively in the absence of CAR; Ad5f30 infected 1% of 3T3 and 0% of
  • Figure 7a Graph depicting the number of eGFP positive HUVECs infected with Ad5 and Ad5f30. A two-fold increase was seen in eGFP positive cells that had been infected with Ad5f30 as compared to Ad5.
  • Figure 7b To be certain that the results shown in Figure 7a did not indicate a difference in titer between the two viruses, both viruses were precipitated with CaPi and the virus:CaPi co-precipitant was used to infect HUVECs.
  • the graph in Figure 7b shows that after CaPi precipitation, over 90%> of the cells were eGFP positive with either virus.
  • Adenovirus has been shown to transduce a large number of cells including lung epithelial cells, muscle cell, endothelial cell, fibroblasts and neuronal cells.
  • the efficiency is variable due to the different levels of CAR expression.
  • Inefficient gene delivery into skeletal muscles, vascular smooth muscle, some endothelial cells and certain tumorogenic cells is a result of low expression of CAR.
  • Adenovirus tropism is the result of specific binding of the virus to the cell to be infected, by means of a cellular receptor.
  • the viral C-terminal portion of fiber or "knob” is responsible for specificity of receptor recognition by the virus.
  • Human coxsackievirus and adenovirus receptor (CAR) interacts with the fiber knob of several adenoviral serotypes (2, 4, 5, 9, 12, 15, 17, 19, 31, 41) (Bergelson, et al, (1997) Science 275:1320-1323; Roelvink, et al, (1998) J Virol. 72:7909-7915; Freimuth, et al, (1999) J. Virol. 73(2): 1392-1398; Wang, et al, (1999) J.
  • adenoviral serotypes have been shown to interact with CAR; however, there are some exceptions namely, Ad3 (Stevenson, et al, (1995). J. Virol. 69:2850-2857) and Ad35 (Shayakhmetov, et al, (2000) J. Virol. 74(6):2567-2583) that do not. It has been demonstrated that some D- serotype viruses utilize CAR 2. Ad30 was not among the serotypes tested. In order to improve the utility of recombinant adenoviral vectors by increasing the therapeutic index, a capsid exhibiting a higher efficiency of transduction was sought. Such a capsid could then be used in conjunction with a gutted adenoviral genome to promote long term transgene expression with minimal immune response.
  • adenoviral serotypes were screened for infection efficiency of primary fetal rat CNS cultures and human umbilical vein epithelial cells (HUVECs) (Chillon, et al, (1999) J. Virol. 73(3):2537-2540). It was determined that Subgroup D viruses exhibit enhanced gene transfer to both culture types. It was thought that this increase in efficiency when compared to Ad5 was due to differences between the viruses in their fiber protein amino acid sequence or length. Ad30 fibers are approximately one-third shorter than those of Ad5. The importance of this difference could be attributed to the two-step process of adenoviral infection.
  • Ad30 fibers may allow higher affinity interactions with cellular a_ integrins to occur. It was reasoned that such tropism could be passed to another adenovirus by replacement of its endogenous fiber sequence with that of the Ad30 fiber sequence.
  • Ad30 fiber protein was responsible for the increase in CNS and HUVEC tropism, it was decided to replace the endogenous fiber sequence of Ad5 with that of Ad30.
  • the ability of the Ad5 genome to tolerate such changes has been demonstrated by several groups (Shayakhmetov, et al, (2000) J Virol. 74(6):2567-2583;Crompton, et al, (1994) J Gen. Virol. 75:133-139; Gall, et al, (1996) J.
  • Ad30 like other adenoviral serotypes (2, 4, 5, 9, 12, 15, 17, 19, 31, 37?, 41) (Bergelson, et al, (1997) Science 275:1320-1323; Roelvink, et al, (1998) J. Virol. 72:7909- 7915; Freimuth, et al, (1999) J. Virol. 73(2): 1392-1398; Wang, etal, (1999) J Virol. 73(3):2559-2562; Zabner, et al, (1999) J. Virol 73(10):8689-8695) could bind CAR.
  • Ad30 was propagated and viral particles were purified. Once purified particles were available, genomic DNA could be isolated and sequence data generated.
  • the fiber gene was sequenced using degenerate primers based on other D-serotype fiber sequences followed by specific primers as the Ad30 sequence data was generated. It was then possible to create a chimeric fiber protein consisting of Ad5 tail and Ad30 shaft/knob by overlapping PCR. This chimeric fiber protein was cloned into the Ad5 backbone replacing the endogenous Ad5 fiber by means of homologous recombination in E coli. A chimeric virus was thus developed that also expressed the reporter gene eGFP. Once propagated this virus was compared to normal Ad5 expressing eGFP in infection studies of cultured cells. It was found that the chimeric virus was less efficient in transduction of CAR positive cells but more efficient in transducing HUVECs.
  • Ad30 fiber gene needed to be cloned.
  • Ad30 genomic DNA was isolated and the fiber gene was amplified by means of degenerate primers based on known D serotype fiber sequences. As sequence data was acquired further specific primers were designed and employed until the entire nucleic acid sequence of the Ad30 fiber gene was known (SEQ ID NO: 12).
  • SEQ ID NO: 1 The amino acid sequence of Ad30 (SEQ ID NO: 1) is shown in Figure 1.
  • Ad30 fiber is quite similar to Ad9 (SEQ ID NO:2) and Adl7 (SEQ ID NO:3), is less similar to Ad3 (SEQ ID NO:4), and is the least similar to Ad5 (SEQ ID NO:5), as shown in Figure 1.
  • This comparison was important in light of recent discoveries regarding the amino acid residues present in the Ad5 knob that are important for binding to CAR (Roelvink, et al, (1999) Science 286:1568-1571; Kirby, et ⁇ /., (2000) J. Virol. 74(6):2804-2813). It was found that Ad30 shares 25%) overall identity of amino acid residues with Ad5 in the fiber protein.
  • Ad30 fiber could be responsible for this difference, a chimeric virus, Ad5RSVeGFPf30, was generated by means of homologous recombination in BJ5183 cells (Anderson, et al, (2000) Gene Ther. 7(12):1034-1038). A chimeric fiber gene was created by overlapping PCR. The Ad5 tail was amplified, as were the A30 shaft and knob. These products were then combined in the second round of PCR amplification to yield a chimeric fiber gene.
  • This gene was cloned into the Ad5 backbone in place of the endogenous Ad5 fiber as depicted in Figure 3.
  • the plasmid containing the chimeric Ad5f30 genome was subjected to sequence analysis to be certain that the chimeric fiber gene was correctly cloned.
  • Western blot analysis of both viruses indicated that Ad5f30 indeed had a shorter fiber corresponding to the appropriate size ( Figure 4).
  • This plasmid was then linearized by restriction digest and transfected into HEK293 cells to generate virus as previously described (Anderson, et al, (2000) Gene Ther. 7(12):1034-1038).
  • Ad5f30 generated CPE much slower than Ad5 (45 hrs. vs. 30 hrs.) and the total particles isolated for Ad5f30 was ⁇ 3 fold less than that for Ad5.
  • plaque assay on HEK293 cells infected with either virus and incubated over several days was performed.
  • the titer of Ad5f30 was 2.5 ' 10 9 pfu/ml and Ad5 was 2 ' 10 10 pfu/ml.
  • titers were performed with viral-CaPi co-precipitants.
  • Ad5 was more efficient in infecting A549 and HeLa cells than the chimeric vector, Ad5f30.
  • A549 cells and HeLa cells were incubated in the presence of 5000 particles per cell of each virus for 1 hour at 37° C. Viral particles were then removed, the cells were washed and then incubated an additional 24 hrs. at 37° C. FACS analysis indicated that Ad5RSVeGFP infected 93% and 95% of A549 and HeLa cells respectively (Fig. 5a.).
  • Ad5RSVeGFPf30 infected only 33% and 29% of A549 and HeLa cells respectively (Fig. 5a.).
  • both adenoviral vectors were precipitated with calcium phosphate (CaPi) (Fasbender, et al, (1998) JClin.Invest. 102(1): 184-193). Such precipitation of adenoviral vectors has been shown to ameliorate fiber dependent cell entry (Fasbender, et al, (1998) JClinJnvest. 102(1): 184- 193).
  • Ad5:CaPi and Ad5f30:CaPi coprecipitates for 30 min at 37°
  • Ad5 infected 95% of A549 and HeLa cells Fig.
  • Ad5f30 infected 85% and 88% of A549 and HeLa cells respectively (Fig. 5b.). These results demonstrate that the chimeric fiber of Ad5f30 is responsible for the difference in tropism initially seen between Ad5 and Ad5f30 as both viruses were shown to be nearly equal in infectivity once the requirement for the Ad5 fiber was removed. The difference in infection efficiencies without CaPi precipitation of the two viruses for A549 and HeLa cells suggested that Ad5f30 infected via a different pathway than that used by Ad5.
  • Ad5 has been shown to infect cells via CAR (Bergelson, et al, (1997) Science 275:1320-1323; Roelvink, et al, (1998) J. Virol. 72:7909-7915; Freimuth, et al, (1999) J. Virol. 73(2):1392-1398; Wang, et al, (1999) J. Virol. 73(3):2559-2562; Zabner, et al, (1999) J. Virol. 73(10):8689-8695).
  • the present inventors obtained, amplified and purified Ad30 wild- type particles. Genomic DNA from these particles was isolated and used to obtain the sequence of the Ad30 fiber protein by means of degenerate primers. When the Ad30 fiber sequence was compared to that of Ad5 fiber significant differences were evident. The shaft of the Ad30 fiber is less than half the length of the Ad5 fiber. Of note also, is the fact that of the seven amino acids shown to be important for CAR binding Ad30 lacks all but two. Those amino acids, conserved between Ad5 and Ad30 fibers, are in the hinge region between shaft and knob and are well conserved among most adenoviral serotypes.
  • Ad30 most likely does not use CAR as its primary receptor. Both viruses were used to infect a number of cell types. Ad5f30 was less efficient in infecting A549 and HeLa cells. A coprecipitant of Ad5f30 and CaPi, however, was very nearly equal in infection efficiency to a coprecipitant of Ad5:CaPi. These results indicated that the chimeric virus was viable and that the differences in infection efficiency were due to the different fibers present on the viral capsids. Both viruses were also tested in 3T3 and CHO cells in the presence or absence of CAR. It is evident from those experiments that the presence of CAR may not play a role in the infection efficiency of Ad5f30.
  • Ad5f30 was two-fold that of Ad5 at all time-points and particle concentrations when the HUVECs are confluent. As confluence of the HUVECs decreases so does the infection efficiency of Ad5f30 relative to that of Ad5. It is possible that in context of the shorter Ad30 fiber the Ad5 penton is better able to mediate viral entry. It is also possible that Ad5f30 uses another receptor present on the cell surface of the HUVEC.
  • fiber protein is responsible for the tropism and infection efficiency exhibited by a virus. Replacement of an endogenous fiber with that of a different serotype alters its infection profile. These fiber proteins are useful for various research and clinical applications.
  • adenoviruses are useful vectors for basic research and for clinical applications. When used in delineating protein function, vectors that contain a given transgene with mutations or alterations to the coding sequence are compared at the same time.
  • Adenoviruses can be made by standard transfection of a shuttle plasmid and viral DNA backbone into HEK 293 cells. Homologous recombination occurs in vivo, and recombinant virus can be isolated and propagated. The major drawback of this technique is that the starting viral DNA backbone, restricted of El containing sequences, must be 100%) free of full-length Ad DNA. Otherwise, varying amounts of wild-type virus are also propagated.
  • adenoviruses can be made via the streamlined method set forth in U.S. Patent Application Serial No. 09/521,524 and in Anderson, et al, (2000) Gene Ther. 7(12):1034-1038.
  • the present invention provides methods of treating a genetic disease or cancer in a mammal by administering a polynucleotide, polypeptide, expression vector, viral particle or cell.
  • a person having ordinary skill in the art of molecular biology and gene therapy would be able to determine, without undue experimentation, the appropriate dosages and routes of administration of the a polynucleotide, polypeptide, expression vector, viral particle or cell used in the novel methods of the present invention.
  • the instant invention provides a cell expression system for expressing exogenous genetic material in a mammalian recipient.
  • the expression system also refened to as a "genetically modified cell,” comprises a cell and an expression vector for expressing the exogenous genetic material.
  • the genetically modified cells are suitable for administration to a mammalian recipient, where they replace the endogenous cells of the recipient.
  • the preferred genetically modified cells are non-immortalized and are non- tumorogenic.
  • the cells are transformed or otherwise genetically modified ex vivo.
  • the cells are isolated from a mammal (such as a human), transformed (i.e., transduced or transfected in vitro) with a vector for expressing a heterologous (e.g., recombinant) gene encoding the therapeutic agent, and then administered to a mammalian recipient for delivery of the therapeutic agent in situ.
  • the mammalian recipient may be a human and the cells to be modified are autologous cells, i.e., the cells are isolated from the mammalian recipient.
  • the cells are transformed or otherwise genetically modified in vivo.
  • the cells from the mammalian recipient are transformed (i.e., transduced or transfected) in vivo with a vector containing exogenous genetic material for expressing a heterologous (e.g., recombinant) gene encoding a therapeutic agent and the therapeutic agent is delivered in situ.
  • exogenous genetic material refers to a nucleic acid or an oligonucleotide, either natural or synthetic, that is not naturally found in the cells; or if it is naturally found in the cells, it is not transcribed or expressed at biologically significant levels by the cells.
  • exogenous genetic material includes, for example, a non-naturally occurring nucleic acid that can be transcribed into anti-sense RNA, as well as a "heterologous sequence” (i.e., a sequence encoding a protein that is not expressed or is expressed at biologically insignificant levels in a naturally-occurring cell of the same type).
  • EPO erythropoietin
  • exogenous genetic material is the introduction of only part of a genetic sequence to create a recombinant sequence, such as combining an inducible promoter with an endogenous coding sequence via homologous recombination.
  • the mammalian recipient has a condition that is amenable to gene replacement therapy.
  • gene replacement therapy refers to administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ.
  • condition amenable to gene replacement therapy embraces conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (i.e., a pathological condition that is not attributable to an inborn defect), cancers and prophylactic processes (i.e., prevention of a disease or of an undesired medical condition).
  • therapeutic agent refers to any agent or material that has a beneficial effect on the mammalian recipient.
  • therapeutic agent embraces both therapeutic and prophylactic molecules having nucleic acid (e.g., antisense RNA) and/or protein components.
  • the condition amenable to gene replacement therapy alternatively can be a genetic disorder or an acquired pathology that is manifested by abnormal cell proliferation, e.g., cancers.
  • the instant invention is useful for delivering a therapeutic agent having anti-neoplastic activity (i.e., the ability to prevent or inhibit the development, maturation or spread of abnormally growing cells), to primary or metastasized tumors, (e.g., ovarian carcinoma, mesothelioma, colon carcinoma).
  • therapeutic agents for treating these and other cancers include, for example, the anti-neoplastic agents provided in Table 3.
  • Emphysema a -antitrypsin
  • Peritoneal sclerosis Fibrinolytic agents e.g., tissue plasminogen activator (t-PA), or single chain urokinase plasminogen activator (scu-PA)
  • Peritonitis Anti-oxidants e.g., Superoxide Dismutase
  • Septic Shock Anti-tl rombotic agents e.g., elastase-resistant form of thrombomodulin (TM)
  • TM thrombomodulin
  • Diabetes mellitus Insulin Pituitary Dwarfism Human growth honnone Thrombosis Hiradin, secreted form of TM Post-Surgical Adhesions
  • Anti-tlirombotic agents e.g., thrombomodulin, hirudin
  • Fibrinolytic agents e.g., TPA, scu-PA
  • Surfactants e.g., TPA, scu-PA
  • a therapeutic agent by a genetically modified cell is not limited to delivery to a particular location in the body in which the genetically modified cells would normally reside.
  • a therapeutic agent secreted by a genetically modified cell within a coelomic cavity could reach the lymphatic network draining that coelomic cavity.
  • the genetically modified cells of the invention are useful for delivering a therapeutic agent, such as an anti-neoplastic agent, to various parts of the body.
  • the condition amenable to gene replacement therapy is a prophylactic process, i. e. , a process for preventing disease or an undesired medical condition.
  • the instant invention embraces a cell expression system for delivering a therapeutic agent that has a prophylactic function (i.e., a prophylactic agent) to the mammalian recipient.
  • a prophylactic function i.e., a prophylactic agent
  • therapeutic agents include: estrogen/progesterone (pregnancy); thyroxine
  • hypothyroidsm hyperthyroidsm
  • agents that stimulate e.g., gamma-interferon, or supplement, e.g., antibodies
  • the immune system response diseases associated with deficiencies of the immune system.
  • the term "therapeutic agent” includes, but is not limited to, the agents listed in Tables 1-3, as well as their variants or functional equivalents.
  • the term “functional equivalent” refers to a molecule (e.g., a peptide or protein) that has the same or an improved beneficial effect on the mammalian recipient as the therapeutic agent of which is it deemed a functional equivalent. Accordingly, the instant invention embraces therapeutic agents encoded by naturally-occuning DNAs, as well as by non-naturally-occurring
  • the exogenous genetic material e.g., a cDNA encoding one or more therapeutic proteins
  • the exogenous genetic material is introduced into the cell ex vivo or in vivo by genetic transfer methods, such as transfection or transduction, to provide a genetically modified cell.
  • Various expression vectors i.e., vehicles for facilitating delivery of exogenous genetic material into a target cell
  • transfection of cells refers to the acquisition by a cell of new genetic material by incorporation of added DNA.
  • transfection refers to the insertion of nucleic acid into a cell using physical or chemical methods.
  • Several transfection techniques are known to those of ordinary skill in the art including: calcium phosphate DNA co-precipitation (Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Ed. E. J. Murray, Humana Press (1991)); DEAE-dextran (supra); electroporation (supra); cationic liposome-mediated transfection (supra); and tungsten particle-faciliated microparticle bombardment (Johnston, S. A., Nature 346:116-111 (1990)).
  • Strontium phosphate DNA co-precipitation (Brash D. E. et al. Molec. Cell. Biol. 7:2031-2034 (1987) is an alternative transfection method.
  • transduction of cells refers to the process of transferring nucleic acid into a cell using virus.
  • a cell that has been transduced with a chimeric DNA virus e.g., a adenovirus carrying a cDNA encoding a therapeutic agent
  • a chimeric DNA virus e.g., a adenovirus carrying a cDNA encoding a therapeutic agent
  • the exogenous genetic material includes the heterologous gene
  • the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity.
  • enhancers is simply any non-translated DNA sequence that works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the exogenous genetic material may introduced into the cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence.
  • An expression vector may include an exogenous promoter element to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.
  • constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth.
  • exemplary constitutive promoters include the promoters for the following genes that encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al, Proc. Natl. Acad. Sci.
  • any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert.
  • inducible promoters Genes that are under the control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions).
  • Inducible promoters include responsive elements (REs) that stimulate transcription when their inducing factors are bound.
  • REs responsive elements
  • Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene.
  • the appropriate promoter constitutive versus inducible; strong versus weak
  • delivery of the therapeutic agent in situ is triggered by exposing the genetically modified cell in situ to conditions for permitting transcription of the therapeutic agent, e.g., by intraperitoneal injection of specific inducers of the inducible promoters that control transcription of the agent.
  • in situ expression by genetically modified cells of a therapeutic agent encoded by a gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducing) metal ions in situ.
  • the amount of therapeutic agent that is delivered in situ is regulated by controlling such factors as: (1) the nature of the promoter used to direct transcription of the inserted gene, (i. e. , whether the promoter is constitutive or inducible, strong or weak); (2) the number of copies of the exogenous gene that are inserted into the cell; (3) the number of transduced/transfected cells that are administered (e.g., implanted) to the patient; (4) the size of the implant (e.g., graft or encapsulated expression system); (5) the number of implants; (6) the length of time the transduced/transfected cells or implants are left in place; and (7) the production rate of the therapeutic agent by the genetically modified cell. Selection and optimization of these factors for delivery of a therapeutically effective dose of a particular therapeutic agent is deemed to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the above-disclosed factors and the clinical profile of the patient.
  • the expression vector may also include a selection gene, for example, a neomycin resistance gene, for facilitating selection of cells that have been transfected or transduced with the expression vector.
  • a selection gene for example, a neomycin resistance gene
  • the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the therapeutic agent(s), the other vector containing a selection gene.
  • a suitable promoter, enhancer, selection gene and/or signal sequence (described below) is deemed to be within the scope of one of ordinary skill in the art without undue experimentation.
  • the therapeutic agent can be targeted for delivery to an extracellular, intracellular or membrane location. If it is desirable for the gene product to be secreted from the cells, the expression vector is designed to include an appropriate secretion "signal" sequence for secreting the therapeutic gene product from the cell to the extracellular milieu.
  • the expression vector can be constructed to include "retention" signal sequences for anchoring the therapeutic agent within the cell plasma membrane.
  • retention signal sequences for anchoring the therapeutic agent within the cell plasma membrane.
  • all membrane proteins have hydrophobic transmembrane regions that stop translocation of the protein in the membrane and do not allow the protein to be secreted.
  • the construction of an expression vector including signal sequences for targeting a gene product to a particular location is deemed to be within the scope of one of ordinary skill in the art without the need for undue experimentation.
  • the instant invention has utility as an expression system suitable for detoxifying intra- and/or extracellular toxins in situ.
  • the therapeutic agent can be targeted for delivery to the extracellular milieu, to the cell plasma membrane or to an intracellular location.
  • the exogenous genetic material containing a gene encoding an intracellular detoxifying therapeutic agent further includes sequences encoding surface receptors for facilitating transport of extracellular toxins into the cell where they can be detoxified intracellularly by the therapeutic agent.
  • the cells can be genetically modified to express the detoxifying therapeutic agent anchored within the cell plasma membrane such that the active portion extends into the extracellular milieu. The active portion of the membrane-bound therapeutic agent detoxifies toxins that are present in the extracellular milieu.
  • the instant invention also embraces agents intended for delivery to the extracellular milieu and/or agents intended to be anchored in the cell plasma membrane.
