- Email: [email protected]
231. Large Scale Production and Scale Up of DNA Plasmid Vectors With Complex Gene Inserts
CLINICAL TRANSLATION OF VECTOR PRODUCTION AND PROTOCOL PREPARATION I 228. Production of Self Complementary AAV in Bioreactors: Comparison of Baculovirus Expression System and Transfection of Suspension Cells
Christine Le Bec,1 Nicolas Marceau,1 Mohammed Rifki,1 Alban Vignaud,1 Matthias Hebben.1 1 Genethon, Evry, France. Gene therapy treatments usually require large amounts of viral vectors, making the manufacturing step one of the main limitations for the clinical development. AAV vectors are originally produced by triple transfection of adherent HEK293 cells, which is a poorly scalable procedure. The introduction of Baculovirus Expression System allowed releasing this bottleneck since the process is transposable in large scale bioreactors and has proven to be efficient for the production of commercial lots of AAV as recently illustrated by the treatment for lipoprotein lipase deficiency (Glybera®). However, the production of self complementary AAVs using Baculovirus has not been investigated yet. We produced a scAAV9 vector using the Baculovirus system in 10L glass bioreactors. In parallel, we developed a production process of the same vector by transfection of HEK293 cells grown in suspension in bioreactors at same scale. A full comparison of scAAV9 produced by the 2 processes was performed: vector productivity, ratio full/empty particles, vector genome profile, and efficacy in an animal model of spinal muscular atrophy. The results showed that the two systems do not generate comparable material for this scAAV9 model despite similar efficacy in vivo. In conclusion, both systems have advantages and drawbacks that must be considered before clinical development.
229. Full Genomic Deep Sequence Analysis of the Genetic Stability of a First-Generation Adenoviral Vector During Serial Passage in Culture
Brian W. Guzik,1 Qin Xiang,2 Katherine Manzanera,1 Steve E. Scherer,2 Estuardo Aguilar-Cordova.1 1 Advantagene, Auburndale, MA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
The use of viral vectors in gene delivery applications offers many advantages, but an inherent challenge in GMP manufacture of these biological agents is DNA sequence variation that may occur during large-scale production. Depending on the biology of the vector system (e.g, lentivirus, adenovirus, alphavirus), mutation rates vary and result in genetic heterogeneity. As the scale of amplification increases, so to would the expected frequency of minor variants. Sequence quality control for clinical grade vectors has typically been performed by a few isolated clones. Advances in modern sequencing techniques during recent years provide broad access to a much deeper analysis of the genetic population across the whole vector genome. The goal of this study is to examine the sequence stability of a first generation adenoviral vector as it is pushed through eleven generations of amplification. The vector used has an HSV thymidine kinase transgene (AdV-tk). It was manufactured under GMP conditions from a master vector bank. A reference bank and sequence were used for comparison. The vector was serially amplified in suspension HEK293 cells. The impact of the vector generation, as well as the producer cell generation was evaluated using modern ‘deep sequencing’ methods to characterize the variability in the isolated vector populations throughout out the passages. DNA was isolated from the vector population at eight stages during the eleven generation passage of the vector population. Genomic vector sequences were determined using Illumina MiSeq technology with an average coverage of 5,000 to 10,000-fold per nucleotide, allowing detection of minor variants. An analysis of
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
the DNA sequence variations observed and their evolution through generations are discussed, in addition to methods and parameter of the sequence analysis. This study provides a base-by-base, per generation analysis of the frequency and quality of changes observed within a first-generation adenoviral vector population during serial passage and provides insight into the critical nature for control of components and process used in the production of complex biologics.
230. Modifying Retroviral Vector Pseudotyped With RRV Envelope Glycoproteins Makes It Utilize Heparan Sulfate as an Attachment Factor
Aditi Kesari,1 David Sanders.1 1 Department of Biological Sciences, Purdue University, West Lafayette, IN.
Retroviruses are popularly used as gene-therapy vectors. However, since retroviruses are limited in the range of cells they can enter and infect, retroviral vectors cannot be used for treating systemic genetic diseases. Hence, they are pseudotyped with envelopes of other viruses that have wide tissue tropism, to form a hybrid virus. In our lab, we pseudotyped Moloney Murine Leukemia Virus (MoMLV), a retrovirus, with the envelope of Ross River Virus (RRV), an alphavirus. Studies have shown that retrovirus pseudotyped with RRV envelope glycoproteins can be successfully used for in vivo gene transfer (Davidson et al. J Virol. 2002 Sep;76(18):9378-88). To further increase the transduction efficiency of this potential gene therapy vector, we substituted amino-acid residues of its RRV-envelope glycoprotein with basic amino-acid residues. This increases the affinity of the virus towards a negatively charged molecule - heparan sulfate, which is present on the surfaces of most cells. Attachment of the virus to the cellular heparan sulfate increases its concentration on the cell surface thereby increasing their chances of cell entry. This increases the transduction efficiency of the virus. Although the cellular heparan sulfate facilitates entry of virus, the heparan sulfate expressed on surfaces of producer cells holds back the pseudotyped virus with mutated envelope glycoproteins thus affecting its release. Hence, one of our future goals would be to develop a producer cell line that doesn’t express heparan sulfate for the generation of such a pseudotyped viral gene-therapy vector.
231. Large Scale Production and Scale Up of DNA Plasmid Vectors With Complex Gene Inserts
Neha Tiwari,1 Jill Beilowitz,1 Carlos Sampson,1 Dorothy Peterson.1 Process Development, VGXI.Inc, The Woodlands, TX.
1
DNA vaccines are showing promising results for the treatment of various diseases including viral infections and cancers during early and late clinical phases. However, for any DNA vaccine to be clinically and commercially successful, it is essential that plasmid vectors containing the gene insert be efficiently produced at large scale utilizing a process that has the capability for scale up. Many plasmid vectors contain gene inserts with highly repetitive sequences or viral sequences which are difficult to be produced in the standard E.coli strain when grown at large scale. We have successfully developed production strategies involving cell line optimization and sequence optimization to produce such plasmid vectors in E.coli at large scale. Plasmid pVGXI1 (4.2kb) failed to grow initially at 10L fermentation scale (OD<15, plasmid yield:0.6g/10L). With cell line optimization, it was successfully grown at 10L fermentation scale (OD>40, plasmid yield 2.4g/10L). The process was efficaciously scaled up to 100L and 400L scale. Another plasmid, pVGXI2 (4.6kb) consisted of high A residue repeats and low GC% in its sequence. We tested it in 3 different E.coli cell lines at 10L scale. It either failed to grow (OD<10) or produced very little plasmid (plasmid yield: 0.7g/10L) in each of the tested cell lines. By S87
PRESIDENTIAL SYMPOSIUM performing sequence optimization, pVGXI2 was grown successfully at 10L scale with high OD (OD>50) and plasmid yield (plasmid yield 3g/10L) in a standard E.coli strain. With the outstanding results of process optimization, both these plasmids were successfully grown with 4 fold increase in fermentation yield and then successfully scaled up. Furthermore, purification of these plasmids yielded high quality DNA products suitable for clinical use.
directed treatment approach to herPAP which may serve as a proofof-principle to extend current HSC-based gene therapy concepts to strategies utilizing more differentiated, long-lived cells.
Presidential Symposium
S. Scaramuzza,1 S. Giannelli,1 F. Ferrua,2 M. C. Castiello,1 M. P. Cicalese,2 A. Assanelli,2 L. Biasco,1 G. Ottaviano,2 M. Casiraghi,2 M. Bosticardo,1 P. Rizzardi,3 A. Finocchi,4 A. Metin,5 M. Albert,6 C. Petrescu,7 P. P. Banerjee,8 J. S. Orange,8 A. Biffi,1,2 F. Ciceri,9 A. Villa,1,10 M. G. Roncarolo,1,2,11 L. Naldini,1,11 A. Aiuti.1,4 1 San Raffaele Telethon Institute (HSR-Tiget), Milan, Italy; 2 Pediatrc Immunology, Scientific Institute HS Raffaele, Milan, Italy; 3MolMed SpA, Milan, Italy; 4Univ. of Rome “Tor Vergata”, Rome, Italy; 5Ankara Dispaki Children’s Hospital, Ankara, Turkey; 6Pediatric Hematology/Oncology, Dr. Von Haunersches Kinderspital der LMU, Munich, Germany; 7Oncohematology, Timisoara Hospital, Timisoara, Romania; 8Texas Children’s Hospital, Baylor College of Medicine, Houston; 9Div. of Hematology, Scientific Institute HS Raffaele, Milan, Italy; 10IRGBCNR, Milan, Italy; 11Univ. Vita-Salute San Raffaele, Milan, Italy.
232. Therapeutic Efficacy of Intratracheally Transplanted Macrophage Progenitor Cells Introduces a Novel Gene-Therapy Approach To Hereditary Pulmonary Alveolar Proteinosis
Nico Lachmann,1,2 Christine Happle,3 Takuji Suzuki,4 Paritha Arumugam,5 Jens Bankstahl,6 Punam Malik,5 Bruce Trapnell,4 Gesine Hansen,3 Thomas Moritz.1,2 1 Institute of Experimental Haematology, Hannover Medical School, Hannover, Germany; 2Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany; 3Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; 4Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH; 5Division of Experimental Hematology and Cancer Biology, Children’s Hospital Medical Center, Cincinnati, OH; 6Institute for Preclinical Molecular Imaging, Hannover Medical School, Hannover, Germany. Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colonystimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce intratracheal (i.t.) transplantation of hematopoietic stem cell (HSC) derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, i.t. transplantation of MPs showed selective pulmonary engraftment of donor cells on flow- and chipcytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as normalisation of lung function parameter and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface markers, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following i.t. transplantation of MPs differentiated from lentivirally corrected Csf2rb-/- HSCs. In vitro these genecorrected HSCs expanded up to 1045-fold, and differentiated into typical macrophages expressing F4/80, CD11b, CD11c, and CD68, as well as Csf2rb mRNA and protein (CD131) with reconstitution of GM-CSF receptor signaling. Significant improved clinical benefit was demonstrated by biomarkers in i.t. transplanted herPAP mice in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, i.t. administration of human CD34+ -derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 cells led to longterm human cell engraftment and reduce alveolar-fluid proteins by 50-70%. CT scans six months after i.t. cell application revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities, and normalisation of the inspiratory to expiratory lung volume ratio. Thus, we here describe a new, cause S88
233. Persistent Multilineage Engraftment and WASP Restored Expression After Lentiviral Mediated CD34+ Cells Gene Therapy for the Treatment of Wiskott-Aldrich Syndrome
Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. Gene therapy (GT) with ex vivo transduced hematopoietic stem cells (HSC) could represent a valid therapeutic option for patients lacking an HLA-identical donor. We developed an approach based on a lentiviral vector (LV) encoding for WAS under the control of the homologous 1.6 kb WAS promoter. A phase I/II clinical trial based on infusion of autologous transduced CD34+ cells and reduced intensity conditioning started in June 2010. Six patients were treated with autologous bone marrow (BM) or mobilized peripheral blood (PB) derived CD34+ cells transduced with highly purified LV. Transduction of clonogenic progenitors was highly efficient (94.4 ± 4.2%), with a mean VCN/genome in bulk CD34+ cells of 2.7 ± 0.98. In the first 4 patients treated (follow up 1.5-3.5 years) engraftment remains stable both in BM cells (VCN in myeloid lineages: 0.36-1.51), clonogenic progenitors (21.9-48.2%), as well as in PB myeloid cells (VCN: 0.24-1.20) and T and B lymphocyte (VCN: 1.47-2.66). Accordingly, WASP expression, measured by flow cytometry, was observed in monocytes (range 23-55%) and in a higher percentage of lymphoid cells (WASP+ cells: 75.4 ± 2.3%). Among T lymphocytes, CD8+ cells and memory T cells showed the highest proportion of WASp expressing cells. TCR driven proliferation was improved also at low doses of anti-CD3 monoclonal antibody, which are typically defective in WAS. TCR repertoire and immunological functions, including NK cytotoxic ability, and Treg function, were also improved after GT. WASP expression was also restored in the majority of platelets (82.1± 5.6%), leading to an increase in their number. All patients are currently clinically well, independent from platelet transfusions. Insertions analyses confirm that hematopoiesis remains highly polyclonal, in the absence of aberrant clonal expansion. In conclusion, the high level of gene transfer obtained with LV-WAS results in stable engraftment of transduced HSC and restored WAS expression even when combined to reduced intensity conditioning. Although a longer observation is required to establish the long-term safety, lentiviral GT represents a promising treatment for WAS.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Christine Le Bec,1 Nicolas Marceau,1 Mohammed Rifki,1 Alban Vignaud,1 Matthias Hebben.1 1 Genethon, Evry, France. Gene therapy treatments usually require large amounts of viral vectors, making the manufacturing step one of the main limitations for the clinical development. AAV vectors are originally produced by triple transfection of adherent HEK293 cells, which is a poorly scalable procedure. The introduction of Baculovirus Expression System allowed releasing this bottleneck since the process is transposable in large scale bioreactors and has proven to be efficient for the production of commercial lots of AAV as recently illustrated by the treatment for lipoprotein lipase deficiency (Glybera®). However, the production of self complementary AAVs using Baculovirus has not been investigated yet. We produced a scAAV9 vector using the Baculovirus system in 10L glass bioreactors. In parallel, we developed a production process of the same vector by transfection of HEK293 cells grown in suspension in bioreactors at same scale. A full comparison of scAAV9 produced by the 2 processes was performed: vector productivity, ratio full/empty particles, vector genome profile, and efficacy in an animal model of spinal muscular atrophy. The results showed that the two systems do not generate comparable material for this scAAV9 model despite similar efficacy in vivo. In conclusion, both systems have advantages and drawbacks that must be considered before clinical development.
229. Full Genomic Deep Sequence Analysis of the Genetic Stability of a First-Generation Adenoviral Vector During Serial Passage in Culture
Brian W. Guzik,1 Qin Xiang,2 Katherine Manzanera,1 Steve E. Scherer,2 Estuardo Aguilar-Cordova.1 1 Advantagene, Auburndale, MA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
The use of viral vectors in gene delivery applications offers many advantages, but an inherent challenge in GMP manufacture of these biological agents is DNA sequence variation that may occur during large-scale production. Depending on the biology of the vector system (e.g, lentivirus, adenovirus, alphavirus), mutation rates vary and result in genetic heterogeneity. As the scale of amplification increases, so to would the expected frequency of minor variants. Sequence quality control for clinical grade vectors has typically been performed by a few isolated clones. Advances in modern sequencing techniques during recent years provide broad access to a much deeper analysis of the genetic population across the whole vector genome. The goal of this study is to examine the sequence stability of a first generation adenoviral vector as it is pushed through eleven generations of amplification. The vector used has an HSV thymidine kinase transgene (AdV-tk). It was manufactured under GMP conditions from a master vector bank. A reference bank and sequence were used for comparison. The vector was serially amplified in suspension HEK293 cells. The impact of the vector generation, as well as the producer cell generation was evaluated using modern ‘deep sequencing’ methods to characterize the variability in the isolated vector populations throughout out the passages. DNA was isolated from the vector population at eight stages during the eleven generation passage of the vector population. Genomic vector sequences were determined using Illumina MiSeq technology with an average coverage of 5,000 to 10,000-fold per nucleotide, allowing detection of minor variants. An analysis of
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
the DNA sequence variations observed and their evolution through generations are discussed, in addition to methods and parameter of the sequence analysis. This study provides a base-by-base, per generation analysis of the frequency and quality of changes observed within a first-generation adenoviral vector population during serial passage and provides insight into the critical nature for control of components and process used in the production of complex biologics.
230. Modifying Retroviral Vector Pseudotyped With RRV Envelope Glycoproteins Makes It Utilize Heparan Sulfate as an Attachment Factor
Aditi Kesari,1 David Sanders.1 1 Department of Biological Sciences, Purdue University, West Lafayette, IN.
