Columbia University

Dana-Farber-Cancer Institute Inc.

Stephen P Goff

Naoko Tanese

Ganjam V Kalpana

Arthur Bank

Dina G Markowitz

William A Haseltine

Jeremy Luban

Liang Tong

Xingqiang Li

Guangxia Gao

Monica J Roth

Yiping Zhu

Shukun Luo

Gilda Tachedjian

Increased viral particle maturation and production can be achieved in various methods for producing viral particles from viral proteins, in general, by inhibiting or preventing Heme Oxygenase 2 (HO-2) from binding to the group-specific antigen (Gag) of the viral proteins, thus allowing delivery of the viral proteins to plasma membranes where they can replicate and mature without interference from HO-2. The increase in viral particle maturation and production can also be achieved by minimizing or eliminating the presence of HO-2 to thus reduce or prevent binding of HO-2 to the group-specific antigen (Gag) of the viral proteins. The invention is particularly applicable to the production of lentiviruses from viral proteins wherein the Matrix domain (MA) of the Gag is myristoylated.

Method for significantly increasing lentiviral production

This invention provides methods of determining whether a compound inhibits HIV-1 reverse transcriptase. This invention provides methods of determining whether a compound inhibits formation of a complex between a p66 and p51 subunit polypeptides of HIV-1 reverse transcriptase. This invention provides a method of determining whether a compound enhances formation of a complex between a p66 and p51 subunit polypeptides of HIV-1 reverse transcriptase. This invention provides methods of determining whether a compound inhibits formation of a complex between two p66 subunit polypeptides of HIV-1 reverse transcriptase. This invention provides methods of determining whether a compound enhances formation of a complex between two p66 subunit polypeptides of HIV-1 reverse transcriptase.

Two hybrid assay that detects HIV-1 reverse transcriptase dimerization

This invention provides isolated nucleic acid molecules encoding envelope-interacting protein-1 and envelope-interacting protein-3. This invention provides fusion proteins comprising EIP-1, EIP-3, or fragments thereof and a second peptide. This invention provides vectors comprising the isolated nucleic acid molecule, encoding mammalian EIP-1 and EIP-3. This invention provides plasmid designated pCGN-EIP-l and pCGN-EIP-3. This invention provides purified mammalian EIP-1 and EIP-3 proteins. This invention provides monoclonal or polyclonal antibodies directed to epitopes of an EIP-1 or an EIP-3. This invention provides a method of increasing transduction efficieny of a retrovirus on target cells comprising: a) incubating an envelope-interacting protein with a retrovirus; and b) transducing the target cells with the retrovirus. This invention provide methods of treating patient with a therapeutic gene comprising use of transducing viruses incubated with an effective amount of an envelope-interacting protein effective to enhance retroviral infectivity. This invention also provides pharmaceutical composition comprising an envelope-interacting protein bound retroviral virion comprising a therapeutic gene and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

EIP-1 and EIP-3 genes, envelope-interacting proteins, EIP-1 and EIP-3

This invention provides a mammalian cell useful for retroviral packaging comprising two plasmids, both of which comprise the 5&prime; long terminal repeat (LTR) sequence from a helper virus, neither of which comprise a functional &psgr; packaging sequence or a 3&prime; LTR from the helper virus, one of which comprises the env gene from the helper virus and the other of which comprises the gag and pol genes from the helper virus. This invention also provides a process for preparing a producer cell useful for transferring a foreign gene into a mammalian cell which comprises treating the above-described mammalian cell with a vector plasmid so as to insert the vector plasmid into the cell and thus create the producer cell, the vector plasmid comprising the foreign gene, a functional &psgr; packaging sequence from the helper virus, both the 5&prime; and 3&prime; LTRS from the helper virus, and a gene encoding a selectable or identifiable phenotypic trait, and recovering the producer cell so created.

Retroviral packaging cell lines and processes of using same

Upon entry into a host cell, retroviruses direct the reverse transcription of the viral RNA genome and the establishment of an integrated proviral DNA. The retroviral integrase protein (IN) is responsible for the insertion of the viral DNA into host chromosomal targets. The IN catalyzes two specific biochemical reactions: (i) cleavage of the 3&prime;termini of the viral DNA to produce 3&prime;-OH ends, and (ii) joining of the two newly generated 3&prime;-termini to the 5&prime;-phosphates on each strand of the target sequence in a concerted strand-transfer reaction. The yeast two-hybrid system was used to identify a novel human gene product, herein designated integrase interactor 1 or INI-1, that binds tightly to the human immunodeficiency virus type 1 (HIV-1) integrase in vitro. Approximately 10,complementary DNAs (cDNAs) of the HL60 macrophage-monocytic cell line were expressed as GAL4AC (activation domain) fusions and tested for coactivation of a reporter gene together with a GAL4DB (DNA binding) IN fusion. Overlapping cDNA clones were identified and their nucleotide sequences ascertained. Nucleotide sequence analysis revealed that INI-1 displays limited amino acid homology to the yeast SNF5 protein, a transcriptional activator required for high-level expression of many disparate cellular genes. Nucleotide sequences encoding the INI-1 gene product will prove useful for the generation of biochemical reagents and the development novel HIV-1 antiviral agents.

Nucleic acids encoding a human immunodeficiency virus type 1 (HIV-1) integrase interactor protein (INI-1)

This invention provides a polypeptide capable of polymerizing i) deoxyribonucleotides; ii) ribonucleotides; or iii) one or more deoxyribonucleotides and one or more ribonucleotides. This invention further provides a purified Moloney murine leukemia virus reverse transcriptase, wherein an amino acid corresponding to position 155 of a wild type Moloney murine leukemia virus reverse transcriptase is a valine. This invention also provides a method of polymerizing a nucleic acid molecule comprising one or more deoxyribonucleotides and one or more ribonucleotides comprising: contacting a polypeptide capable of polymerizing i) deoxyribonucleotides; ii) ribonucleotides; or iii) one or more deoxyribonucleotides and one or more ribonucleotides with deoxyribonucleotides and ribonucleotides under conditions permitting incorporation of deoxyribonucleotides and ribonucleotides into a nucleic acid molecule. This invention still further provides a method of converting a DNA polymerase into an RNA polymerase comprising: a) structurally modifying the DNA polymerase; b) contacting the structurally modified DNA polymerase of step (a) with deoxyribonucleotides and ribonucleotides under conditions permitting incorporation of deoxyribonucleotides and ribonucleotides into a nucleic acid molecule; and c) detecting polymerization of ribonucleotides in the nucleic acid molecule resulting from step (b), the detection of ribonucleotides in the polymerized nucleic acid molecule indicating that the structurally modified DNA polymerase has been converted into an RNA polymerase. This invention also provides a nucleic acid molecule polymerized by the above-described method of polymerizing a nucleic acid molecule comprising one or more deoxyribonucleotides and one or more ribonucleotides.

Reverse transcriptase of moloney murine leukemia virus with RNA

Upon entry into a host cell, retroviruses direct the reverse transcription of the viral RNA genome and the establishment of an integrated proviral DNA. The retroviral integrase protein (IN) is responsible for the insertion of the viral DNA into host chromosomal targets. The IN catalyzes two specific biochemical reactions: (i) cleavage of the 3'termini of the viral DNA to produce 3'-OH ends, and (ii) joining of the two newly generated 3'-termini to the 5'-phosphates on each strand of the target sequence in a concerted strand-transfer reaction. The yeast two-hybrid system was used to identify a novel human gene product, herein designated integrase interactor 1 or INI-1, that binds tightly to the human immunodeficiency virus type 1 (HIV-1) integrase in vitro. Approximately 10.sup.6 complementary DNAs (cDNAs) of the HL60 macrophage-monocytic cell line were expressed as GAL4AC (activation domain) fusions and tested for coactivation of a reporter gene together with a GAL4DB (DNA binding) IN fusion. Overlapping cDNA clones were identified and their nucleotide sequences ascertained. Nucleotide sequence analysis revealed that INI-1 displays limited amino acid homology to the yeast SNF5 protein, a transcriptional activator required for high-level expression of many disparate cellular genes. The INI-1 gene product will prove useful for the generation of biochemical reagents and the development novel HIV-1 antiviral agents.

Identification and characterization of a novel human protein designated

The human immunodeficiency virus type 1 (HIV-1) gag gene product is capable of directing the assembly of virion particles independent of other viral elements. The Gag protein also plays an important role during the early stages of viral replication. Employing the yeast two-hybrid system, a cDNA expression library was screened and two host proteins identified. These proteins, designated cyclophilins A and B (CyPsA and B), interacted specifically with the HIV-1 Gag polyprotein Pr55.sup.gag. Glutathione S-transferase-CyP fusion proteins bind tightly to Pr55.sup.gag in vitro. Cyclosporin A (CsA) efficiently disrupts the Gag-CyPA binding interaction. The identification of novel compounds capable of abrogating this protein-protein interaction employing the disclosed screening assay will facilitate the development of HIV-1 antiviral agents.

Screening method for the identification of compounds capable of

The invention relates to a mammalian cell line useful for retroviral packaging comprising two plasmids, the first such plasmid comprising in 5' to 3' order: a DNA sequence comprising a 5' long terminal repeat (LTR); a mutated .psi. packaging sequence; a DNA sequence comprising the encoding portion of the Moloney murine leukemia virus (MULV) env gene; and the second such plasmid comprising in 5' to 3' order: a DNA sequence comprising a 5' long terminal repeat (LTR); a mutated .psi. packaging sequence; a DNA sequence comprising the encoding portion of the Moloney murine leukemia virus (MULV) gag and pol genes; a selectable marker; and an origin of replication. The invention also relates to processes for preparing a producer cell line useful for transferring a gene of interest into recipient mammalian cells in vitro.

Retroviral packaging cell lines and process of using same

This invention describes pHRT25, a plasmid containing a modified pol gene of the Human Immunodeficiency Virus Type 1 (HIV-1), formerly HTLV-III, under control of an inducible trp promoter. Methods of expressing reverse transcriptase activity using pHRT25 in E. coli are described.

Expression of human immunodeficiency virus (HIV) reverse transcriptase

This invention provides a plasmid identified as pHRTRX2 and deposited in E. coli HB101 under ATCC Accession No. 67828. The invention also provides for a polypeptide having HIV reverse transcriptase activity and for a method by which the polypeptide can be prepared which comprises growing a host cell comprising the plasmid pHRTRX2 under suitable conditions permitting production of the polypeptide and recovering the resulting polypeptide.

This invention provides a plasmic which, when introduced into a suitable host cell and grown under appropriate conditions, effects expression of a gene on the plasmid and production of a polypeptide having reverse transcriptase activity. The plasmid is a double-stranded DNA molecule which includes in a 5' to 3' order the following: a DNA sequence which includes an inducible promoter; a DNA sequence which includes an ATG initiation condon; the central portion of the Moloney murine leukemia virus (MuLV) pol gene, said central portion including a DNA sequence which encodes the polypeptide having reverse transcriptase activity; a DNA sequence which contains a gene associated with a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell; and a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell. The invention also concerns a method for recovering purified enzymatically-active polypeptide having reverse transcriptase activity, the polypeptide being encoded by the plasmid pB6 B15.23, from a suitable host cell e.g., E. coli HB101 producing the polypeptide. Finally, the invention concerns use of the polypeptide to prepare complementary DNA (cDNA).

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Tenafly, NJ, United States of America

Stephen P Goff