  • the selection and optimization of a particular expression vector for expressing a specific gene product in an isolated cell is accomplished by obtaining the coding sequence, such as with one or more appropriate control regions (e.g., promoter, insertion sequence); preparing a vector construct comprising the vector into which is inserted the coding sequence; transfecting or transducing cultured cells in vitro with the vector construct; and detennining whether the gene product is present in the cultured cells.
  • appropriate control regions e.g., promoter, insertion sequence
  • the adenovirus is used as an expression vector for transformation of cells.
  • the adenovirus is frequently responsible for respiratory tract infections in humans and thus appears to have an avidity for the epithelium of the respiratory tract (Straus, S., The Adenovirus, H. S. Ginsberg, Editor, Plenum Press, New York, P. 451-496 (1984)).
  • the adenovirus is infective in a wide range of cell types, including, for example, muscle and endothelial cells (Larrick, J. W. and Burck, K. L., Gene Therapy. Application of Molecular Biology, Elsevier Science Publishing Co., Inc., New York, p. 71-104 (1991)).
  • the adenovirus also has been used as an expression vector in muscle cells in vivo (Quantin, B., et al, Proc. Natl. Acad. Sci. USA 89:2581-2584 (1992)).
  • the adenovirus genome is adaptable for use as an expression vector for gene therapy, i.e., by removing the genetic information that controls production of the virus itself (Rosenfeld, M. A., et al, Science 252:431434 (1991)). Because the adenovirus functions in an extrachromosomal fashion, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis.
  • the instant invention also provides various methods for making and using the above-described genetically-modified cells. In particular, the invention provides a method for genetically modifying cell(s) of a mammalian recipient ex vivo and administering the genetically modified cells to the mammalian recipient.
  • the cells are autologous cells, i.e., cells isolated from the mammalian recipient.
  • autologous cells i.e., cells isolated from the mammalian recipient.
  • isolated means a cell or a plurality of cells that have been removed from their naturally-occurring in vivo location. Methods for removing cells from a patient, as well as methods for maintaining the isolated cells in culture are known to those of ordinary skill in the art.
  • the instant invention also provides methods for genetically modifying cells of a mammalian recipient in vivo.
  • the method comprises introducing an expression vector for expressing a heterologous gene product into cells of the mammalian recipient in situ by, for example, injecting the vector into the recipient.
  • the preparation of genetically modified cells contains an amount of cells sufficient to deliver a therapeutically effective dose of the therapeutic agent to the recipient in situ.
  • the detennination of a therapeutically effective dose of a specific therapeutic agent for a known condition is within the scope of one of ordinary skill in the art without the need for undue experimentation.
  • one of ordinary skill would consider the condition of the patient, the severity of the condition, as well as the results of clinical studies of the specific therapeutic agent being administered.
  • genetically modified cells are not already present in a pharmaceutically acceptable carrier they are placed in such a carrier prior to administration to the recipient.
  • pharmaceutically acceptable carriers include, for example, isotonic saline and other buffers as appropriate to the patient and therapy.
  • the genetically modified cells are administered by, for example, intraperitoneal injecting or implanting the cells or a graft or capsule containing the cells in a target cell-compatible site of the recipient.
  • target cell-compatible site refers to a structure, cavity or fluid of the recipient into which the genetically modified cell(s), cell graft, or encapsulated cell expression system can be implanted, without triggering adverse physiological consequences. More than one recombinant gene can be introduced into each genetically modified cell on the same or different vectors, thereby allowing the expression of multiple therapeutic agents by a single cell.
  • the instant invention further embraces a cell graft.
  • the graft comprises a plurality of the above-described genetically modified cells attached to a support that is suitable for implantation into a mammalian recipient.
  • the support can be formed of a natural or synthetic material.
  • an encapsulated cell expression system includes a capsule suitable for implantation into a mammalian recipient and a plurality of the above-described genetically modified cells contained therein.
  • the capsule can be formed of a synthetic or naturally-occurring material. The formulation of such capsules is known to one of ordinary skill in the art.
  • the encapsulated cells remain isolated (i.e., not in direct physical contact with the site) following implantation.
  • the encapsulated system is not limited to a capsule including genetically-modified non-immortalized cells, but may contain genetically modified immortalized cells. The following examples are intended to illustrate but not limit the invention.
  • ATCC modified Eagle medium
  • FCS fetal calf serum
  • A549 (ATCC) cells were maintained in RPMI 1640 medium-10% FCS-1%> glutamine-penicillin-streptomycin.
  • Human embryonic kidney-293 (HEK293) cells were maintained in DMEM-10%> FCS-1% glutamine-penicillin-streptomycin, as were NIH3T3 (ATCC) cells.
  • Chinese hamster ovary (CHO) (ATCC) cells were maintained in DMEM F12 medium supplemented with 1% penicillin-streptomycin and 10%> FCS.
  • Human umbilical vein epithelial cells were harvested from umbilical chords and then maintained in M199 medium-20% FCS-1% BME vitamins-2% BME amino acids-1% glutamine-penicillin-streptomycin on plates previously coated with fibronectin (Sigma F-0895, St. Louis, MO).
  • Ad30 VR-273 was purchased from the ATCC and subsequently amplified by infection of 293 cells. Viral particles were banded in CsCl gradients, dialyzed, and stored in 100 ml aliquots at -80° C.
  • Ad5CMVhCAR produced by the University of Iowa Gene Transfer Vector Core, was a kind gift from Dr. Mike Welsh, University of Iowa.
  • Viral DNA from purified Ad30 particles was isolated by standard protease treatment and ethanol precipitation methods.
  • Degenerate primers to the 5' and 3' ends of the fiber gene were designed by means of comparison of the known sequences of four'D-serotype viruses, adenovirus types 8, 9,15 and 17. They are 5 '-CGGGATCCGCCACCATGTCAAAGAGGCTCCGG-3 ' (AdDfiberF) (SEQ ID NO:6) and 5'-
  • Ad5 The endogenous fiber sequence of Ad5 (nt 31042 to 32787) was replaced with Ad30 sequence by overlapping PCR.
  • the Ad30 fiber was amplified such that it contained the Ad5 tail (nt 31042 to 31189, the first 147 nts.).
  • Overlapping primers specific for the tail/shaft boundary containing 18 base-pairs of Ad5 and 18 base-pairs of Ad30 sequence were generated.
  • two DNA fragments corresponding to the Ad5 tail region (nt 1 to 147 - nt 31042 to 31189) using primers 5 ' -
  • Primers to the 5 ' and 3 ' ends were designed to incorporate restriction enzyme recognition sites, BamHl and Spel respectively.
  • the Ad5 tail and Ad30 shaft/knob products were purified by agarose gel electrophoresis, mixed together and used as a template for the second phase of the overlapping PCR reaction, using Ad5forBamHl and Ad30fRevSpel to amplify the entire chimeric 5/30 fiber.
  • the 1119-bp-long chimeric 5/30 fiber product containing the Ad5 tail and the Ad30 shaft and knob domains, was purified by agarose gel electrophoresis, digested with Ndel and Spel and ligated into a plasmid containing bases 29509 - 33096 of the Ad5 genome, pBS-5/30.
  • This plasmid linearized by Notl and BamHl, and pTG3602/RSV-eGFP/Swal (Xia, et al, (2000) J. Virol. 74(23):11359-11366), linearized by Swal to drive homologous recombination in the region of fiber, were used to co-transform the RecA + E.
  • Lysates of Ad5RSV-eGFPf30 were used to infect a twenty plates of 293 cells. CPE was evident 40 hours post-infection. Virus was harvested and purified by standard methods as described previously. The control virus, Ad5RSV-eGFP, with non-recombinant fiber was similarly generated.
  • Ad5RSVeGFP and Ad5RSVeGFPf30 (2xl0 10 particles) were boiled at 95° C. for 15 min in Laemli buffer and fractionated by SDS-PAGE. Proteins were transfened to nitrocellulose membranes, blocked with 5% skim milk in PBS-0.1% Tween for 1 hr. at RT, and incubated with a monoclonal antibody to the N-terminus of Ad5 (4D2.5 was a gift of Jeffrey Engler) (Mullis, et al, (1990) J. Virol. 64(11):5317-5323), diluted 1 to 2500 inPBS-0.1% Tween overnight at 4° C. The membrane was then washed 4x5 min.
  • HeLa, A549, HUVEC, CHO and 3T3 cells were infected with both viral serotypes.
  • HeLa and A549 cells were plated 24 hrs. previous to infection at a density of 3x10 5 cells per 60mm dish.
  • HUVECs were plated at a density of ⁇ lxl0 6 per 60mm dish, which had been previously coated with fibronectin 24 hrs. prior to infection.
  • All media was removed from the cells and replaced with 1ml of fresh media containing 5000 pt./cell of the virus to be tested. Cells were incubated in the presence of virus for 1 hr. at 37° C.
  • CHO cells and 3T3 cells were plated and prepared in a similar manner; however, a total of 500 particles per cell was used and the cells were incubated with the virus for only 30 minutes.
  • Example 6 CaPi mediated infections Four ml of lxl 0 12 pts/ml Ad5RSV-eGFP or Ad5RSV-eGFPf30 were added to one ml of MEM, vortexed lightly and then precipitated by the addition of 25ml of 1M CaCl 2 , lightly vortexed and then incubated at RT for 20 min. Media was removed from 60mm dishes of A549, HeLa and HUVECs, prepared as above and 1 ml of MEM containing the Ad-CaPi precipitant was added to each dish. Cells were incubated with the viral precipitant at a concentration of 500 particles/cell for 30 minutes at 37° C, washed with fresh media, and then incubated with 3 mis fresh media for an additional 24 hrs at 37° C prior to FACs analysis.
  • CHO and 3T3 cells were transfected with Ad5CMVhCAR previously prepared with CaPi as described above. Twenty four hours after CAR transfection, cells were infected with 500 particles/cell of either virus for 1 half hour at 37° C. Cells were washed and incubated an additional 24 hrs. at 37° C before FACs analysis.
  • Infected cells were detached from dishes by incubation with Trypsin for 5 min at 37° C, spun down, resuspended in media with propidium iodide (PPI) added, and subjected to FACs analysis for the expression of eGFP. All analyses were performed on a Becton Dickinson flow cytometer (San Jose, CA) equipped with a 488-nm ion argon laser.
  • PPI propidium iodide
  • Those cells analyzed for CAR expression were detached from dishes with EDTA, spun down and resuspended in 1%> FBS/PBS at 2xl0 5 cells/ml and incubated with monoclonal antibodies (mAbs) against CAR (RmcB) (Bergelson, et al, (1997) Science 275: 1320-1323; Hsu, . et al, (1988) J. Virol. 62(5): 1647- 1652) for 45 min at 37° C. These cells were then spun down, washed and resuspended as previously with R-phycoerytluin-conjugated goat anti-mouse secondary antibodies, Jackson ImmunoResearch 115-116-146, for .45 min. at 4° C. Cells were then subjected to FACs analysis as above.
  • the coxsackivirus- adenovirus receptor protein can function as a cellular attachment protein for adenovirus serotypes from subgroups A, C, D, E, and F. J. Virol. 72:7909-7915.
  • HCAR and MCAR The human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc.Natl.Acad.Sci. U.S.A. 94:3352-3356.
  • the murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses. J. Virol. 72:415-419.
  • Adenovirus type 5 and 7 capsid chimera Fiber replacement alters receptor tropism without affecting primary immune neutralization epitopes. J. Virol 70:2116-2123.

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Abstract

La présente invention concerne une séquence d'acides aminés d'une fibre d'adénovirus de sérotype 30 (Ad30). La présente invention concerne également des polynucléotides et des vecteurs d'expression codant une fibre Ad30 et des particules virales et des cellules renfermant ces vecteurs d'expression. La présente invention concerne également des méthodes de traitement de maladies génétiques ou de cancers chez un mammifère, faisant intervenir les polynucléotides, polypeptides, vecteurs d'expression, particules virales et cellules de la présente invention.
PCT/US2002/000362 2001-01-09 2002-01-09 Adenovirus de serotype 30 (ad30) Ceased WO2002083902A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111251A3 (fr) * 2003-06-11 2005-03-03 Scripps Research Inst Proteines fibreuses modifiees pour une liaison efficace a un recepteur
FR2860004A1 (fr) * 2003-09-18 2005-03-25 Roussy Inst Gustave Nouveau vecteur adenoviral pour l'infection de cellules deficientes ou depourvues en recepteurs car
CN107058359A (zh) * 2017-04-14 2017-08-18 北京交通大学 一种抗呼吸道合胞病毒药物的高通量筛选方法和应用

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US5877011A (en) * 1996-11-20 1999-03-02 Genzyme Corporation Chimeric adenoviral vectors
US20030017138A1 (en) * 1998-07-08 2003-01-23 Menzo Havenga Chimeric adenoviruses

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111251A3 (fr) * 2003-06-11 2005-03-03 Scripps Research Inst Proteines fibreuses modifiees pour une liaison efficace a un recepteur
FR2860004A1 (fr) * 2003-09-18 2005-03-25 Roussy Inst Gustave Nouveau vecteur adenoviral pour l'infection de cellules deficientes ou depourvues en recepteurs car
WO2005026337A3 (fr) * 2003-09-18 2005-07-28 Roussy Inst Gustave Vecteur adenoviral pour l’infection de cellules deficientes en recepteur car
CN107058359A (zh) * 2017-04-14 2017-08-18 北京交通大学 一种抗呼吸道合胞病毒药物的高通量筛选方法和应用

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