Retroviruses are popularly used as gene-therapy vectors. However, since retroviruses are limited in the range of cells they can enter and infect, retroviral vectors cannot be used for treating systemic genetic diseases. Hence, they are pseudotyped with envelopes of other viruses that have wide tissue tropism, to form a hybrid virus. In our lab, we pseudotyped Moloney Murine Leukemia Virus (MoMLV), a retrovirus, with the envelope of Ross River Virus (RRV), an alphavirus. Studies have shown that retrovirus pseudotyped with RRV envelope glycoproteins can be successfully used for in vivo gene transfer (Davidson et al. J Virol. 2002 Sep;76(18):9378-88). To further increase the transduction efficiency of this potential gene therapy vector, we substituted amino-acid residues of its RRV-envelope glycoprotein with basic amino-acid residues. This increases the affinity of the virus towards a negatively charged molecule - heparan sulfate, which is present on the surfaces of most cells. Attachment of the virus to the cellular heparan sulfate increases its concentration on the cell surface thereby increasing their chances of cell entry. This increases the transduction efficiency of the virus. Although the cellular heparan sulfate facilitates entry of virus, the heparan sulfate expressed on surfaces of producer cells holds back the pseudotyped virus with mutated envelope glycoproteins thus affecting its release. Hence, one of our future goals would be to develop a producer cell line that doesn’t express heparan sulfate for the generation of such a pseudotyped viral gene-therapy vector.
231. Large Scale Production and Scale Up of DNA Plasmid Vectors With Complex Gene Inserts
Neha Tiwari,1 Jill Beilowitz,1 Carlos Sampson,1 Dorothy Peterson.1 Process Development, VGXI.Inc, The Woodlands, TX.
1
DNA vaccines are showing promising results for the treatment of various diseases including viral infections and cancers during early and late clinical phases. However, for any DNA vaccine to be clinically and commercially successful, it is essential that plasmid vectors containing the gene insert be efficiently produced at large scale utilizing a process that has the capability for scale up. Many plasmid vectors contain gene inserts with highly repetitive sequences or viral sequences which are difficult to be produced in the standard E.coli strain when grown at large scale. We have successfully developed production strategies involving cell line optimization and sequence optimization to produce such plasmid vectors in E.coli at large scale. Plasmid pVGXI1 (4.2kb) failed to grow initially at 10L fermentation scale (OD<15, plasmid yield:0.6g/10L). With cell line optimization, it was successfully grown at 10L fermentation scale (OD>40, plasmid yield 2.4g/10L). The process was efficaciously scaled up to 100L and 400L scale. Another plasmid, pVGXI2 (4.6kb) consisted of high A residue repeats and low GC% in its sequence. We tested it in 3 different E.coli cell lines at 10L scale. It either failed to grow (OD<10) or produced very little plasmid (plasmid yield: 0.7g/10L) in each of the tested cell lines. By S87
PRESIDENTIAL SYMPOSIUM performing sequence optimization, pVGXI2 was grown successfully at 10L scale with high OD (OD>50) and plasmid yield (plasmid yield 3g/10L) in a standard E.coli strain. With the outstanding results of process optimization, both these plasmids were successfully grown with 4 fold increase in fermentation yield and then successfully scaled up. Furthermore, purification of these plasmids yielded high quality DNA products suitable for clinical use.
directed treatment approach to herPAP which may serve as a proofof-principle to extend current HSC-based gene therapy concepts to strategies utilizing more differentiated, long-lived cells.
Presidential Symposium
S. Scaramuzza,1 S. Giannelli,1 F. Ferrua,2 M. C. Castiello,1 M. P. Cicalese,2 A. Assanelli,2 L. Biasco,1 G. Ottaviano,2 M. Casiraghi,2 M. Bosticardo,1 P. Rizzardi,3 A. Finocchi,4 A. Metin,5 M. Albert,6 C. Petrescu,7 P. P. Banerjee,8 J. S. Orange,8 A. Biffi,1,2 F. Ciceri,9 A. Villa,1,10 M. G. Roncarolo,1,2,11 L. Naldini,1,11 A. Aiuti.1,4 1 San Raffaele Telethon Institute (HSR-Tiget), Milan, Italy; 2 Pediatrc Immunology, Scientific Institute HS Raffaele, Milan, Italy; 3MolMed SpA, Milan, Italy; 4Univ. of Rome “Tor Vergata”, Rome, Italy; 5Ankara Dispaki Children’s Hospital, Ankara, Turkey; 6Pediatric Hematology/Oncology, Dr. Von Haunersches Kinderspital der LMU, Munich, Germany; 7Oncohematology, Timisoara Hospital, Timisoara, Romania; 8Texas Children’s Hospital, Baylor College of Medicine, Houston; 9Div. of Hematology, Scientific Institute HS Raffaele, Milan, Italy; 10IRGBCNR, Milan, Italy; 11Univ. Vita-Salute San Raffaele, Milan, Italy.
