- Email: [email protected]
1041. Lentiviral Vector Tropism to Skin Tissue
TISSUE ENGINEERING & OTHER CELL THERAPIES II We think this vaccine manufacturing process and its associated release testing can be accomplished in a reasonable timeframe to be clinically relevant in advance cancer patients. We will present data on the tumor harvest / purification process as well as the xenograft vaccine quality control testing plan.
1039. Gene Delivery to Bone Marrow Cells Identifies Cells That Migrate to the Vessels of the CNS and Allows for Gene Delivery to CNS Vasculature
Jean-Pierre Louboutin,1 Alena A. Chekmasova,1 Beverly S. Reyes,2 Elisabeth J. van Bockstaele,2 David S. Strayer.1 1 Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA; 2Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA. Using bone marrow-directed gene transfer and recombinant SV40derived vectors, we previously demonstrated that bone marrowderived cells may function as progenitors of CNS cells in adult animals. To accomplish this, SV40-derived gene delivery vectors, carrying marker epitopes (FLAG or AU1), appended to carrier protein, were injected directly into femoral bone marrow (BM). Ttransgene expression was examined in the blood and the brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. Transduction was limited to cells in the femoral marrow at the time of injection. An average of 5% of peripheral blood cells, and 25% of femoral marrow cells, were FLAG+ throughout the study. In the CNS, FLAGexpressing cells were mainly detected in the dentate gyrus (DG) and the periventricular subependymal zone (PSZ). Though absent before 1 month and rare at 4 months, DG and PSZ FLAG+ cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggested that a population(s) of cells resident within adult BM can migrate to the brain and differentiate into CNS-specific cells. We then tested whether BM progenitor cells could migrate in other areas of the CNS. A rSV40 vector, carrying RevM10 with a carboxyl-terminal AU1 epitope was injected into the femoral BM. Transgene (RevM10. AU1) expression was examined in the blood 4 months after injection. Controls animals received intramarrow SV(BUGT), a control vector. Transgene-positive cells were found in the vessels of the striatum 4 months after injection of SV(RevM10.AU1). AU1-positive cells were mainly localized in laminin- or CD31-positive structures (markers of brain vessels). These results suggest that BM-derived, transgene expressing cells can migrate to brain vessels. They also suggest that intramarrow gene delivery may provide an avenue to deliver genes to the vascular endothelium.
1040. The New Regulation of Advanced Therapy Medicinal Products in the European Union
Sol Ruiz.1 1 Biologics and Biotechnology, Spanish Medicines Agency, Madrid, Spain. Since the EMEA (European Medicines Agency) was created in 1995 marketing authorization applications (MAA) for biotechnological products (i.e. those obtained using rDNA technology) are assessed following the so called centralized procedure coordinated by the EMEA. A positive outcome of this evaluation results in the marketing authorization of the medicinal product in all members states of the European Union (EU). Gene therapy products are included in this category and, therefore, any MAA follows the same procedure. However, the situation is different for cell therapy products and their classification varies greatly among member states. Furthermore, if a cell therapy product is combined with a medical device, the situation Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
is even more complex as medical devices fall under a different regulation than medicinal products. This is the reason for the European Commission’s initiative to develop a new law for medicinal products based on gene therapy, cell therapy and tissue engineering defined in the regulation as advanced therapy medicinal products (ATMP). The main objective of this new legislation (Regulation (EC) no 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products) that is applicable from 30 December 2008 is to improve patients’ safe access to advanced therapies by increasing the research, development and authorisation of these products and more specifically: to guarantee a high level of health protection for European patients treated with ATMP; to harmonise market access and ensure the free movement of these products by establishing a tailored and comprehensive regulatory framework for their authorisation, supervision and post-authorisation vigilance; to foster the competitiveness of European undertakings operating in this field, in particular small and medium-sized enterprises; and to provide overall legal certainty, while allowing for sufficient flexibility at the technical level, in order to keep pace with the evolution of science and technology. The key measures included in the proposal are: A centralised marketing authorisation procedure for all ATMP, to benefit from the pooling of expertise at the European level and direct access to the EU market; A new and multidisciplinary expert committee (Committee for Advanced Therapies, CAT), within the EMEA, gathering European experts in many different areas to assess the quality, safety and efficacy of ATMP and follow scientific developments in the field; Tailored technical requirements, which are adapted to the particular characteristics of these products; Strengthened requirements for risk management and traceability; A system of low-cost, top-quality scientific advice provided by the EMEA; Special financial and administrative incentives for small and medium-sized enterprises. These incentives include the scientific evaluation (certification) of quality and non-clinical data to facilitate further product development. The CAT had its first meeting in January 2009. An update on the ongoing activities of the CAT and the implementation of the new regulation will be presented.
1041.
Lentiviral Vector Tropism to Skin Tissue
Nikolai Kunicher,1 Haya Falk,1 Barak Yaacov,1 Tomer Tzur,2 Amos Panet.1 1 Virology, Hebrew University, Jerusalem, Israel; 2Plastic and Aesthetic Surgery, The Hadassah Medical Center, Jerusalem, Israel. Background: Skin diseases are attractive targets for gene therapy as the tissue is easily accessible for the evaluation of efficacy and toxicity. Understanding viral tropism to human skin is a prerequisite for the treatment of coetaneous diseases with viral vectors. Using a novel ex-vivo system of organ culture, we have studied the factors that determined viral tropism to the epidermal and dermal cells in the skin. We have applied in these studies a lentiviral vector enveloped by several surface proteins that utilize different cell receptors. Methods: Organ cultures of skin were infected with lentiviral vectors pseudotyped with different envelopes and vector tropism was evaluated using histological and molecular methods. Results: At low viral titers infection of mice and human skin tissues was targeted to the basal membrane zone (BMZ), in between the dermis and the epidermis layers. Infection of separated epidermis and dermis layers indicated very little infection in the epidermal layer. Yet, analysis of the viral receptor distribution in the two skin layers indicated expression of the receptor in cells of both the dermis and epidermis. To investigate the level at which the virus is blocked within the epidermis, different stages of viral infection were analyzed. We found that the lentiviral vector was able to enter epidermal cells, normal reverse transcription takes place but no integration occurs. Immuno-histochemical analysis of the infected BMZ showed that early progenitor keratinocytes (as determined by P-63 marker) were resistant to infection, while S395
TISSUE ENGINEERING & OTHER CELL THERAPIES II other cells such as fibroblasts were infected. Several keratinocytes differentiation markers (such as keratin 14 and 15) were used to investigate the origin of the infected cells in the hair follicle. Our results show that infection occurred in Transit Amplifying Cells in the follicle bulge area but early progenitors were resistant to infection. Conclusion: We show here for the first time that viral vector tropism in a solid tissue is determined by several factors, distinct of the cell receptor, among them intra cellular components. Furthermore, our results indicate that susceptibility of keratinocytes to infection with lentiviral vector depends on their differentiation stage.
1042. Novel Role of Substance P as Inducing Growth Factor for Expansion of CD34+ CD38- Cord Blood Cells
Somayeh Shahrokhi,1 Kamran Alimoghaddam,2 Massoumeh Ebtekar,1 Ali Akbar Pourfathollah,1 Maryam Kheirandish,3 Alireza Ardjmand,2 Ardeshir Ghavamzadeh.2 1 Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran; 2 Hematology, Oncology and Stem Cell Transplantation Research Center of Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; 3Research Center, Department of Immunology, Iranian Blood Transfusion Organization, Tehran, Islamic Republic of Iran.
Introduction: Ex vivo expansion of Hematopoietic stem cells (HSCs) of Umbilical Cord Blood (UCB) has been progressively interested. Most experiments evaluated proliferative inducing activity in HSCs, diverse range of cytokines; however, there is little evidence from other regulatory factors such as neuropeptides. According to existence of Substance P (SP) as well as its receptor on cord blood CD34+cells, we aimed to explore novel approach of HSCs expansion, using Substance P (SP). Material and Methods: CD34+ purified from cord blood, were cultured in a serum-free liquid culture system. Different concentration and combination of SP used in combination with cytokine cocktail of SCF, FL, TPO, IL3 and IL6. Phenotypic and functional analysis of the cell growth in culture was performed by Flowcytometry and Colonogenic assay. Results: Our results showed more than 2 fold total expansion of cytokine-neuropeptide treated cells in comparison to cells cultivated just in conventional cytokine cocktail(56 versus 24), Also, selective expansion of CD34+CD38cells observed in 10-9 M of SP treated cells for 7 day. Conclusions: Our findings suggest that SP neuropeptides could act as a novel supplement for cytokine cocktails to expand UCB-HSC. Considering role of other growth factor such as neuropeptides along with cytokines may enable us to succeed in dealing with the difficulties in ex vivo expansion of cord blood cells.
S396
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
1039. Gene Delivery to Bone Marrow Cells Identifies Cells That Migrate to the Vessels of the CNS and Allows for Gene Delivery to CNS Vasculature
Jean-Pierre Louboutin,1 Alena A. Chekmasova,1 Beverly S. Reyes,2 Elisabeth J. van Bockstaele,2 David S. Strayer.1 1 Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA; 2Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA. Using bone marrow-directed gene transfer and recombinant SV40derived vectors, we previously demonstrated that bone marrowderived cells may function as progenitors of CNS cells in adult animals. To accomplish this, SV40-derived gene delivery vectors, carrying marker epitopes (FLAG or AU1), appended to carrier protein, were injected directly into femoral bone marrow (BM). Ttransgene expression was examined in the blood and the brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. Transduction was limited to cells in the femoral marrow at the time of injection. An average of 5% of peripheral blood cells, and 25% of femoral marrow cells, were FLAG+ throughout the study. In the CNS, FLAGexpressing cells were mainly detected in the dentate gyrus (DG) and the periventricular subependymal zone (PSZ). Though absent before 1 month and rare at 4 months, DG and PSZ FLAG+ cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggested that a population(s) of cells resident within adult BM can migrate to the brain and differentiate into CNS-specific cells. We then tested whether BM progenitor cells could migrate in other areas of the CNS. A rSV40 vector, carrying RevM10 with a carboxyl-terminal AU1 epitope was injected into the femoral BM. Transgene (RevM10. AU1) expression was examined in the blood 4 months after injection. Controls animals received intramarrow SV(BUGT), a control vector. Transgene-positive cells were found in the vessels of the striatum 4 months after injection of SV(RevM10.AU1). AU1-positive cells were mainly localized in laminin- or CD31-positive structures (markers of brain vessels). These results suggest that BM-derived, transgene expressing cells can migrate to brain vessels. They also suggest that intramarrow gene delivery may provide an avenue to deliver genes to the vascular endothelium.
1040. The New Regulation of Advanced Therapy Medicinal Products in the European Union
Sol Ruiz.1 1 Biologics and Biotechnology, Spanish Medicines Agency, Madrid, Spain. Since the EMEA (European Medicines Agency) was created in 1995 marketing authorization applications (MAA) for biotechnological products (i.e. those obtained using rDNA technology) are assessed following the so called centralized procedure coordinated by the EMEA. A positive outcome of this evaluation results in the marketing authorization of the medicinal product in all members states of the European Union (EU). Gene therapy products are included in this category and, therefore, any MAA follows the same procedure. However, the situation is different for cell therapy products and their classification varies greatly among member states. Furthermore, if a cell therapy product is combined with a medical device, the situation Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
is even more complex as medical devices fall under a different regulation than medicinal products. This is the reason for the European Commission’s initiative to develop a new law for medicinal products based on gene therapy, cell therapy and tissue engineering defined in the regulation as advanced therapy medicinal products (ATMP). The main objective of this new legislation (Regulation (EC) no 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products) that is applicable from 30 December 2008 is to improve patients’ safe access to advanced therapies by increasing the research, development and authorisation of these products and more specifically: to guarantee a high level of health protection for European patients treated with ATMP; to harmonise market access and ensure the free movement of these products by establishing a tailored and comprehensive regulatory framework for their authorisation, supervision and post-authorisation vigilance; to foster the competitiveness of European undertakings operating in this field, in particular small and medium-sized enterprises; and to provide overall legal certainty, while allowing for sufficient flexibility at the technical level, in order to keep pace with the evolution of science and technology. The key measures included in the proposal are: A centralised marketing authorisation procedure for all ATMP, to benefit from the pooling of expertise at the European level and direct access to the EU market; A new and multidisciplinary expert committee (Committee for Advanced Therapies, CAT), within the EMEA, gathering European experts in many different areas to assess the quality, safety and efficacy of ATMP and follow scientific developments in the field; Tailored technical requirements, which are adapted to the particular characteristics of these products; Strengthened requirements for risk management and traceability; A system of low-cost, top-quality scientific advice provided by the EMEA; Special financial and administrative incentives for small and medium-sized enterprises. These incentives include the scientific evaluation (certification) of quality and non-clinical data to facilitate further product development. The CAT had its first meeting in January 2009. An update on the ongoing activities of the CAT and the implementation of the new regulation will be presented.
1041.
Lentiviral Vector Tropism to Skin Tissue
Nikolai Kunicher,1 Haya Falk,1 Barak Yaacov,1 Tomer Tzur,2 Amos Panet.1 1 Virology, Hebrew University, Jerusalem, Israel; 2Plastic and Aesthetic Surgery, The Hadassah Medical Center, Jerusalem, Israel. Background: Skin diseases are attractive targets for gene therapy as the tissue is easily accessible for the evaluation of efficacy and toxicity. Understanding viral tropism to human skin is a prerequisite for the treatment of coetaneous diseases with viral vectors. Using a novel ex-vivo system of organ culture, we have studied the factors that determined viral tropism to the epidermal and dermal cells in the skin. We have applied in these studies a lentiviral vector enveloped by several surface proteins that utilize different cell receptors. Methods: Organ cultures of skin were infected with lentiviral vectors pseudotyped with different envelopes and vector tropism was evaluated using histological and molecular methods. Results: At low viral titers infection of mice and human skin tissues was targeted to the basal membrane zone (BMZ), in between the dermis and the epidermis layers. Infection of separated epidermis and dermis layers indicated very little infection in the epidermal layer. Yet, analysis of the viral receptor distribution in the two skin layers indicated expression of the receptor in cells of both the dermis and epidermis. To investigate the level at which the virus is blocked within the epidermis, different stages of viral infection were analyzed. We found that the lentiviral vector was able to enter epidermal cells, normal reverse transcription takes place but no integration occurs. Immuno-histochemical analysis of the infected BMZ showed that early progenitor keratinocytes (as determined by P-63 marker) were resistant to infection, while S395
TISSUE ENGINEERING & OTHER CELL THERAPIES II other cells such as fibroblasts were infected. Several keratinocytes differentiation markers (such as keratin 14 and 15) were used to investigate the origin of the infected cells in the hair follicle. Our results show that infection occurred in Transit Amplifying Cells in the follicle bulge area but early progenitors were resistant to infection. Conclusion: We show here for the first time that viral vector tropism in a solid tissue is determined by several factors, distinct of the cell receptor, among them intra cellular components. Furthermore, our results indicate that susceptibility of keratinocytes to infection with lentiviral vector depends on their differentiation stage.
1042. Novel Role of Substance P as Inducing Growth Factor for Expansion of CD34+ CD38- Cord Blood Cells
Somayeh Shahrokhi,1 Kamran Alimoghaddam,2 Massoumeh Ebtekar,1 Ali Akbar Pourfathollah,1 Maryam Kheirandish,3 Alireza Ardjmand,2 Ardeshir Ghavamzadeh.2 1 Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran; 2 Hematology, Oncology and Stem Cell Transplantation Research Center of Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; 3Research Center, Department of Immunology, Iranian Blood Transfusion Organization, Tehran, Islamic Republic of Iran.
Introduction: Ex vivo expansion of Hematopoietic stem cells (HSCs) of Umbilical Cord Blood (UCB) has been progressively interested. Most experiments evaluated proliferative inducing activity in HSCs, diverse range of cytokines; however, there is little evidence from other regulatory factors such as neuropeptides. According to existence of Substance P (SP) as well as its receptor on cord blood CD34+cells, we aimed to explore novel approach of HSCs expansion, using Substance P (SP). Material and Methods: CD34+ purified from cord blood, were cultured in a serum-free liquid culture system. Different concentration and combination of SP used in combination with cytokine cocktail of SCF, FL, TPO, IL3 and IL6. Phenotypic and functional analysis of the cell growth in culture was performed by Flowcytometry and Colonogenic assay. Results: Our results showed more than 2 fold total expansion of cytokine-neuropeptide treated cells in comparison to cells cultivated just in conventional cytokine cocktail(56 versus 24), Also, selective expansion of CD34+CD38cells observed in 10-9 M of SP treated cells for 7 day. Conclusions: Our findings suggest that SP neuropeptides could act as a novel supplement for cytokine cocktails to expand UCB-HSC. Considering role of other growth factor such as neuropeptides along with cytokines may enable us to succeed in dealing with the difficulties in ex vivo expansion of cord blood cells.
S396
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy