Lentiviral Hematopoietic Stem and Progenitor Cell Gene Therapy for Wiskott-Aldrich Syndrome (WAS): Up to 8 Years of Follow up in 17 Subjects Treated Since 2010

Background: Wiskott-Aldrich syndrome (WAS) is a rare, X-linked, life-threatening primary immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP). WASP-deficient immune cells have compromised immunological synapse formation, cell migration and cytotoxicity. Thus, WAS is characterized by development of recurrent or severe infections, eczema, and increased risk of autoimmunity and malignancies. In addition, WASP deficiency results in microthrombocytopenia, leading to severe bleeding episodes. When a suitable donor is available, WAS can be treated by hematopoietic stem cell transplant (HSCT), but HSCT can be impeded by complications such as graft versus host disease, rejection and autoimmunity. Importantly, HSCT may carry higher risks in older children (>2-5 yrs) [Shin et al, 2012; Moratto et al, 2011]. An alternative approach is gene therapy (GT). We previously reported interim results of a Phase I/II clinical trial (NCT01515462) in 8 subjects treated with OTL-103, a drug product composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a lentiviral vector (LV) encoding human WASP cDNA under the control of the endogenous promoter [Ferrua et al, 2019]. We now report updated results on the safety and efficacy of OTL-103 in 17 subjects treated at San Raffaele Hospital as part of the same clinical trial or expanded access programs (EAP) with up to 8 yrs follow up (FU). Methods: NCT01515462: As described in Ferrua et al, 8 male subjects (mean age at GT: 4.8 yrs, range 1.1-12.4) were treated with OTL-103. The source of autologous CD34+ HSPCs was bone marrow (BM; n=5), mobilized peripheral blood (mPB; n=2) or both (n=1). As part of a reduced-intensity conditioning regimen, rituximab was given 22 days prior and busulfan + fludarabine during the week before OTL-103 infusion. At time of reporting, all subjects had ≥3 yrs FU (range: 3-8 yrs). EAP: 9 male subjects (11.2 yrs, 1.4-35.1) received identical treatment to subjects in the clinical trial; autologous CD34+ HSPCs source was mPB in all subjects. At time of reporting, subjects had a median of 1.4 yrs FU (range: 0.1-3.0 yrs) with 6/9 having ≥1 yr FU. Results: At last FU for all subjects (median: 3.0 yrs, range 0.1-8.0), overall survival was 94% (16/17). One EAP subject died 4.5 mo post-GT, due to deterioration of an underlying neurodegenerative condition considered unrelated to OTL-103 by investigator. To date, there have been no reports of insertional oncogenesis or replication-competent LV. While most subjects experienced adverse events (AEs) due to the reduced-intensity conditioning regimen (mainly mild or moderate), there were no reports of AEs related to OTL-103. Efficacy endpoints analyses were performed on surviving patients with ≥1 yr FU. Evidence of engraftment of genetically corrected HSPCs and LV+ colonies in BM was observed within 3 mo and persisted up to 8 yrs - the longest published FU of LV vector durability to date (Figure). WASP expression was restored after GT, shown by increases in the fraction of WASP+ lymphocytes and platelets (PLT) within 3 mo and maintained thereafter (Table). After GT, PLT counts improved, leading to a reduction of frequency and severity of bleeding events. Independence from PLT transfusions and absence of severe bleeding events were observed in all subjects by 9 mo FU (Table). Immune function improved; all evaluable patients discontinued immunoglobulin supplementation after GT (median time to discontinuation: 0.9 years after GT, range: 0.2-5 years). Furthermore, reduction in severe infection rate was observed post-GT, suggestive of immune reconstitution (Table). The decrease in bleeding events and severe infection rates occurred despite the integration of subjects into normal daily activities. Eczema progressively resolved or was reduced compared to baseline. Conclusions: This combined analysis of 17 subjects treated in a clinical trial or EAP with up to 8 yrs FU demonstrates that GT continues to be an effective treatment for WAS. All surviving subjects achieved high levels of multilineage engraftment, sustained restoration of WASP expression in lymphocytes and PLTs, improved PLT counts, and fewer bleeding events. A significant reduction in severe infection rate suggests reconstitution of immune function. Importantly, clinical benefit was also attained in older subjects (>5 yrs), a group considered at higher risk when treated with allogeneic HSCT. Disclosures Jones: Orchard Therapeutics: Employment, Equity Ownership. Dott:Orchard Therapeutics: Employment, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

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Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome

Science ◽

10.1126/science.1233151 ◽

2013 ◽

Vol 341 (6148) ◽

pp. 1233151 ◽

Cited By ~ 671

Author(s):

Alessandro Aiuti ◽

Luca Biasco ◽

Samantha Scaramuzza ◽

Francesca Ferrua ◽

Maria Pia Cicalese ◽

...

Keyword(s):

Gene Therapy ◽

Lentiviral Vector ◽

Ex Vivo ◽

Conditioning Regimen ◽

Immune Functions ◽

Gene Encoding ◽

Hematopoietic Stem ◽

Wiskott Aldrich Syndrome ◽

Vector Integration ◽

Lentiviral Gene Therapy

Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.

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Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

Blood ◽

10.1182/blood-2019-128805 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 607-607 ◽

Cited By ~ 1

Author(s):

Bernhard Gentner ◽

Maria Ester Bernardo ◽

Francesca Tucci ◽

Erika Zonari ◽

Francesca Fumagalli ◽

...

Keyword(s):

Gene Therapy ◽

Phase I ◽

Board Of Directors ◽

Financial Support ◽

Joint Venture ◽

Ex Vivo ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Allogeneic hematopoietic stem cell transplantation (HSCT) performed early in life is the current standard of care for patients with severe type 1 mucopolysaccharidosis (Hurler disease), a metabolic disorder caused by mutations in the alpha-L-iduronidase (IDUA) gene, leading to impaired breakdown of glycosaminoglycans (GAG). Secretion of IDUA by donor-derived hematopoietic cells may cross-correct non-hematopoietic cells, slowing progression of tissue damage and cognitive decline. Nevertheless, Hurler patients undergoing HSCT manifest substantial residual disease burden, e.g. on the skeleton and central nervous system (CNS). We conducted a phase I/II clinical study (NCT03488394) to test whether infusion of autologous CD34+ hematopoietic stem and progenitor cells (HSPC) transduced ex vivo with a lentiviral vector coding for the IDUA gene was feasible, safe and capable of restoring enzymatic activity in the patients' blood and tissues, up to supraphysiologic levels. The trial originally planned to enroll 6 Hurler patients with preserved neurocognitive function (DQ/IQ>70) that had no access to a suitable allogeneic donor. Sample size has recently been increased to 8 patients. By July 2019, six patients have been treated at a median age of 24 months (range: 14-34), with a median follow up of 4 months (range: 1-13). In all patients, we collected a high number of autologous HSPC by leukapheresis following mobilization with lenograstim and plerixafor, resulting in drug products with a median of 21 million CD34+ cells/kg (range: 13-29). Transduction efficiency was high with a median above 80% and a vector copy number (VCN) of 1.7 (range: 1.0-5.2), employing a shortened, 2 day transduction protocol that included prostaglandin E2. All patients showed rapid hematopoietic recovery following myeloablative conditioning with busulfan (targeted to an AUC of 80mg*h/L), fludarabine (160mg/sqm) and rituximab (375mg/sqm). Median duration of grade 4 neutropenia associated with conditioning was 15.5 days (range: 13-19). Also associated with conditioning, Grade 3 thrombocytopenia lasted 4 days, while only 2 out of 6 patients experienced a platelet drop below 20,000/mcL on a single day, in the absence of transfusion support. Adverse events were mild and compatible with myeloablative conditioning, with the exception of patient 3 who experienced an anaphylactic reaction on day+12, which promptly responded to antihistamines, IV fluids and steroids. All evaluable patients showed sustained, supraphysiologic blood IDUA activity (dried blood spot), which was on average 3 fold above the upper limit of normal (evaluable patients: n=5 at 1 month, n=4 at 2 months, n=3 at 3 months). Notably, in n=4 Hurler patients treated with allogeneic HSCT, we detected IDUA activity that ranged within the lowest quartile of normal in spite of full donor chimerism, suggesting substantial gain achieved by overexpressing IDUA in ex vivo genetically-modified autologous HSPC. Urinary GAG excretion fell to normal levels within 3-6 months. IDUA activity was also detected in the cerebrospinal fluid (CSF) of treated patients, accompanied by a logfold reduction in CSF GAGs in the 2 patients with longest follow up. This suggests that gene therapy accomplishes full metabolic correction of tissues, including the CNS. Gene therapy did not induce antibodies against the IDUA protein, while pre-existing antibodies induced by enzyme replacement therapy before gene therapy rapidly disappeared. Patient 1 who reached the 1-year follow-up demonstrated a stable cognitive score, improved findings on brain and spine MRI, resumed growth velocity and an improvement of his skeletal phenotype. The preliminary results from our phase I/II study compare favorably with the standard of care in terms of safety and efficacy, and highlight the potential of genetic engineering of HSPC grafts for therapeutic gain-of-function. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Parini:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; BioMarin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Ultragenyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Orphan Europe: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support. Naldini:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

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Immune Reconstitution After Gene Therapy Approaches in Patients With X-Linked Severe Combined Immunodeficiency Disease

Frontiers in Immunology ◽

10.3389/fimmu.2020.608653 ◽

2020 ◽

Vol 11 ◽

Author(s):

Elena Blanco ◽

Natalia Izotova ◽

Claire Booth ◽

Adrian James Thrasher

Keyword(s):

Gene Therapy ◽

T Cell ◽

Nk Cells ◽

Immune Reconstitution ◽

Lymphoblastic Leukemia ◽

Conditioning Regimen ◽

Cell Recovery ◽

Hematopoietic Stem ◽

Conditioning Treatment ◽

Immunodeficiency Disease

X-linked severe immunodeficiency disease (SCID-X1) is an inherited, rare, and life-threating disease. The genetic origin is a defect in the interleukin 2 receptor γ chain (IL2RG) gene and patients are classically characterized by absence of T and NK cells, as well as presence of partially-functional B cells. Without any treatment the disease is usually lethal during the first year of life. The treatment of choice for these patients is hematopoietic stem cell transplantation, with an excellent survival rate (>90%) if an HLA-matched sibling donor is available. However, when alternative donors are used, the success and survival rates are often lower. Gene therapy has been developed as an alternative treatment initially using γ-retroviral vectors to correct the defective γ chain in the absence of pre-conditioning treatment. The results were highly promising in SCID-X1 infants, showing long-term T-cell recovery and clinical benefit, although NK and B cell recovery was less robust. However, some infants developed T-cell acute lymphoblastic leukemia after the gene therapy, due to vector-mediated insertional mutagenesis. Consequently, considerable efforts have been made to develop safer vectors. The most recent clinical trials using lentiviral vectors together with a low-dose pre-conditioning regimen have demonstrated excellent sustained T cell recovery, but also B and NK cells, in both children and adults. This review provides an overview about the different gene therapy approaches used over the last 20 years to treat SCID-X1 patients, particularly focusing on lymphoid immune reconstitution, as well as the developments that have improved the process and outcomes.

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Therapeutic Effect of HOXB4-Expanded Stem Cells in Mice with Beta Thalassemia Given a Non-Myeloablative Conditioning Regimen.

Blood ◽

10.1182/blood.v108.11.341.341 ◽

2006 ◽

Vol 108 (11) ◽

pp. 341-341

Author(s):

Silvia Bakovic ◽

Patricia M. Rosten ◽

Connie J. Eaves ◽

R. Keith Humphries

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Ex Vivo ◽

Globin Gene ◽

Conditioning Regimen ◽

Beta Thalassemia ◽

Mouse Bone Marrow ◽

Myeloablative Conditioning ◽

Hematopoietic Stem

Abstract The ultimate promise of gene therapy for patients with hemoglobinopathies depends on the development of safe strategies for achieving 2 goals. One is to obtain efficient and permanent correction of the gene defect in autologous hematopoietic stem cells (HSCs). The second is to develop methods for the pre-transplant amplification of transduced HSCs to high levels to ensure that they will outcompete the large residual endogenous HSC population remaining in non-myeloablated hosts (e.g. previous experiments have shown that a minimum of ~5 × 106 normal adult mouse bone marrow (BM) cells (~500 HSC) is required to achieve a level of chimerism of 20% in mice given 200 cGy). The ability of HOXB4 to promote HSC self-renewal divisions in short term culture prior to their use as transplants offers an attractive approach to achieve this latter goal. As a first test we transduced day-4 5FU BM cells from normal mice with a MSCV-HOXB4-IRES-GFP or control MSCV-IRES-GFP virus and then transplanted the cells either before or after 7 days maintenance in vitro into normal recipients given 250 cGy. Mice transplanted with an estimated 50 HSCs immediately after transduction with either virus reached equivalent low levels of chimerism (~10%) showing that HOXB4 does not impart an in vivo selective growth advantage under sublethal conditions. After ex vivo culture, the GFP transduced cells yielded an even lower level of chimerism (~5%), in contrast recipients of cultured HOXB4-transduced cells attained much higher stable levels of lympho-myeloid chimerism (~50%), indicative of a marked expansion of the HSCs pre-transplant and their retention of robust competitive repopulating potential. We then applied this approach to a gene therapy model of severe β-thalassemia in mice bearing a homozygous deletion of the β-major globin gene (β-MDD). To model a transplant of genetically corrected cells, BM cells were harvested from day-4 5FU pre-treated congenic wild-type donors and transduced with the HOXB4 virus. Cells were then cultured for 10 days and the progeny of 200K starting cells transplanted into 3 β-MDD and 4 normal recipients given 200 cGy. Transplantation of 500K freshly harvested day-4 5FU BM cells into 4 similarly conditioned control mice failed to produce significant chimerism (1–3% at 5 months). In contrast, all 4 control recipients of ex vivo expanded HOXB4-transduced cells exhibited significant stable chimerism (21±6% at 5 months). Similar levels of chimerism were also achieved in all 3 β-MDD recipients (18–76%), one of which was sustained at 34% at 5 months (52% in the RBCs). This was associated with substantial improvement in the Hct (36% vs 23% in untreated β-MDD), Hb (10.5 vs 5 g/dl) and RBC morphology. Southern blot analyses performed on 53 individual in vitro-expanded myeloid colonies generated from FACS-selected GFP+ marrow cells from this mouse 2 months post-transplant showed 19 distinct integration patterns indicating reconstitution from polyclonal expanded HSCs. This conclusion was further confirmed by proviral integration site analyses, which identified 13 separate integration sites from 9 colonies that had unique proviral patterns. These data demonstrate the curative potential of ex vivo expanded HSCs in a preclinical model of β-thalassemia treated with non-myeloablative conditioning. They also underscore the potential of HOXB4 as a potent tool to achieve the HSC expansions required.

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Efficacy of Gene Therapy for Wiskott-Aldrich-Syndrome

Blood ◽

10.1182/blood.v118.21.165.165 ◽

2011 ◽

Vol 118 (21) ◽

pp. 165-165

Author(s):

Christian J. Braun ◽

Kaan Boztug ◽

Manfred Schmidt ◽

Michael H Albert ◽

Adrian Schwarzer ◽

...

Keyword(s):

Gene Therapy ◽

T Cell ◽

Progenitor Cells ◽

Nk Cell ◽

Myeloid Cells ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Wiskott Aldrich Syndrome ◽

Life Threatening

Abstract Abstract 165FN2 CB and KB contributed equally and should be considered aequo loco. Wiskott-Aldrich-Syndrome (WAS) is a rare and life-threatening immune-disorder characterized by autoimmunity, microthrombocytopenia, immunodeficiency and susceptibility to lymphoma. WAS is caused by mutations in the WAS gene which encodes WASP, a key regulator of actin polymerization exclusively expressed in hematopoietic cells. WASP deficiency causes defects in lymphocytes, myeloid cells, and platelets. We here report a comprehensive analysis of ten patients treated by hematopoietic stem cell gene therapy between 2006 and 2009 (median follow up time 29.6 months, range 15 to 58 months). Patients were mobilized with G-CSF alone (3/10) or G-CSF combined with anti-CXCR4/AMD3100 (7/10), conditioned with busulfan (8mg/kg body weight) and received between 2.8×106 and 24.9×106 cells/kg bw, with a median transduction efficacy of 52%. Upon transplantation of retrovirus-transduced WASP-expressing progenitor cells, the proportion of corrected platelets and lymphocytes increased steadily over time reaching 85–90% and 80–85%, respectively. Interestingly, also myeloid cells showed a continuously increasing percentage of WASP-expressing fractions (20 to 70%). Due to transplantation of insufficient numbers of WASP-transduced HSC, one patient failed to engraft. He had no evidence of corrected myeloid/hematopoietic progenitor cells and continued to suffer from life-threatening infections and autoimmunity. He was successfully treated by haploidentical HSCT. All other patients had marked improvement of their clinical status. Bleeding diatheses, susceptibility to infections, and autoimmunity resolved. Patients had evidence of significant and sustained increase in platelet counts (p=0.01) together with a reconstituted WASP expression and normalization of thrombocyte size (p<0.001). After gene therapy, we observed a normalization of the T cell compartment (T cell proliferative responses and TCR Vb spectratyping), NK cell function (assessed by in vitro cytotoxicity and formation of NK cell immunological synapse). The B cell compartment showed consistent expression of WASP. In 4 of 7 patients, IgG substitution could be discontinued. These patients and also those without initial IgG substitution mounted a specific immune response to vaccines, as evidenced by determination of specific antibody titers. One patient developed a T-cell acute lymphoid leukemia 488 days after gene therapy associated with vector integration close to the LMO2 locus. Chemotherapeutic treatment induced remission that is documented since d33 after initiation of induction therapy. Long-term follow up observation indicated that gene therapy for WAS, although not without toxicity, is feasible and provides an effective alternative treatment strategy to allogeneic HSC transplantation. Disclosures: Baum: Patent office: Patents & Royalties.

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Vincristine Sulfate Liposomes Injection (Marqibo®) Facilitates Durable Remissions and Potentially Curative Hematopoietic Stem Cell Transplantation in Adults with Advanced, Relapsed and/or Refractory Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.4235.4235 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4235-4235

Author(s):

Gary J. Schiller ◽

John Lister ◽

Leonard T. Heffner ◽

Stuart L. Goldberg ◽

Lloyd E. Damon ◽

...

Keyword(s):

Clinical Trial ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

Salvage Therapy ◽

Lymphoblastic Leukemia ◽

Vincristine Sulfate ◽

Employment Equity ◽

Dose Intensification ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract Abstract 4235 A durable response in advanced, relapsed and/or refractory adult acute lymphoblastic leukemia (ALL) may be defined as remission that results in a meaningful prolongation of life or response that facilitates “bridging” to a subsequent, potentially curative, hematopoietic stem cell transplantation (HSCT). Vincristine sulfate liposomes injection (VSLI, Marqibo®) is a sphingomyelin/cholesterol (SM/Chol) nanoparticle formulation of standard vincristine sulfate (VCR) designed to facilitate dose intensification, prolonged drug delivery and enhanced lymphoid malignancy penetration and concentration without increased toxicity. Recently, VSLI was evaluated in a multi-institutional, Phase 1/2 (VSLI-06; NCT00144963) clinical trial and a multi-national, Phase 2 (HBS407; NCT00495079) clinical trial in a combined 101 adults (median age 31 years [range 18 to 83 years]) with advanced, relapsed and/or refractory ALL. All but 1 patient had Philadelphia chromosome negative disease. Thirteen patients (13%) had extramedullary disease, 37 (37%) had undergone a prior HSCT, and 100% had received at least one prior line of therapy including standard VCR. Study VSLI-06 (N = 36) was a dose-ascending trial of weekly VSLI (1.5 to 2.4 mg/m2) combined with pulse dexamethasone. Study HBS407 was a single-arm trial of weekly single-agent VSLI at the maximum tolerated dose established in VSLI-06 of 2.25 mg/m2. Overall, 19 (19%) patients received VSLI as a first salvage therapy, 57 (56%) patients received VSLI as a second salvage therapy, and 25 (25%) patients received VSLI as a third or greater salvage therapy. All patients had to be deemed ineligible for immediate HSCT in order to enroll in VSLI-06 or HBS407. In the combined study population, the overall response rate (complete remission [CR], CR with incomplete hematologic recovery [CRi], partial remission [PR], and bone marrow blast response [BMB]) was 31% (95% CI: 22–41) with a 20% (95% CI: 13–29) rate of CR+CRi. Despite delivering intensified individual (2.8–5.5 mg) and cumulative (up to 70.1 mg) doses of VCR, VSLI had a similar safety profile to that reported for the approved dose of standard VCR. VSLI enabled bridging to a post-VSLI HSCT in 12 of 65 (18%) patients in HBS407 and 5 of 36 (14%) patients in VSLI06 for a total of 17 of 101 (17%). All 17 post-VSLI HSCT patients were under the age of 60 years. Three of 12 post-VSLI HSCT patients from HBS407 remain alive at greater than 28, 33, and 35 months following VSLI, respectively. All 12 patients lived for greater than 100 days after post-VSLI HSCT. Long-term survival (greater than 12 months) was achieved in 27% of those able to receive post-VSLI HSCT. These outcomes, that are important to patients, may reflect the effectiveness of the VCR dose intensification facilitated by VSLI. The neuropathy associated with the dose intensified VCR administered as VSLI was predictable, manageable, and comparable to that published for standard VCR. The lack of early, pre-day 100, mortality following post-VSLI HSCT suggests that the sphingomyelin-based liposomal formulation did not adversely affect subsequent transplantation procedures. In conclusion, VSLI produced both clinically important endpoints of prolonged survival and achievement of response allowing for a bridge to HSCT for advanced, relapsed and/or refractory ALL. Disclosures: Schiller: Talon Therapeutics: Research Funding. Silverman:Talon Therapeutics: Employment, Equity Ownership. Deitcher:Talon Therapeutics: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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In Vivo Selection Of Genetically Manipulated Hematopoietic Stem Cells For Platelet Gene Therapy Of Hemophilia A

Blood ◽

10.1182/blood.v122.21.2329.2329 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2329-2329

Author(s):

Qizhen Shi ◽

Jocelyn A. Schroeder ◽

David A. Wilcox ◽

Robert R. Montgomery ◽

Yingyu Chen

Keyword(s):

Gene Therapy ◽

Hemophilia A ◽

Genetically Modified ◽

Conditioning Regimen ◽

Selective Treatment ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Fviii Activity ◽

Selection Of

Abstract Our previous studies have demonstrated that targeting FVIII expression to platelets (2bF8) by lentiviral (LV) gene delivery to hematopoietic stem cells (HSCs) corrects bleeding diathesis in hemophilia A mice with or without inhibitors. Although the bleeding diathesis is improved in transduced recipients, the transduction efficiency using our current 2bF8 LV, is only about 10%, resulting a median level of platelet-FVIII (Plt-F8) of 1.5 mU/108 platelets even thought a myeloablative conditioning regimen was employed. It has been shown in clinical trials that efficient stem cell gene transfer and myeloablation is not required when there is a powerful selective advantage to the genetically modified cells. We hypothesize that incorporating a drug-resistance gene into the 2bF8 LV construct will allow for in vivo selection of 2bF8 LV-transduced cells which will result in the increase of therapeutic levels of Plt-F8 for hemophilia A gene therapy and reduce the potential for genotoxicity. To address our hypothesis, we constructed a new lentiviral vector, pWPT-2bF8/MGMT, which harbors dual genes, the 2bF8 gene and a drug-resistance gene, the MGMTP140K cassette. To explore the feasibility of the MGMT-based in vivo selection system, HSCs from FVIIInull mice were transduced with 2bF8/MGMT LV at an MOI (multiplicity of infectious) of 1, which is 1/10 of the MOI used for our regular 2bF8 LV transduction, and transplanted into littermates pre-conditioned with a non-myeloablative regimen, 660 cGy total body irradiation (TBI). We chose a low MOI because one of the goals of using the MGMT selection system is to reduce the potential for genotoxicity. After bone marrow (BM) reconstitution, the recipients were treated with O6-benzylguanine (BG) followed by 1, 3-bis-2 chloroethyl-1-nitrosourea (BCNU) monthly for 3 or 4 times. As determined by a chromogenic assay on platelet lysates, functional Plt-F8 expression in recipients was only 0.22 ± 0.15 mU/108 platelets before the drug treatment, but remarkably increased to 4.33 ± 5.48 mU/108 platelets (n = 16) after BG/BCNU drug-selective treatments. The levels of Plt-F8 in the untreated transduced control group remained low over the study period. FVIII activity was not detected in the plasma in any of the recipients even with Plt-F8 as high as 22 mU/108 platelets. The average copy number of 2bF8/MGMT proviral DNA per cell was determined by quantitative real-time PCR. 2bF8 proviral DNA was barely detectable (0.01 ± 0.02 copies/cell) in recipients before drug-selective treatment, but it increased to 0.42 ± 0.15 copies/cell after BG/BCNU treatments, confirming that 2bF8/MGMT genetically modified cells were effectively enriched in vivo after drug-selective treatment. When the tail clip survival test was used to assess phenotypic correction of the FVIIInull coagulation defect, 15 of 16 treated animals survived the tail clip challenge; in contrast, none of the untransduced FVIIInull control mice survived. When ROTEM analysis was used to determine the whole blood clotting time (CT), the CT was shortened from 3043 ± 728 seconds (n = 7) to 931± 273 seconds (n = 6) (P < 0.0001) in treated transduced recipients when compared to FVIIInull mice. There was no significant difference between wild type (722 ± 270 seconds, n = 7) and treated recipients. To ensure sustained Plt-F8 expression in BG/BCNU treated transduced recipients, some primary recipients were sacrificed 9 months after transplantation and BM mononuclear cells were transplanted into secondary recipients. Platelet lysate FVIII activity assays showed that the levels of Plt-F8 in secondary recipients were similar to those in primary recipients, confirming that long-term repopulating HSCs were successfully genetically modified by 2bF8/MGMT LV. When a low intensity pre-conditioning regimen of 440 cGy TBI was used, the levels of Plt-F8 increased from 0.06 ± 0.12 mU/108 platelets to 1.86 ± 2.06 mU/108 platelets after BG/BCNU drug-selective treatment. It is notable that no anti-FVIII inhibitory antibodies were detected in the treated recipients even after rhFVIII challenge, indicating that immune tolerance was induced in the treated animals. In contrast, all FVIIInull mice under the same challenge developed various levels of inhibitors. Taken together, we have established a powerful in vivo selective system that allows us to enrich 2bF8 LV-transduced cells and to enhance platelet-FVIII expression for hemophilia A gene therapy. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Stem Cell Gene Therapy For Wiskott- Aldrich Syndrome

Blood ◽

10.1182/blood.v122.21.718.718 ◽

2013 ◽

Vol 122 (21) ◽

pp. 718-718

Author(s):

Maximilian GW Witzel ◽

Christian J. Braun ◽

Kaan Boztug ◽

Anna Paruzynski ◽

Michael H. Albert ◽

...

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Insertional Mutagenesis ◽

Residual Disease ◽

Hematopoietic Stem ◽

Wiskott Aldrich Syndrome ◽

Cell Gene ◽

Stem Cell Gene ◽

Stem Cell Gene Therapy

Abstract Wiskott Aldrich Syndrome (WAS) is a life-threatening primary immunodeficiency disorder characterized by susceptibility to infections, hemorrhage and malignancies. We here report the clinical, immunological, and molecular results of ten patients treated with transfusion of autologous genetically corrected hematopoietic stem cells between 2006 and 2009. After hematopoietic stem cell gene therapy with a gamma-retroviral vector, we observed phenotypical and functional reconstitution of lymphoid, myeloid cell lines and platelet numbers in 9/10 patients. Of note, patients had sustained correction of T-cell proliferation, immunological synapse formation and NK-cell mediated lysis, capacity to generate specific antibodies, and platelet production and size. All nine patients with sustained stem cell engraftment had marked clinical improvement with respect to infections, bleeding, and autoimmunity. Comprehensive analysis of retroviral insertions sites revealed a polyclonal hematopoiesis with more than 120.000 recurring loci. However, 5/10 of patients developed T-ALL (mean 1054.2d post GT; range 488-1813d post GT), each associated with a retroviral insertion in close proximity to the LMO2 locus. In 3/5 of patients with T-ALL (treatment according to AEIOP 2009 protocol) allogeneic HSCT due to poor initial response, minimal residual disease relapse, or cytological relapse was indicated. Further, one patient developed MDS like bone marrow changes and consecutively AML (1165d post GT), HSCT was performed in remission. Retrospectively, MDS1 clonal contribution to insertion sites showed a slow increase over the period of 22 month in this patient. GT has proven to revert the cellular and clinical phenotype of WAS but remains associated with considerable risk for insertional mutagenesis. Refining gene therapy strategies is an important goal for patients with WAS. Disclosures: Naundorf: EUFETS GmbH: Employment. Kühlcke:EUFETS GmbH: Employment.

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