232. Therapeutic Efficacy of Intratracheally Transplanted Macrophage Progenitor Cells Introduces a Novel Gene-Therapy Approach To Hereditary Pulmonary Alveolar Proteinosis
Nico Lachmann,1,2 Christine Happle,3 Takuji Suzuki,4 Paritha Arumugam,5 Jens Bankstahl,6 Punam Malik,5 Bruce Trapnell,4 Gesine Hansen,3 Thomas Moritz.1,2 1 Institute of Experimental Haematology, Hannover Medical School, Hannover, Germany; 2Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany; 3Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; 4Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH; 5Division of Experimental Hematology and Cancer Biology, Children’s Hospital Medical Center, Cincinnati, OH; 6Institute for Preclinical Molecular Imaging, Hannover Medical School, Hannover, Germany. Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colonystimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce intratracheal (i.t.) transplantation of hematopoietic stem cell (HSC) derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, i.t. transplantation of MPs showed selective pulmonary engraftment of donor cells on flow- and chipcytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as normalisation of lung function parameter and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface markers, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following i.t. transplantation of MPs differentiated from lentivirally corrected Csf2rb-/- HSCs. In vitro these genecorrected HSCs expanded up to 1045-fold, and differentiated into typical macrophages expressing F4/80, CD11b, CD11c, and CD68, as well as Csf2rb mRNA and protein (CD131) with reconstitution of GM-CSF receptor signaling. Significant improved clinical benefit was demonstrated by biomarkers in i.t. transplanted herPAP mice in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, i.t. administration of human CD34+ -derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 cells led to longterm human cell engraftment and reduce alveolar-fluid proteins by 50-70%. CT scans six months after i.t. cell application revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities, and normalisation of the inspiratory to expiratory lung volume ratio. Thus, we here describe a new, cause S88
233. Persistent Multilineage Engraftment and WASP Restored Expression After Lentiviral Mediated CD34+ Cells Gene Therapy for the Treatment of Wiskott-Aldrich Syndrome
Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. Gene therapy (GT) with ex vivo transduced hematopoietic stem cells (HSC) could represent a valid therapeutic option for patients lacking an HLA-identical donor. We developed an approach based on a lentiviral vector (LV) encoding for WAS under the control of the homologous 1.6 kb WAS promoter. A phase I/II clinical trial based on infusion of autologous transduced CD34+ cells and reduced intensity conditioning started in June 2010. Six patients were treated with autologous bone marrow (BM) or mobilized peripheral blood (PB) derived CD34+ cells transduced with highly purified LV. Transduction of clonogenic progenitors was highly efficient (94.4 ± 4.2%), with a mean VCN/genome in bulk CD34+ cells of 2.7 ± 0.98. In the first 4 patients treated (follow up 1.5-3.5 years) engraftment remains stable both in BM cells (VCN in myeloid lineages: 0.36-1.51), clonogenic progenitors (21.9-48.2%), as well as in PB myeloid cells (VCN: 0.24-1.20) and T and B lymphocyte (VCN: 1.47-2.66). Accordingly, WASP expression, measured by flow cytometry, was observed in monocytes (range 23-55%) and in a higher percentage of lymphoid cells (WASP+ cells: 75.4 ± 2.3%). Among T lymphocytes, CD8+ cells and memory T cells showed the highest proportion of WASp expressing cells. TCR driven proliferation was improved also at low doses of anti-CD3 monoclonal antibody, which are typically defective in WAS. TCR repertoire and immunological functions, including NK cytotoxic ability, and Treg function, were also improved after GT. WASP expression was also restored in the majority of platelets (82.1± 5.6%), leading to an increase in their number. All patients are currently clinically well, independent from platelet transfusions. Insertions analyses confirm that hematopoiesis remains highly polyclonal, in the absence of aberrant clonal expansion. In conclusion, the high level of gene transfer obtained with LV-WAS results in stable engraftment of transduced HSC and restored WAS expression even when combined to reduced intensity conditioning. Although a longer observation is required to establish the long-term safety, lentiviral GT represents a promising treatment for WAS.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy