Lentiviral Hematopoietic Stem Cell Gene Research Articles

Gene and Cellular Therapies for Leukodystrophies.

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber's amaurosis, epidermolysis bullosa, Duchenne's muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results. Gene therapy offers the possibility for overexpression of secretable enzymes that can be released and through uptake, allow cross-correction of affected cells. Here, we discuss some of the leukodystrophies that have demonstrated strong potential for gene therapy interventions, such as X-linked adrenoleukodystrophy (X-ALD), and metachromatic leukodystrophy (MLD), which have reached clinical application. We further discuss the advantages and disadvantages of ex vivo lentiviral hematopoietic stem cell gene therapy, an approach for targeting microglia-like cells or rendering cross-correction. In addition, we summarize ongoing developments in the field of in vivo administration of recombinant adeno-associated viral (rAAV) vectors, which can be used for direct targeting of affected cells, and other recently developed molecular technologies that may be applicable to treating leukodystrophies in the future.

Regulatable Lentiviral Hematopoietic Stem Cell Gene Therapy in a Mouse Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent neuroprotective properties in preclinical models of Parkinson's disease (PD), but challenges in GDNF delivery have been reported from clinical trials. To address this barrier, we developed a hematopoietic stem cell transplantation-based macrophage-mediated GDNF therapy platform. Here, we introduced a regulatable lentiviral vector (LV-MSP-Tet-Off-hGDNF) to allow the expression of human GDNF (hGDNF) to be adjusted or stopped by oral administration of doxycycline (Dox). C57BL/6J mice were lethally irradiated with head protection and then transplanted with syngeneic bone marrow cells transduced with either the hGDNF-expressing vector or a corresponding GFP-expressing vector, LV-MSP-Tet-Off-GFP. Suppression of vector gene expression was achieved through administration of Dox in drinking water. To create a toxin-induced Parkinsonian model, mice were injected in two cycles with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to yield nigral cell/striatal dopamine loss and behavioral deficits. During the presence of Dox in the drinking water, plasma GDNF was at a basal level, whereas during the absence of Dox, plasma GDNF was significantly elevated, indicating reliable regulation of therapeutic gene expression. Midbrain GDNF levels were altered in parallel, although these did not return completely to basal levels during the periods of Dox withdrawal. Motor activities of the MPTP-Tet-off-hGDNF group were comparable to those of the Tet-off-GFP (subject to no MPTP treatment) group, but substantially better than those of the MPTP-Tet-off-GFP group. Interestingly, the improvement in motor activities was sustained during the Dox-withdrawn periods in MPTP-Tet-off-hGDNF animals. Neuroprotection by therapeutic GDNF expression was further evidenced by significant amelioration of nigral tyrosine hydroxylase loss after both the first and second MPTP treatment cycles. These data suggest that neurotrophic factor expression can be upregulated to achieve efficacy or downregulated in case of off-target effects or adverse events, a feature that may eventually increase the acceptance of this potentially neuroprotective/disease-modifying PD therapy.

Update on safety and efficacy of lentiviral hematopoietic stem cell gene therapy (HSC-GT) for metachromatic leukodystrophy (MLD)

Update on safety and efficacy of lentiviral hematopoietic stem cell gene therapy (HSC-GT) for metachromatic leukodystrophy (MLD)

Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Immune and Lysoso- mal Enzyme Deficiencies

Rare diseases affect millions of people worldwide. Many of those are inherited disorders resulting in chronic disability and requiring cost-intensive care. Hematopoietic stem cell gene therapy has been developed over more than 20 years. At the state of the art, gene therapy is within reach for diseases in which (i) the genetic defect is identified, (ii) the diagnosis is made sufficiently early for a meaningful therapeutic intervention, (iii) a specific animal model is available for efficacy and safety evaluation. Appropriate therapeutic transgenes should also comply with certain biological criteria. Third-generation lentiviral vectors have been made self-inactivating (SIN) by deletion of enhancer regions from the LTR sequences thus reducing the risk of influencing nearby genes, resulting in favorable safety profiles. At the present time, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and lysosomal storage disorders. We discuss initial clinical trials using these vectors for selected metabolic storage disorders, which include adrenoleukodystrophy, metachromatic leukodystrophy, Hurler (MPS I), Pompe (GSD II), and Fabry diseases. This brief review summarizes the development and current clinical implementation of these approaches.

166. Lentiviral Hematopoietic Stem Cell Gene Therapy for Sjögren-Larsson Syndrome

Sjogren-Larsson syndrome (SLS) is a rare autosomal recessive disorder characterized by scaling skin (ichthyosis), mental retardation, and spasticity. SLS is caused by mutations in the ALDH3A2 gene, which encodes fatty aldehyde dehydrogenase (FALDH), an enzyme that is involved in the oxidation of fatty aldehyde and fatty alcohol. In SLS, FALDH deficiency and impaired fatty aldehyde oxidation results in lipid accumulation, which is responsible for the symptoms. Spurred by previous hematopoietic stem cell gene therapy trials for multi-systemic diseases, such as Adrenoleukodystrophy (ALD), Metachromatic leukodystrophy (MLD), and Cystinosis, we investigated the plausibility of hematopoietic stem cell gene transfer to correct FALDH deficiency and alleviate the phenotype in SLS. We designed the hematopoietic stem cell gene therapy using a lentiviral vector containing human ALDH3A2 cDNA based on our previous MND-WASP (Wiskott-Aldrich syndrome protein) vector design. The lentiviral vector contains the normal human ALDH3A2 coding sequence which was derived from PCR products using human mobilized peripheral blood CD34+ cDNA. The vector was tested for FALDH protein expression in transfected human HEK293T cells by western blot and the enzyme activity was confirmed using HPLC-MS/UV. A transplantation experiment was performed using the Aldh3a2-/- KO mouse model, where the lineage negative BM cells from CD45.1 Aldh3a2-/- KO mice (C57BL/6 background) were sorted and transduced with a lentiviral vector followed by transplantation into lethally irradiated CD45.2 Aldh3a2-/- KO mice (C57BL/6 background). These mice are being monitored for cell engraftment, vector copy numbers, and any phenotypic changes. An initial analysis for the grafts showed 56% transduction in the MND(a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer)-GFP group and 31% transduction in the MND-ALDH3A2 group based on the CFU-C data. Flow cytometric analysis of peripheral blood on these mice at 6 and 12 post- transplant week showed 32% transduction in MND-GFP group. Further analysis including vector copy numbers and end point analysis for various tissues including liver, spleen, and brain are underway. This work provides the foundation for moving forward with potential lentiviral hematopoietic stem cell gene therapy for non-hematological disorders.

Safety and Clinical Benefit of Lentiviral Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, recurrent infections, eczema, autoimmunity and increased susceptibility to malignancies. Allogeneic hematopoietic stem cell transplantation (HSCT) is a recognized curative treatment for WAS, but is still associated with transplant-related complications and long-term morbidity, particularly in the absence of fully matched donors. In April 2010, we initiated a phase I/II clinical trial with hematopoietic stem cell (HSC) gene therapy (GT) for WAS. The investigational medicinal product (IMP) consists of autologous CD34+ HSC engineered with a lentiviral vector (LV) driving the expression of WAS cDNA from an endogenous 1.6 kb human WAS promoter (LV-WAS), infused after a reduced intensity conditioning (RIC) based on anti-CD20 mAb, targeted busulfan and fludarabine. We previously reported early follow up (FU) results from the first 3 patients (Aiuti et al., Science 2013). Seven patients (Zhu score ≥3) have now been treated at a median age of 1.9 years (1.1 - 11.1). As of May 2015, all patients are alive with a median FU of 3.2 years (0.7 - 5.0). CD34+ cell source was bone marrow (BM) (n=5), mobilized peripheral blood (MPB) (n=1) or both (n=1). IMP dose ranged between 7.0 and 14.1 x106 CD34+/kg, containing on average 94.4 ± 3.5% transduced clonogenic progenitors and a mean vector copy number (VCN)/genome in bulk CD34+ cells of 2.7 ± 0.8. No adverse reactions were observed after IMP infusion and RIC was well tolerated. Median duration of severe neutropenia was 19 days; granulocyte-colony stimulating factor was administered to 1 patient.In the first 6 treated patients with FU >2 years, we observed robust and persistent engraftment of gene corrected cells. At the most recent FU, transduced BM progenitors ranged between 20.7 and 59.7%, and LV-transduced cells were detected in multiple lineages, including PB granulocytes (VCN 0.34 - 0.93) and lymphocytes (VCN 1.18 - 2.73). WAS protein expression, measured by flow-cytometry, was detected in the majority of PB platelets [mean ± standard deviation (SD), 71.4 ± 14.0%], monocytes (63.3 ± 18.5%) and lymphocytes (78.9 ± 14.9%). Lymphocyte subset counts were normal in most patients and proliferative response to anti-CD3 mAb was in the normal range in all 6 patients.After immune reconstitution, a marked reduction in the annualized estimated rate of severe infections was observed, as compared with baseline (figure 1A). The first 6 treated patients discontinued anti-infective prophylaxis and no longer require a protected environment. Four patients stopped immunoglobulin supplementation and 2 of them developed specific antibodies after vaccination. Eczema resolved in 4 patients and remains mild in 2. No clinical manifestations of autoimmunity were observed ≥1 year after GT in accordance with improved B-cell development and decreased autoantibody production. All patients became platelet transfusion independent at a median of 4 months after GT (range: 1.0 - 8.7). Mean platelet counts progressively increased after treatment (mean ± SD: before GT, 13.4 ± 7.8 x109/l; 24-30 month FU, 45.8 ± 22.0 x109/l; 36-42 month FU, 57.0 ± 18.7 x109/l).The frequency and the severity of bleeding events decreased after the 1st year of FU. No severe bleedings were recorded after treatment (figure 1B).Quality of life improved in all patients after GT. From the 2nd year of FU, the number of hospitalizations for infections decreased and no hospitalizations due to bleeding were observed after treatment.The seventh patient treated, who received MPB derived CD34+ cells only, showed the fastest platelet recovery with the highest level of transduced myeloid cell engraftment, and is clinically well.No Serious Adverse Events (SAE) related to the IMP were observed. The most frequent SAE were related to infections (85%), occuring mainly during the 1st year of FU.Importantly, no evidence of abnormal clonal proliferations emerged after GT and the LV integration profile show a polyclonal pattern, with no skewing for proto-oncogenes.In conclusion, this updated report in 7 WAS patients show that GT is well tolerated and leads to a sustained clinical benefit. The high level of gene transfer obtained with LV-WAS results in robust engraftment of transduced HSC, even when combined with RIC. Prolonged FU will provide additional information on the long-term safety and clinical efficacy of this treatment. [Display omitted] DisclosuresVilla:Fondazione Telethon: Research Funding. Dott:GlaxoSmithKline: Consultancy. van Rossem:GlaxoSmithKline: Employment. Naldini:Salk Institute: Patents & Royalties: Lentiviral vectors; San Raffaele Telethon Institute: Patents & Royalties: Lentiviral vector technology; GlaxoSmithKline: Other: GSK licensed gene therapies developed at my Institute and the Institute receives milestone payments; Sangamo Biosciences: Research Funding; Biogen: Research Funding; Genenta Sciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Aiuti:GlaxoSmithKline (GSK): Other: PI of clinical trial which is financially sponsored by GSK; Fondazione Telethon: Research Funding.

Lentiviral Hematopoietic Stem Cell Gene Therapy for Older Patients with X-Linked Severe Combined Immunodeficiency

Background: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the IL2RG gene that encodes the common gamma chain (gc). Hematopoietic stem cell (HSC) transplant can be curative, but for infants without a matched sibling donor, haploidentical parental hematopoietic stem cell transplant (HSCT) without myeloconditioning is life-saving but restores only T-cell immunity. In infants with SCID-X1, autologous HSC transduced with murine gamma retrovirus (gRV) carrying gc infused without conditioning also only effectively restores T-cell immunity. Similar gene therapy of older children with SCID-X1 who have persistent immune defects despite haploidentical transplant in infancy failed to achieve significant gene marking or sustained improvement of T-cell immunity. Furthermore, the gRV gene therapy in the infants also resulted in vector-associated leukemia in 25% of patients. Here, we use a lentiviral vector (CL20-i4-EF1a hgcOPT) containing a 400bp insulator fragment from the chicken beta-globin locus within the self-inactivating long-terminal repeat (LTR), driven by the eukaryotic elongation factor alpha (EF1alpha) promoter to express a codon-optimized gc cDNA. Herein, we provide the initial data on subjects enrolled and treated in this study (NIAID protocol 11-I-0007).Subjects and Methods: SCID-X1 Subjects >2years of age with persistent immune deficiency and medical problems undergo G-CSF and plerixafor mobilized peripheral blood apheresis and CD34 isolation. For transduction, CD34+ cells are prestimulated for one day in SCF, FLT-3L, and TPO (100ng/ml), followed by daily exposure to vector for 6-8 hours daily on 2 consecutive days. On day 3, transduced cells were harvested and infused. Prior to cell infusion, subjects receive i. v. busulfan 6mg/kg total. Subjects are monitored for hematologic engraftment, immunological phenotype and functional profile, as well as gene marking in sorted immune cell lineages. Vector integration site analysis by modified linear amplification-mediated PCR and high-throughput deep sequencing, as well as replication-competent lentivirus (RCL) assays are performed for safety monitoring.Results: Five (23, 24,7, 16 and 10 year old) subjects (P1-5, respectively) with worsening immune dysfunction and complex medical problems, with IgG supplementation dependence despite one or more prior haploidentical HSC transplants have been treated with a follow up period of 30-, 27, 4, 1 and <1 months, respectively (as of abstract submission). Busulfan conditioning was well tolerated with no serious adverse events. In the 2 older patients with significantly longer follow-up, we observe stable engraftment of gc-expressing cells with expansion of gene corrected T-, B-, and NK-cells. Gene marking in the myeloid lineages stabilize by a year following treatment to 8-10% (=0.1 vector genome (vg)/cell), while continuing to increase in B- (38%/0.38 vg/cell), T- (13-55%/0.13-0.55vg/cell) and NK-(56-76%/0.56-0.76vg/cell) cells. Early gene marking in P3 is comparable at 6% or 0.06vg/cell in myeloid lineages with increasing IgM production. Chimerism studies of their T-cells show increasing host cell contribution that reflects gradual replacement of the donor T-cell graft over time. Increase in NK-cells in P2 corresponds to an improvement in chronic warts. Both P1 and P2 produce IgG and antigen-specific responses with protective titer response to immunization, and clearance of chronic norovirus, and resolution of protein-losing enteropathy. Vector tracking confirms diverse repertoire of gene insertion sites and absence of oncogene-targeted clones observed in earlier gRV gene therapy.Conclusion: Lentiviral-gene therapy with reduced-intensity conditioning appears safe and achieves unprecendented pan-immunologic correction of T, B, and NK cell compartments due to engraftment of relatively high levels of transduced HSCs in the bone marrow. This is the first demonstration of the use of gene therapy to salvage failed allogeneic HSCT in older SCID-X1 patients. DisclosuresNo relevant conflicts of interest to declare.

Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders.

After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease.

A possible turning point in the hematopoietic stem cell gene therapy for primary immunodeficiency diseases? Lentiviral vectors could take the place of retroviral vectors

Evaluation of: Aiuti A, Biasco L, Scaramuzza S et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341(6148), 1233151 (2013).Wiskott–Aldrich syndrome (WAS), an X-linked primary immunodeficiency disease (PID) with unique and characteristic features, had been considered to be a good candidate for gene therapy. In 2010, hematopoietic stem cell (HSC) gene therapy, using a retroviral vector, was performed for WAS patients; however, concerns remain regarding the long-term safety of this therapy as several patients with PID developed myeloproliferative diseases due to insertional mutagenesis related to HSC gene therapy using retroviral vectors. Aiuti et al. first reported HSC gene therapy for WAS using a lentiviral vector and compared the safety and efficacy of the two therapies in the context of the same disease background. They undertook a detailed study of the vector integration sites and concluded that lentiviral HSC gene therapy was safer than retroviral gene therapy.