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541. Detargeted Integration of MLV-Based Vectors By Integrase Mutants That Disrupt BET Protein Interaction
RNA VIRUS VECTORS II 539. Atonal Adenoviral Gene Therapy Results in Hair Cell Regeneration and Partial Recovery of Auditory Function After Ototoxin Injury
Susan C. Stevenson,1 Chi Hsu,2 Chi-Hse Teng,1 Hinrich Staecker,3 Douglas E. Brough,2 Lloyd Klickstein.1 1 Novartis Institutes for Biomedical Research, Cambridge, MA; 2 GenVec, Inc., Gaithersburg, MD; 3University of Kansas Medical Center, Kansas City, KS. Sensorineural hearing loss and balance dysfunction result from loss of inner ear sensory hair cells and/or auditory nerves. Sensory cells can be damaged by pharmacological agents, loud noises, infections or simply aging; loss of these cells is permanent as they do not regenerate. No pharmacologic agents exist to restore hearing and balance function. The atonal gene, ATOH1, is a bHLH transcription factor that is required for differentiation of sensory hair cells. An adenoviral gene therapy approach has been developed to express ATOH1 locally in the inner ear to regenerate sensory hair cells. Adenoviral vectors expressing the ATOH1 mouse homolog (Math1) have been evaluated previously in multiple rodent models and reported to result in regeneration of hair cells and restoration of hearing or balance function. The objective of this study was to compare directly the ATOH1 human homolog, Hath1 with Math1 for its ability to promote hair cell regeneration and hearing recovery in an adult mouse model of hearing loss. Ad5.GFAP.Hath1 consists of an E1, E3, E4-deleted adenovirus 5 (Ad5) vector engineered to express Hath1 under the control of the GFAP (glial fibrillary acidic protein) promoter, which will limit expression to the inner ear supporting cells. Ad5.GFAP.Math1 is an identical vector except it expresses Math1. The in vivo adult mouse model of kanamycin/ furosemide-induced hearing loss was used to evaluate the efficacy of the Hath1- or Math1-expressing vectors. Ten days after ototoxin exposure, hearing loss was confirmed by the increase in auditory brain stem response (ABR) thresholds across all frequencies compared to normal mice. Subsequent pathological examination of the cochlea showed >90% inner hair cell loss only at the base, corresponding to high frequencies. The adenoviral vectors encoding Hath1 or Math1 were injected directly into the inner ear via the posterior semi-circular canal. Three doses were evaluated for each vector: high (5.5 x 108 vp), medium (5.5 x 107 vp) and low dose (5.5 x 106 vp). At 3 days post administration, vector delivery and ATOH1 RNA expression were verified by PCR analysis. The effect of adenoviral-mediated expression of ATOH1 on ABR thresholds was determined at 1 and 2 months post vector administration compared to injured control and untreated mice. At 1 month, all treatment groups showed similar ABR thresholds. At 2 months, there was a partial restoration of auditory function. The restoration was most prominent (p < 0.05) at the high frequency of 32 kHz with a 13 to 18 dB threshold recovery found in mice treated with the high and medium dose of Math1 and with the high dose of Hath1. The hearing improvements found at 32 kHz correlated with the recovery of inner hair cells in the basal region of the cochlea. These data support the hypothesis that local expression of ATOH1 may provide a therapeutic means of restoring auditory function related to sensory hair cell loss and that administration of Ad5.GFAP.Hath1 to patients with sensorineural hearing loss may be beneficial.
RNA Virus Vectors II 540. Safety Evaluation of a Novel Insulated SIN. LV in Stringent In Vivo Genotoxicity Assays
Monica Volpin,1,2 Daniela Cesana,1 Andrea Calabria,1 Fabrizio Benedicenti,1 Odile Cohen Haguenauer,3 Eugenio Montini.1 1 Telethon Institute for Gene Therapy, Milan, Italy; 2Università Vita-Salute San Raffaele, Milan, Italy; 3Ecole Normale Supérieure de Cachan, Cachan, France.
We tested if the inclusion of chromatin insulators (CI) in selfinactivating (SIN) lentiviral vectors (LVs) could increase their safety profile. To do this we challenged the enhancer-blocking ability of a validated CI by cloning it into the Long Terminal Repeats (LTRs) of a SIN.LV harboring the strong Spleen Focus Forming Virus enhancerpromoter driving GFP expression (INS.SIN.LV). The insulated LV and its non-insulated parental LV (SIN.LV) were systemically injected in newborn tumor-prone Cdkn2a-/- and Cdkn2+/- mice (1-2.5x108 TU/mouse). In these mouse models vector-induced genotoxicity results in an accelerated onset of hematopoietic tumors in vectortreated mice vs. mock controls. Both vectors caused accelerated tumor onset in Cdkn2a-/- mice when compared to mock controls and although mice injected with the insulated LV displayed a slightly delayed tumor onset vs. SIN. LV-treated mice we could not detect any significant improvement in the mouse survival. Interestingly, the Common Integrations Sites (CIS) analysis in tumor infiltrated tissues showed that the genotoxicity of the insulated LV was caused mainly by the inactivation of tumor suppressors such as Pten, while the non-insulated LV induced early tumor onset by activating oncogenes such as Map3k8. Thus, the genotoxicity observed in Cdkn2a-/- mice treated with the insulated LV was not due to inefficient insulating activity but rather to an escape mechanism represented by the inactivation of tumor-suppressors. Here we show that also in heterozygous Cdkn2a+/- mice, SIN.LV and INS.SINLV treatments resulted to be genotoxic (median survival: 392 and 429.5 days respectively vs. 534 days of mock-treated mice). Molecular analysis of 2037 integration sites retrieved from 10 SIN.LV-marked tumors identified Sfi1 as predominant CIS, that was targeted by 9 integrations present all along the gene body and in opposite orientation to gene transcription direction, suggesting that this CIS gene might be activated by enhancer-mediated overexpression. Differently, preliminary integration site analysis on 518 integrations retrieved from INS.SIN.LV-induced tumors from 5 Cdkn2a+/- mice show a new set of targeted CIS, headed by Fggy gene (hit by 5 integrations) and no integrations targeting Sfi1, Pten or Map3k8. These preliminary results suggest that the inclusion of the CI induced also in Cdkn2a+/- mice a skewing of the genes that are involved in vector-induced tumor formation. Taken together our results from Cdkn2a-/- and Cdkn2a+/mice demonstrate the ability of CI to block enhancer-mediated overexpression of oncogenes in vivo and promote the use of insulator elements in future gene therapy applications to reduce the risks associated with this mechanism of insertional mutagenesis.
541. Detargeted Integration of MLV-Based Vectors By Integrase Mutants That Disrupt BET Protein Interaction
Sara El Ashkar,1 Jan De Rijck,1 Jonas Demeulemeester,1 Sofie Vets,1 Katarina Cermakova,1 Christine de Kogel,1 Zeger Debyser,1 Rik Gijsbers.1 1 Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium. Stable integration of the viral DNA in the host cell genome makes retrovirus-derived vector particles attractive tools for gene therapy. Retroviral integration is however not random, with lentiviruses S210
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
RNA VIRUS VECTORS II mainly integrating in the body of active transcription units, and gammaretroviruses, like the Moloney Murine Leukemia Virus (MLV), favouring transcription start sites (TSS) and CpG islands. The latter explains the activation of oncogenes and the development of acute leukemia in a subset of patients in several clinical trials using MLV vector-based gene therapy. We unveiled the bromodomain and extraterminal (BET) family proteins (BRD2, BRD3 and BRD4) as specific cellular binding partners of MLV integrase (IN), directing MLV integration (1). BET protein interaction specifically occurs through the unstructured C-terminal end of MLV IN. Here, we report that deletion of the MLV IN C-terminal end or specific mutation of W390 results in viral vectors that no longer bind BET-proteins in vitro, yet integrate as efficiently as vectors carrying wild-type IN. Integration site analysis showed detargeted integration, shifted away from RefSeq and oncogene TSS, CpG islands and DNaseI-hypersensitive sites, thereby reducing the potential for insertional mutagenesis. We are currently evaluating the genotoxic profile of these viral vectors. These results explain the molecular mechanism of gammaretroviral integration targeting and open perspectives for engineered retroviral vectors with a safer integration site profile. (1) De Rijck J., de Kogel C. et al. 2013, Cell Reports 5, 886–894.
542. A Novel Non-Restrictive, Semi-Quantitative Method for Vector Insertion Site Analysis Based on Random Shearing of Genomic DNA
Sheng Zhou,1 Chunlao Tang,1 Samuel Rapp,1 Melissa A. Bonner,1 Suk See De Ravin,2 Harry L. Malech,2 Brian P. Sorrentino.1 1 St. Jude Children’s Research Hospital, Memphis, TN; 2NIAID, National Institutes of Health, Bethesda, MD.
Analysis of integrating vector insertion sites (VIS) and measurement of clonal abundance based on vector insertion sites has important applications in monitoring dominant clones in clinical gene therapy. Currently available methods for VIS analysis may not be quantitative and can miss dominant vector insertion sites. We have developed a novel method for VIS analysis based on random shearing of genomic DNA, inclusion of a defined internal control sample containing 19 insertion sites, and minimum PCR amplification. The VIS control was generated by isolating a high copy number clone of human Jurkat T cells that had been transduced with our clinical lentiviral vector for SCID-X1 gene therapy. The genomic DNA from the clone was randomly sheared by sonication into 300-500bp fragments. The vector/genomic DNA junctions were linear-amplified for 50 cycles using a biotinylated primer specific to the viral LTR followed by purification with streptavidin magnetic beads. The purified product was subject to 12 cycles of PCR to incorporate Illumina adaptors. Size-selected 300-600bp fragments from the final PCR product were then sequenced using the MiSeq sequencer. There were a total of 19 copies of the vector genome inserted into 15 unique chromosomal locations, with 8 copies oriented as head-to-head juxtaposed pairs on opposite strands of 4 unique chromosomal locations. The read counts for majority of the insertions (13/19) occurred at approximately equal frequencies showing that this method is relatively quantitative. We next evaluated this assay using three experimental samples with unknown VIS. Two were genomic DNA preparations from normal human CD34+ cells transduced with a lentiviral vector and one was CD16+ cells sorted from peripheral blood of a SCID-X1 trial participant undergoing gene therapy. These highly polyclonal DNA samples were mixed with DNA from the 19 copy Jurkat clone at a ratio of 49:1 and VIS results are summarized in the table below. All 19 VIS from the Jurkat line were retrieved at relatively high frequencies despite being diluted to 2% with highly polyclonal samples, demonstrating the sensitivity of the assay. In all cases, there were no sequence reads from the experimental samples that exceeded any internal control VIS. This comparison demonstrates that none of the 3 complex samples had any clones exceeding 2% of the cells Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
in the polyclonal samples. This result demonstrates how spiking an appropriate internal control to samples from patients undergoing gene therapy can be used for quantitative clonal size determination. In summary, we have developed a novel sensitive, semi-quantitative method for high-throughput vector insertion site analysis and show its application for analysis of samples from human gene therapy trials.
543. Biodistribution of LV-TSTA Transduced Rat Bone Marrow Cells Used for “Ex-Vivo” Regional Gene Therapy for Bone Repair
Farhang Alaee,1 Cynthia Bartholomae,2 Osamu Sugiyama,3 Mandeep Virk,1 Hicham Drissi,1 Manfred Schmidt,2 Jay R. Lieberman.3 1 Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington; 2Department of Translational Oncology, German Cancer Research Center, Heidelberg, Germany; 3Department of Orthopaedic Surgery, University of Southern California, Los Angeles.
Our aim was to determine the biodistribution of lentiviral transduced cells placed in a rat femoral defect. We evaluated two different “ex vivo” approaches using either “same day” cells or cultured bone marrow cells transduced with a LV based two-step transcriptional activation system overexpressing GFP (LV-TSTA-GFP). In the “same day” strategy, the mononuclear cell layer was isolated from the rat bone marrow cells (SD-RBMCs). 15x106 transduced cells were placed in femoral defects of syngeneic rats in the same day. In the “cultured bone marrow” strategy, the cells (C-RBMCs) were harvested, expanded in tissue culture and were transduced overnight with the LV-TSTA-GFP. 5x106 transduced cells were implanted in the rat defects. Animals were sacrificed at 4, 14, 28 and 56 days after surgery (n=5). DNA from defect site and internal organs was extracted for quantitation of viral copies. Large scale integration site analysis using linear amplification mediated PCR (LAM-PCR) was conducted on 4d and 56d samples. In SD-RBMC animals viral copies were detectable in the defect site at 4 days (1.95x108- 7.24x108 copies/μg). The copy numbers declined to <50-8451 copies/μg at 56d. Viral copies were minimally detected in blood, testis, brain and the GI. Heart and spleen were positive only in one animal and copies were undetectable in the lung, kidney and BM at all time points. No consistent pattern of organ involvement was noticed. In the C-RBMC animals, 6.41x104- 6.75x105 copies/ μg were detected in the defect site at 4 days. There was a decline in the viral copies at 14d (<50-3142 copies/μg) and 28d (998-6731 copies/μg), but at 56d there was a relative rise (about 100 fold) in the number of the viral vectors in the defect site of 4 animals compared to the previous time points (18360-2.58x105 copies/μg). The pattern of tissue distribution was non-specific and levels were detectable in blood more commonly than other organs. No viral copies were detected in BM, testis, heart and lung. No histological abnormalities were noted in either group. Integration site analysis revealed 630 unique insertion sites (IS). The majority of IS (590 IS) was identified in C-RBMC group (579 IS at 4d; 14 IS at 56d). In contrast, merely 40 unique IS were identified in the SD-RBMC group (13 IS at 4d; 27 IS at 56d). Common IS analyses on the combined IS data set (630 IS) showed a low frequency of CIS detection (8 CIS of 2nd order) and the absence of genes previously linked to adverse events in clinical gene therapy studies.
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Susan C. Stevenson,1 Chi Hsu,2 Chi-Hse Teng,1 Hinrich Staecker,3 Douglas E. Brough,2 Lloyd Klickstein.1 1 Novartis Institutes for Biomedical Research, Cambridge, MA; 2 GenVec, Inc., Gaithersburg, MD; 3University of Kansas Medical Center, Kansas City, KS. Sensorineural hearing loss and balance dysfunction result from loss of inner ear sensory hair cells and/or auditory nerves. Sensory cells can be damaged by pharmacological agents, loud noises, infections or simply aging; loss of these cells is permanent as they do not regenerate. No pharmacologic agents exist to restore hearing and balance function. The atonal gene, ATOH1, is a bHLH transcription factor that is required for differentiation of sensory hair cells. An adenoviral gene therapy approach has been developed to express ATOH1 locally in the inner ear to regenerate sensory hair cells. Adenoviral vectors expressing the ATOH1 mouse homolog (Math1) have been evaluated previously in multiple rodent models and reported to result in regeneration of hair cells and restoration of hearing or balance function. The objective of this study was to compare directly the ATOH1 human homolog, Hath1 with Math1 for its ability to promote hair cell regeneration and hearing recovery in an adult mouse model of hearing loss. Ad5.GFAP.Hath1 consists of an E1, E3, E4-deleted adenovirus 5 (Ad5) vector engineered to express Hath1 under the control of the GFAP (glial fibrillary acidic protein) promoter, which will limit expression to the inner ear supporting cells. Ad5.GFAP.Math1 is an identical vector except it expresses Math1. The in vivo adult mouse model of kanamycin/ furosemide-induced hearing loss was used to evaluate the efficacy of the Hath1- or Math1-expressing vectors. Ten days after ototoxin exposure, hearing loss was confirmed by the increase in auditory brain stem response (ABR) thresholds across all frequencies compared to normal mice. Subsequent pathological examination of the cochlea showed >90% inner hair cell loss only at the base, corresponding to high frequencies. The adenoviral vectors encoding Hath1 or Math1 were injected directly into the inner ear via the posterior semi-circular canal. Three doses were evaluated for each vector: high (5.5 x 108 vp), medium (5.5 x 107 vp) and low dose (5.5 x 106 vp). At 3 days post administration, vector delivery and ATOH1 RNA expression were verified by PCR analysis. The effect of adenoviral-mediated expression of ATOH1 on ABR thresholds was determined at 1 and 2 months post vector administration compared to injured control and untreated mice. At 1 month, all treatment groups showed similar ABR thresholds. At 2 months, there was a partial restoration of auditory function. The restoration was most prominent (p < 0.05) at the high frequency of 32 kHz with a 13 to 18 dB threshold recovery found in mice treated with the high and medium dose of Math1 and with the high dose of Hath1. The hearing improvements found at 32 kHz correlated with the recovery of inner hair cells in the basal region of the cochlea. These data support the hypothesis that local expression of ATOH1 may provide a therapeutic means of restoring auditory function related to sensory hair cell loss and that administration of Ad5.GFAP.Hath1 to patients with sensorineural hearing loss may be beneficial.
RNA Virus Vectors II 540. Safety Evaluation of a Novel Insulated SIN. LV in Stringent In Vivo Genotoxicity Assays
Monica Volpin,1,2 Daniela Cesana,1 Andrea Calabria,1 Fabrizio Benedicenti,1 Odile Cohen Haguenauer,3 Eugenio Montini.1 1 Telethon Institute for Gene Therapy, Milan, Italy; 2Università Vita-Salute San Raffaele, Milan, Italy; 3Ecole Normale Supérieure de Cachan, Cachan, France.
We tested if the inclusion of chromatin insulators (CI) in selfinactivating (SIN) lentiviral vectors (LVs) could increase their safety profile. To do this we challenged the enhancer-blocking ability of a validated CI by cloning it into the Long Terminal Repeats (LTRs) of a SIN.LV harboring the strong Spleen Focus Forming Virus enhancerpromoter driving GFP expression (INS.SIN.LV). The insulated LV and its non-insulated parental LV (SIN.LV) were systemically injected in newborn tumor-prone Cdkn2a-/- and Cdkn2+/- mice (1-2.5x108 TU/mouse). In these mouse models vector-induced genotoxicity results in an accelerated onset of hematopoietic tumors in vectortreated mice vs. mock controls. Both vectors caused accelerated tumor onset in Cdkn2a-/- mice when compared to mock controls and although mice injected with the insulated LV displayed a slightly delayed tumor onset vs. SIN. LV-treated mice we could not detect any significant improvement in the mouse survival. Interestingly, the Common Integrations Sites (CIS) analysis in tumor infiltrated tissues showed that the genotoxicity of the insulated LV was caused mainly by the inactivation of tumor suppressors such as Pten, while the non-insulated LV induced early tumor onset by activating oncogenes such as Map3k8. Thus, the genotoxicity observed in Cdkn2a-/- mice treated with the insulated LV was not due to inefficient insulating activity but rather to an escape mechanism represented by the inactivation of tumor-suppressors. Here we show that also in heterozygous Cdkn2a+/- mice, SIN.LV and INS.SINLV treatments resulted to be genotoxic (median survival: 392 and 429.5 days respectively vs. 534 days of mock-treated mice). Molecular analysis of 2037 integration sites retrieved from 10 SIN.LV-marked tumors identified Sfi1 as predominant CIS, that was targeted by 9 integrations present all along the gene body and in opposite orientation to gene transcription direction, suggesting that this CIS gene might be activated by enhancer-mediated overexpression. Differently, preliminary integration site analysis on 518 integrations retrieved from INS.SIN.LV-induced tumors from 5 Cdkn2a+/- mice show a new set of targeted CIS, headed by Fggy gene (hit by 5 integrations) and no integrations targeting Sfi1, Pten or Map3k8. These preliminary results suggest that the inclusion of the CI induced also in Cdkn2a+/- mice a skewing of the genes that are involved in vector-induced tumor formation. Taken together our results from Cdkn2a-/- and Cdkn2a+/mice demonstrate the ability of CI to block enhancer-mediated overexpression of oncogenes in vivo and promote the use of insulator elements in future gene therapy applications to reduce the risks associated with this mechanism of insertional mutagenesis.
541. Detargeted Integration of MLV-Based Vectors By Integrase Mutants That Disrupt BET Protein Interaction
Sara El Ashkar,1 Jan De Rijck,1 Jonas Demeulemeester,1 Sofie Vets,1 Katarina Cermakova,1 Christine de Kogel,1 Zeger Debyser,1 Rik Gijsbers.1 1 Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium. Stable integration of the viral DNA in the host cell genome makes retrovirus-derived vector particles attractive tools for gene therapy. Retroviral integration is however not random, with lentiviruses S210
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
RNA VIRUS VECTORS II mainly integrating in the body of active transcription units, and gammaretroviruses, like the Moloney Murine Leukemia Virus (MLV), favouring transcription start sites (TSS) and CpG islands. The latter explains the activation of oncogenes and the development of acute leukemia in a subset of patients in several clinical trials using MLV vector-based gene therapy. We unveiled the bromodomain and extraterminal (BET) family proteins (BRD2, BRD3 and BRD4) as specific cellular binding partners of MLV integrase (IN), directing MLV integration (1). BET protein interaction specifically occurs through the unstructured C-terminal end of MLV IN. Here, we report that deletion of the MLV IN C-terminal end or specific mutation of W390 results in viral vectors that no longer bind BET-proteins in vitro, yet integrate as efficiently as vectors carrying wild-type IN. Integration site analysis showed detargeted integration, shifted away from RefSeq and oncogene TSS, CpG islands and DNaseI-hypersensitive sites, thereby reducing the potential for insertional mutagenesis. We are currently evaluating the genotoxic profile of these viral vectors. These results explain the molecular mechanism of gammaretroviral integration targeting and open perspectives for engineered retroviral vectors with a safer integration site profile. (1) De Rijck J., de Kogel C. et al. 2013, Cell Reports 5, 886–894.
542. A Novel Non-Restrictive, Semi-Quantitative Method for Vector Insertion Site Analysis Based on Random Shearing of Genomic DNA
Sheng Zhou,1 Chunlao Tang,1 Samuel Rapp,1 Melissa A. Bonner,1 Suk See De Ravin,2 Harry L. Malech,2 Brian P. Sorrentino.1 1 St. Jude Children’s Research Hospital, Memphis, TN; 2NIAID, National Institutes of Health, Bethesda, MD.
Analysis of integrating vector insertion sites (VIS) and measurement of clonal abundance based on vector insertion sites has important applications in monitoring dominant clones in clinical gene therapy. Currently available methods for VIS analysis may not be quantitative and can miss dominant vector insertion sites. We have developed a novel method for VIS analysis based on random shearing of genomic DNA, inclusion of a defined internal control sample containing 19 insertion sites, and minimum PCR amplification. The VIS control was generated by isolating a high copy number clone of human Jurkat T cells that had been transduced with our clinical lentiviral vector for SCID-X1 gene therapy. The genomic DNA from the clone was randomly sheared by sonication into 300-500bp fragments. The vector/genomic DNA junctions were linear-amplified for 50 cycles using a biotinylated primer specific to the viral LTR followed by purification with streptavidin magnetic beads. The purified product was subject to 12 cycles of PCR to incorporate Illumina adaptors. Size-selected 300-600bp fragments from the final PCR product were then sequenced using the MiSeq sequencer. There were a total of 19 copies of the vector genome inserted into 15 unique chromosomal locations, with 8 copies oriented as head-to-head juxtaposed pairs on opposite strands of 4 unique chromosomal locations. The read counts for majority of the insertions (13/19) occurred at approximately equal frequencies showing that this method is relatively quantitative. We next evaluated this assay using three experimental samples with unknown VIS. Two were genomic DNA preparations from normal human CD34+ cells transduced with a lentiviral vector and one was CD16+ cells sorted from peripheral blood of a SCID-X1 trial participant undergoing gene therapy. These highly polyclonal DNA samples were mixed with DNA from the 19 copy Jurkat clone at a ratio of 49:1 and VIS results are summarized in the table below. All 19 VIS from the Jurkat line were retrieved at relatively high frequencies despite being diluted to 2% with highly polyclonal samples, demonstrating the sensitivity of the assay. In all cases, there were no sequence reads from the experimental samples that exceeded any internal control VIS. This comparison demonstrates that none of the 3 complex samples had any clones exceeding 2% of the cells Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
in the polyclonal samples. This result demonstrates how spiking an appropriate internal control to samples from patients undergoing gene therapy can be used for quantitative clonal size determination. In summary, we have developed a novel sensitive, semi-quantitative method for high-throughput vector insertion site analysis and show its application for analysis of samples from human gene therapy trials.
543. Biodistribution of LV-TSTA Transduced Rat Bone Marrow Cells Used for “Ex-Vivo” Regional Gene Therapy for Bone Repair
Farhang Alaee,1 Cynthia Bartholomae,2 Osamu Sugiyama,3 Mandeep Virk,1 Hicham Drissi,1 Manfred Schmidt,2 Jay R. Lieberman.3 1 Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington; 2Department of Translational Oncology, German Cancer Research Center, Heidelberg, Germany; 3Department of Orthopaedic Surgery, University of Southern California, Los Angeles.
Our aim was to determine the biodistribution of lentiviral transduced cells placed in a rat femoral defect. We evaluated two different “ex vivo” approaches using either “same day” cells or cultured bone marrow cells transduced with a LV based two-step transcriptional activation system overexpressing GFP (LV-TSTA-GFP). In the “same day” strategy, the mononuclear cell layer was isolated from the rat bone marrow cells (SD-RBMCs). 15x106 transduced cells were placed in femoral defects of syngeneic rats in the same day. In the “cultured bone marrow” strategy, the cells (C-RBMCs) were harvested, expanded in tissue culture and were transduced overnight with the LV-TSTA-GFP. 5x106 transduced cells were implanted in the rat defects. Animals were sacrificed at 4, 14, 28 and 56 days after surgery (n=5). DNA from defect site and internal organs was extracted for quantitation of viral copies. Large scale integration site analysis using linear amplification mediated PCR (LAM-PCR) was conducted on 4d and 56d samples. In SD-RBMC animals viral copies were detectable in the defect site at 4 days (1.95x108- 7.24x108 copies/μg). The copy numbers declined to <50-8451 copies/μg at 56d. Viral copies were minimally detected in blood, testis, brain and the GI. Heart and spleen were positive only in one animal and copies were undetectable in the lung, kidney and BM at all time points. No consistent pattern of organ involvement was noticed. In the C-RBMC animals, 6.41x104- 6.75x105 copies/ μg were detected in the defect site at 4 days. There was a decline in the viral copies at 14d (<50-3142 copies/μg) and 28d (998-6731 copies/μg), but at 56d there was a relative rise (about 100 fold) in the number of the viral vectors in the defect site of 4 animals compared to the previous time points (18360-2.58x105 copies/μg). The pattern of tissue distribution was non-specific and levels were detectable in blood more commonly than other organs. No viral copies were detected in BM, testis, heart and lung. No histological abnormalities were noted in either group. Integration site analysis revealed 630 unique insertion sites (IS). The majority of IS (590 IS) was identified in C-RBMC group (579 IS at 4d; 14 IS at 56d). In contrast, merely 40 unique IS were identified in the SD-RBMC group (13 IS at 4d; 27 IS at 56d). Common IS analyses on the combined IS data set (630 IS) showed a low frequency of CIS detection (8 CIS of 2nd order) and the absence of genes previously linked to adverse events in clinical gene therapy studies.
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