- Email: [email protected]
624. Correction of Mutant Fanconi Anemia Gene by Homologous Recombination in Human Hematopoietic Cells Using Adeno-Associated Virus Vector
Hematologic - Transduction, Engraftment and Transgene Expression MMC (p < 0.0002 for 20 nM MMC). Lineage depleted BM cells from FancA -/- mice were transduced using the brief overnight procedure. A transduction efficiency of 76% of colony forming cells was obtained. In summary, a lentiviral vector with a functional FancA transgene was developed which achieves efficient transduction of human and murine hematopoietic progenitors using a brief transduction protocol. Reduction of oxidative stress during ex vivo culture may also enhance the viability and engraftment potential of FA stem cells.
623. Exogenous Telomerase RNA Component (TERC) Alone Can Enhance Proliferative Potential and Telomere Length in B Lymphocyte Lines from Dyskeratosis Congenita Patients
Michael J. Kirwan, Richard Beswick, Amit C. Nathwani, Amanda J. Walne,1 Tom Vulliamy,1 Colin Casimir,3 Inderjeet Dokal.1 1 Academic Department of Paediatrics, Barts and The London School of Medicine and Dentistry, London, United Kingdom; 2 Department of Haematology, University College London, London, United Kingdom; 3School of Life Sciences, Kingston University, London, United Kingdom. 1
1
2
Dyskeratosis congenita (DC) is an inherited multi-system disorder characterised by muco-cutaneous abnormalities, bone marrow failure and a predisposition to malignancy. Bone marrow failure is the principal cause of mortality and is thought to be the result of premature cell death in the haematopoietic compartment. DC cells age prematurely and tend to have short telomeres which may account, at least in part, for the premature cell death and rapid decline in haematopoiesis seen in patients. The telomerase complex is important in maintaining telomeres and DC patients have mutations in genes that encode several of its components including the telomerase reverse transcriptase, TERT, and RNA component, TERC, and the nucleolar proteins dyskerin and NOP10. Previous studies in DC have used fibroblasts transduced with TERT but we wished to study haematopoietic cells to get closer to the mechanism of the pathology observed in vivo. B lymphocyte lines are typically difficult to establish from DC patients and tend to grow poorly by comparison to those derived from normal, healthy volunteers. We transduced B lymphocyte lines established from patients with TERC and DKC1 (dyskerin) mutations with wild type TERC and DKC1-bearing lentiviral vectors. We found that transduction with exogenous TERC alone was capable of improving the survival and thus overall growth of mutant lines over a prolonged period, regardless of their mutation and also resulted in telomere elongation. However, exogenous DKC1 had no beneficial effect even on DKC1 mutant lines. This study is the first of its kind in DC lymphocytes and the first to demonstrate that transduction with TERC alone can improve cell survival and lengthen telomeres without the need for exogenous TERT. The effects on telomere length and cell survival provided by exogenous, wild type TERC, regardless of the underlying pathological mutation, directly implicate telomerase dysfunction as a cause of DC symptoms and as a therapeutic target in cells that would normally express telomerase in vivo.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
624. Correction of Mutant Fanconi Anemia Gene by Homologous Recombination in Human Hematopoietic Cells Using Adeno-Associated Virus Vector
Kittiphong Paiboonsukwong,1 Fumi Ohbayashi,1 Haruka Shiiba,1 Takayuki Yamashita,2 Kohnosuke Mitani.1 1 Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan; 2Laboratory of Molecular Genetics, Department of Molecular and Cellular Biology, Institute of Molecular and Cellular Regulation, Gunma University, Gunma, Japan. Adeno-associated virus (AAV) vectors have been utilized to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which would overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. Here, we report an application of AAV-mediated gene targeting to disrupt the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus in a normal human B-lymphoblastoid cell line (BCL) and to correct a mutation in the Fanconi anemia group A (FANCA) gene in BCL derived from a Fanconi anemia (FA) patient. We first examined the transduction efficiency of normal BCL cells by using a self-complementary AAV vector encoding for GFP (scAAV-GFP) packaged into eight AAV serotypes (AAV-1, -2, -3, -4, -7, -8, -9, and -10). At a multiplicity of infection (MOI) of 1 x 105 vector genomes/cell, approximately 40% and 50% of BCL cells were transduced and became GFP-positive by AAV-1 and AAV-2, respectively, while the rest of serotypes achieved a percentage of positive cells lower than 10%. This result suggests that AAV-2, which is known to transduce a wide range of cell types in vitro, also efficiently transduces BCL. After infection of normal BCL cells with an AAV-2/HPRT1 targeting vector at an MOI of 300 and selection with G418 and thereafter with 6-thioguanine, we estimated the chromosomal integration and HPRT1-knockout frequencies to be 7.3 x 10-6 and 2.1 x 10-6 per infected cell, respectively. Gene targeting events by HR were confirmed by Southern blot analyses. Furthermore, we constructed an AAV-2/FANCA targeting vector designed to correct a mutant allele on the FANCA gene of BCL cells established from a FA patient. In this cell lines, a maternal allele of FANCA has a deletion of C in exon 27 (designated 2546delC), which is the most common pathogenic mutation among Japanese FANCA patients. BCL cells infected with this vector, which encodes a 4.4 kb normal FANCA sequence surrounding exon 27, at an MOI of 1 x 104 and selected for with 10-20 nM of mitomycin C (MMC) produced MMC-resistant cell clones at a frequency of 1.6 x 10-4 per infected cell (about 1 out of 6400 infected cells). Subsequent analyses by PCR-based RFLP and sequencing showed the genotypic correction of the FANCA gene in these MMC-resistant clones. The FANCA-corrected clones had restored FANCA expression as revealed by Western blot analyses. Cellular hypersensitivity to MMC, which is the characteristic of FA cells, was also corrected to a normal level in these clones. In summary, this study showed that AAV-2 vectors are able to efficiently transduce and target a gene by HR in EBV-transformed human BCL derived from normal and FA patients. This strategy may therefore be applicable for gene correction in hematopoietic stem cells. As the gene targeting efficiency is improved, AAV-based gene repair therapy in various inherited hematopoietic diseases could become a reality.
S233
623. Exogenous Telomerase RNA Component (TERC) Alone Can Enhance Proliferative Potential and Telomere Length in B Lymphocyte Lines from Dyskeratosis Congenita Patients
Michael J. Kirwan, Richard Beswick, Amit C. Nathwani, Amanda J. Walne,1 Tom Vulliamy,1 Colin Casimir,3 Inderjeet Dokal.1 1 Academic Department of Paediatrics, Barts and The London School of Medicine and Dentistry, London, United Kingdom; 2 Department of Haematology, University College London, London, United Kingdom; 3School of Life Sciences, Kingston University, London, United Kingdom. 1
1
2
Dyskeratosis congenita (DC) is an inherited multi-system disorder characterised by muco-cutaneous abnormalities, bone marrow failure and a predisposition to malignancy. Bone marrow failure is the principal cause of mortality and is thought to be the result of premature cell death in the haematopoietic compartment. DC cells age prematurely and tend to have short telomeres which may account, at least in part, for the premature cell death and rapid decline in haematopoiesis seen in patients. The telomerase complex is important in maintaining telomeres and DC patients have mutations in genes that encode several of its components including the telomerase reverse transcriptase, TERT, and RNA component, TERC, and the nucleolar proteins dyskerin and NOP10. Previous studies in DC have used fibroblasts transduced with TERT but we wished to study haematopoietic cells to get closer to the mechanism of the pathology observed in vivo. B lymphocyte lines are typically difficult to establish from DC patients and tend to grow poorly by comparison to those derived from normal, healthy volunteers. We transduced B lymphocyte lines established from patients with TERC and DKC1 (dyskerin) mutations with wild type TERC and DKC1-bearing lentiviral vectors. We found that transduction with exogenous TERC alone was capable of improving the survival and thus overall growth of mutant lines over a prolonged period, regardless of their mutation and also resulted in telomere elongation. However, exogenous DKC1 had no beneficial effect even on DKC1 mutant lines. This study is the first of its kind in DC lymphocytes and the first to demonstrate that transduction with TERC alone can improve cell survival and lengthen telomeres without the need for exogenous TERT. The effects on telomere length and cell survival provided by exogenous, wild type TERC, regardless of the underlying pathological mutation, directly implicate telomerase dysfunction as a cause of DC symptoms and as a therapeutic target in cells that would normally express telomerase in vivo.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
624. Correction of Mutant Fanconi Anemia Gene by Homologous Recombination in Human Hematopoietic Cells Using Adeno-Associated Virus Vector
Kittiphong Paiboonsukwong,1 Fumi Ohbayashi,1 Haruka Shiiba,1 Takayuki Yamashita,2 Kohnosuke Mitani.1 1 Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan; 2Laboratory of Molecular Genetics, Department of Molecular and Cellular Biology, Institute of Molecular and Cellular Regulation, Gunma University, Gunma, Japan. Adeno-associated virus (AAV) vectors have been utilized to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which would overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. Here, we report an application of AAV-mediated gene targeting to disrupt the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus in a normal human B-lymphoblastoid cell line (BCL) and to correct a mutation in the Fanconi anemia group A (FANCA) gene in BCL derived from a Fanconi anemia (FA) patient. We first examined the transduction efficiency of normal BCL cells by using a self-complementary AAV vector encoding for GFP (scAAV-GFP) packaged into eight AAV serotypes (AAV-1, -2, -3, -4, -7, -8, -9, and -10). At a multiplicity of infection (MOI) of 1 x 105 vector genomes/cell, approximately 40% and 50% of BCL cells were transduced and became GFP-positive by AAV-1 and AAV-2, respectively, while the rest of serotypes achieved a percentage of positive cells lower than 10%. This result suggests that AAV-2, which is known to transduce a wide range of cell types in vitro, also efficiently transduces BCL. After infection of normal BCL cells with an AAV-2/HPRT1 targeting vector at an MOI of 300 and selection with G418 and thereafter with 6-thioguanine, we estimated the chromosomal integration and HPRT1-knockout frequencies to be 7.3 x 10-6 and 2.1 x 10-6 per infected cell, respectively. Gene targeting events by HR were confirmed by Southern blot analyses. Furthermore, we constructed an AAV-2/FANCA targeting vector designed to correct a mutant allele on the FANCA gene of BCL cells established from a FA patient. In this cell lines, a maternal allele of FANCA has a deletion of C in exon 27 (designated 2546delC), which is the most common pathogenic mutation among Japanese FANCA patients. BCL cells infected with this vector, which encodes a 4.4 kb normal FANCA sequence surrounding exon 27, at an MOI of 1 x 104 and selected for with 10-20 nM of mitomycin C (MMC) produced MMC-resistant cell clones at a frequency of 1.6 x 10-4 per infected cell (about 1 out of 6400 infected cells). Subsequent analyses by PCR-based RFLP and sequencing showed the genotypic correction of the FANCA gene in these MMC-resistant clones. The FANCA-corrected clones had restored FANCA expression as revealed by Western blot analyses. Cellular hypersensitivity to MMC, which is the characteristic of FA cells, was also corrected to a normal level in these clones. In summary, this study showed that AAV-2 vectors are able to efficiently transduce and target a gene by HR in EBV-transformed human BCL derived from normal and FA patients. This strategy may therefore be applicable for gene correction in hematopoietic stem cells. As the gene targeting efficiency is improved, AAV-based gene repair therapy in various inherited hematopoietic diseases could become a reality.
S233