Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells

Abstract

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR).Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo r ), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo r alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo r transduction of primary human CD34 ؉ hematopoietic progenitor cells from normal bone marrow was alsodocumented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34 ؉ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo r alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV-and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

“…The integration patterns of the proviral DNA were distinct in all of the clones examined. Using PCR assays with a neo-and chromosome 19-specific primer pair (27,32,38), we did not observe proviral genomic integration at the chromosome 19 target site previously characterized for the wt AAV genome (17,18,38) (data not shown). These results are consistent with previously published reports by members of our laboratory (27,32) and others ( 14) which state that unlike the wt AAV genome, recombinant AAV genomes integrate randomly in human cells.…”

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

“…The integration patterns of the proviral DNA were distinct in all of the clones examined. Using PCR assays with a neo-and chromosome 19-specific primer pair (27,32,38), we did not observe proviral genomic integration at the chromosome 19 target site previously characterized for the wt AAV genome (17,18,38) (data not shown). These results are consistent with previously published reports by members of our laboratory (27,32) and others ( 14) which state that unlike the wt AAV genome, recombinant AAV genomes integrate randomly in human cells.…”

Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

“…Ecotropic Moloney MLV use the murine cationic amino acid transporter MCAT-1 as a cellular receptor. Most nonrodent cells are not permissive for ecotropic MLV infection; however, these cells become permissive if they express MCAT-1 (5,8,28,51,58,67,68). Since avian cells lack a functional receptor for ecotropic murine retroviruses, they are not efficiently infected by ecotropic murine retroviruses.…”

Polarized AAVR expression determines infectivity by AAV gene therapy vectors

“…These obstacles to gene transfer and gene editing can be overcome by employing more efficient vectors. Attempts to increase vector efficiency have addressed stages throughout the viral-cycle, often focusing on the manipulation of cell-surface receptors and the capsid elements they recognize 45–47 . Our previous efforts used directed evolution to produce AAV2.5T, a novel vector with tropism for human airway epithelia 5 .…”

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

“…The integration patterns of the proviral DNA were distinct in all of the clones examined. Using PCR assays with a neo-and chromosome 19-specific primer pair (27,32,38), we did not observe proviral genomic integration at the chromosome 19 target site previously characterized for the wt AAV genome (17,18,38) (data not shown). These results are consistent with previously published reports by members of our laboratory (27,32) and others ( 14) which state that unlike the wt AAV genome, recombinant AAV genomes integrate randomly in human cells.…”

Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

“…Ecotropic Moloney MLV use the murine cationic amino acid transporter MCAT-1 as a cellular receptor. Most nonrodent cells are not permissive for ecotropic MLV infection; however, these cells become permissive if they express MCAT-1 (5,8,28,51,58,67,68). Since avian cells lack a functional receptor for ecotropic murine retroviruses, they are not efficiently infected by ecotropic murine retroviruses.…”

Polarized AAVR expression determines infectivity by AAV gene therapy vectors

“…These obstacles to gene transfer and gene editing can be overcome by employing more efficient vectors. Attempts to increase vector efficiency have addressed stages throughout the viral-cycle, often focusing on the manipulation of cell-surface receptors and the capsid elements they recognize 45–47 . Our previous efforts used directed evolution to produce AAV2.5T, a novel vector with tropism for human airway epithelia 5 .…”

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

“…The integration patterns of the proviral DNA were distinct in all of the clones examined. Using PCR assays with a neo-and chromosome 19-specific primer pair (27,32,38), we did not observe proviral genomic integration at the chromosome 19 target site previously characterized for the wt AAV genome (17,18,38) (data not shown). These results are consistent with previously published reports by members of our laboratory (27,32) and others ( 14) which state that unlike the wt AAV genome, recombinant AAV genomes integrate randomly in human cells.…”

Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

“…Ecotropic Moloney MLV use the murine cationic amino acid transporter MCAT-1 as a cellular receptor. Most nonrodent cells are not permissive for ecotropic MLV infection; however, these cells become permissive if they express MCAT-1 (5,8,28,51,58,67,68). Since avian cells lack a functional receptor for ecotropic murine retroviruses, they are not efficiently infected by ecotropic murine retroviruses.…”

Polarized AAVR expression determines infectivity by AAV gene therapy vectors

“…These obstacles to gene transfer and gene editing can be overcome by employing more efficient vectors. Attempts to increase vector efficiency have addressed stages throughout the viral-cycle, often focusing on the manipulation of cell-surface receptors and the capsid elements they recognize 45–47 . Our previous efforts used directed evolution to produce AAV2.5T, a novel vector with tropism for human airway epithelia 5 .…”