Gene-corrected Cells Research Articles

Gene Correction by Homologous Recombination With Zinc Finger Nucleases in Primary Cells From a Mouse Model of a Generic Recessive Genetic Disease

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34+ cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor (SCF), and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC exhibited increased colony formation compared to 21% oxygen without NAC (P < 0.03), demonstrated increased resistance to mitomycin C compared to green fluorescent protein (GFP )-transduced controls (P < 0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Progress and prospects: gene therapy for inherited immunodeficiencies

Haematopoietic stem cell transplantation (HSCT) is now widely used to treat primary immunodeficiencies (PID). For patients with specific disorders (severe combined immunodeficiency (SCID)-X1, adenosine deaminase deficiency (ADA)-SCID, X-chronic granulomatous disease (CGD) and Wiskott-Aldrich Syndrome (WAS)) who lack a suitable human leukocyte antigen (HLA)-matched donor, gene therapy has offered an important alternative treatment option. The success of gene therapy can be attributed, in part, to the selective advantage offered to gene-corrected cells, the avoidance of graft-versus-host disease and to the use of pre-conditioning in patients with chemotherapy to facilitate engraftment of corrected cells. Adverse events have been encountered and this has led to detailed characterization of retroviral vector integration profiles. A new generation of self-inactivating retroviral and lentiviral vectors have been designed to address these safety concerns, and are at an advanced stage of preparation for the next phase of clinical testing.

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

AbstractOverexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)–matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

Successful Low-Risk Hematopoietic Cell Therapy in a Mouse Model of Type 1 Gaucher Disease

Hematopoietic stem cell-based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, optimization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. In this study we have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a low-risk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of the type 1 GD mouse. Our results demonstrate that a median wild-type (WT) cell engraftment of 7%, corresponding to GCase activity levels above 10 nmoles/hour and mg protein, was sufficient to reverse pathology in bone marrow and spleen in the GD mouse. Moreover, we applied nonmyeloablative doses of busulfan as a pretransplant conditioning regimen and show that even WT cell engraftment in the range of 1%-10% can confer a beneficial therapeutical outcome in this disease model. Taken together, our data provide encouraging evidence for the possibility of developing safe and efficient conditioning protocols for diseases that require only a low level of normal or gene-corrected cells for a permanent and beneficial therapeutic outcome.

A Possible Approach for Stem Cell Gene Therapy of Fanconi Anemia

Fanconi anemia (FA) is an inherited chromosomal recessive syndrome characterized by cellular hypersensitivity to DNA crosslinking agents and bone marrow failure, which cause aplastic anemia, and an increased incidence of malignancy. 13 complementation groups are currently discovered, and 13 distinct genes have been cloned (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FNACI, FANCJ, FANCL, FANCM, FANCN). Stem cells can theoretically divide to other cells without limit as long as a person is still alive. The stem cells that form blood and immune cells are known as hematopoietic stem cells. Hematopoietic stem cells can be acquired from a Fanconi anemia patient, whereas genomic DNA can be obtained easily from blood cells of a normal person. Normal genes also can be synthesised by PCR method. Normal genomic DNA will be delivered into a patient's stem cells via microinjection or transfection after enzyme digestion; the defective genes might be repaired by homologous genetic recombination. The gene-corrected stem cells can be transplanted into the same patient finally. It is possible that human genomic DNA to be considered as materials for homologous genetic recombination to repair defective genes in vivo. This might be an efficient method for gene therapy, which has no or less immunological rejection for Fanconi anemia and some genetic diseases. Several related observations and experiments are discussed to support this possible means of stem cell gene therapy of Fanconi anemia.

Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity

Precise manipulations of complex genomes by zinc-finger nucleases (ZFNs) depend on site-specific DNA cleavage, which requires two ZFN subunits to bind to two target half-sites separated by a spacer of 6 base pairs (bp). ZFN subunits consist of a specific DNA-binding domain and a nonspecific cleavage domain, connected by a short inter-domain linker. In this study, we conducted a systematic analysis of 11 candidate-based linkers using episomal and chromosomal targets in two human cell lines. We achieved gene targeting in up to 20% of transfected cells and identified linker variants that enforce DNA cleavage at narrowly defined spacer lengths and linkers that expand the repertoire of potential target sites. For instance, a nine amino acid (aa) linker induced efficient gene conversion at chromosomal sites with 7- or 16-bp spacers, whereas 4-aa linkers had activity optima at 5- and 6-bp spacers. Notably, single aa substitutions in the 4-aa linker affected the ZFN activity significantly, and both gene conversion and ZFN-associated toxicity depended on the linker/spacer combination and the cell type. In summary, both sequence and length of the inter-domain linker determine ZFN activity and target-site specificity, and are therefore important parameters to account for when designing ZFNs for genome editing.

Patient-Specific Induced Pluripotent Stem Cells in Hurler Syndrome

Hurler syndrome (HS; mucopolysaccharidosis type I) is caused by severe mutations in the iduronidase (IDUA) gene, leading to multi-organ system dysfunction due to the toxic accumulation of glycosaminoglycans. Although allogeneic hematopoietic cell transplantation (HCT) has been shown to provide the IDUA protein and to reverse many of the manifestations of HS, allogeneic HCT is associated with significant morbidity and mortality. We hypothesized that an advantageous alternative strategy may be to induce gene-corrected autologous pluripotent cells to become hematopoietic stem cells, which then provide the missing IDUA enzyme. Because patient-specific embryonic stem cell isolation is not practical, recent strategies have been developed that reprogram adult cells to acquire pluripotency. Such induced pluripotent stem (iPS) cells can be created from fibroblasts or mesenchymal stromal cells (MSCs). As a first step in testing of iPS cells for gene-corrected HS treatment, we isolated host MSCs from the bone chips of a 9-year-old boy with HS who had undergone spinal surgery 8 years after successful allogeneic HCT. HS-MSCs expressed no IDUA, confirming a lack of contamination from either donor-derived hematopoietic cells or MSCs. To create HS-iPS cells, HS-MSCs were transduced with viral vectors carrying reprogramming transcription factors (OCT4, SOX2, KLF4, and c-MYC) that are typically associated with pluripotency and expressed at high levels in embryonic but not adult stem cells. Transduced cells were cultured on supportive stroma of irradiated mouse embryo fibroblasts. Within several weeks, colonies of iPS cells emerged from the two-dimensional culture. When compared to MSCs, the HS-iPS cells showed persistent mRNA expression of OCT3/4 and SOX2 and transient mRNA expression of c-MYC and KLF4, which is expected to occur in the wild-type iPS cells. HS-iPS cells expressed protein markers characteristic of reprogrammed immature cells: OCT3/4, NANOG, stage-specific embryonic antigens (SSEA) 3 and 4, tumor rejection antigens (TRA) 1–60 and 1–81, and alkaline phosphatase. HS-iPS cells had normal male karyotype as determined by chromosomal G-banding. As a second step in creating gene-corrected HS-iPS cells, we employed the non-viral Sleeping Beauty (SB) transposon system (because of the less random pattern of genome integration when compared to viral vectors). Human HS-iPS cells were co-nucleofected with an SB transposon that harbored the human IDUA gene and an expression cassette of the green fluorescent protein (GFP) along with an SB transposase plasmid that provides the enzymatic machinery necessary for integration into TA dinucleotide sites within the genome. Two weeks after nucleofection 10%-15% of HS-iPS cells expressed GFP. Total glycosaminoglycans (a hallmark of the biochemical defect in HS) in unsorted cultures were decreased to wild-type levels. IDUA expression in unsorted cultures was approximately 10% of wild-type IDUA levels, which is within the range sufficient for phenotypic rescue in HS patients after allogeneic HCT. Experiments are ongoing, and data will be presented in regards to: a) map transposon insertions in the genome to prove stable transgenesis by transposition; b) characterization of the differentiation potential of the corrected HS-iPS cells into various mesodermal lineages relevant to rescue of the clinical phenotype associated with HS (hematopoietic, chondrogenic, and osteogenic); c) assessment of development and consequences of cellular pathology in numerous tissue types affected by IDUA deficiency. To our knowledge these are the first data to report that autologous iPS cells can be obtained from HS patients. In summary, HS-iPS cells present an opportunity to use the hematopoietic progeny of gene-corrected autologous cells clinically in a manner that may preclude the immunologic complications of allogeneic transplantation.

Gene therapy of Diamond Blackfan anemia CD34 + cells leads to improved erythroid development and engraftment following transplantation

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia caused by mutations in ribosomal protein (RP) genes. Our aim is to develop gene therapy for DBA patients with mutations in RPS19. We previously demonstrated that RPS19 gene transfer partially corrects erythroid development in vitro. In this study, we asked if RPS19 gene transfer corrects erythroid development in unsorted cells transplanted to immunodeficient mice and if the RPS19-corrected fraction has a proliferative advantage after transplantation. We further determined if high level of RPS19 expression is required for correction. Mobilized peripheral blood CD34(+) cells were transduced by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus envelope. Vectors containing two different promoters with different RPS19 transgene expression levels were compared. Transduced cells were transplanted to immunocompromised nonobese diabetic/severe combined immunodeficient-beta2 microglobulin null mice in order to assess therapeutic effects of RPS19 gene transfer in vivo. We show that correction of erythroid development requires high RPS19 expression. The corrected fraction of unselected DBA cells have a survival advantage in vivo, suggesting that successful gene therapy may only require correction of a fraction of the patient cells. Our findings are fundamental for development of clinical gene therapy for DBA because they demonstrate increased engraftment of RPS19-transduced cells without selection of gene-corrected cells prior to transplantation, an essential prelude to studying long-term therapeutic effects in emerging animal models for DBA.

A novel competitive repopulation strategy to quantitate engraftment of ex vivo manipulated murine marrow cells in submyeloablated hosts

Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts. Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation-conditioned hosts using a "three-way" marking system, so that test, competitor, and host cell chimerism could be reliably determined posttransplantation. We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced approximately 10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay. Murine marrow cells transduced using a clinically applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.

Gene therapy for chronic granulomatous disease

Patients with chronic granulomatous disease (CGD) cannot generate reactive oxygen metabolites, and suffer from severe recurrent infections and dysregulated inflammation. Haematopoietic stem cell transplantation is the only established option for definitive cure for patients with a suitable donor and is indicated when conventional prophylaxis and therapy with antimicrobial medication fail. Gene therapy has the potential to cure CGD, and several clinical trials have been conducted since 1997. Whereas initial studies resulted in low and short-term engraftment of CGD-corrected cells, recent trials demonstrated clinical benefit when engraftment was enhanced by busulfan conditioning prior to infusion of gene-corrected cells. However, the progress in gene therapy has been hampered by the appearance of insertional mutagenesis causing leukaemia in a trial for patients with X-linked severe combined immunodeficiency and by the emergence of dominant clones in a recent trial for the X-linked form of CGD. These findings stimulated the development of modified vector systems that demonstrate reduced genotoxicity in vitro and in animal models. New gene therapy protocols that allow efficient gene transfer and durable expression but limit the risk for insertional mutagenesis are envisioned to become an important therapeutic option for patients with CGD.

Comparative Results of Gene Therapy for Adenosine Deaminase Deficiency with or without PEG-ADA Withdrawal and Myelosuppressive Chemotherapy.

Adenosine deaminase (ADA) deficiency is a form of severe combined immunodeficiency (SCID) that has long been considered a good candidate for gene therapy (GTx). In 2001–2002, we treated 4 ADA-SCID patients in a clinical trial evaluating the efficacy of 2 different retroviral vectors while continuing enzyme replacement with pegylated bovine ADA (PEG-ADA). No chemotherapy was used. All patients have been monitored for 6 years. No treatment-related serious adverse events occurred. A mild transient elevation in absolute lymphocyte count (ALC) was seen in 2 patients early post-treatment, however, no durable immunologic changes were observed. Low levels (0.1–0.7%) of vector-marked peripheral blood mononuclear cells (PBMCs) persist in 2 patients treated at the age of 4–5 years. All patients remain on PEG-ADA, prophylactic antibiotics and intravenous immunoglobulins. In 2004, we revised the protocol in order to facilitate engraftment and selective advantage of gene-corrected cells by withdrawing PEG-ADA and giving busulfan (75 mg/m^2) before GTx. In November 2005, a first patient was treated who developed unexpected prolonged bone marrow (BM) aplasia. Cytogenetics revealed trisomy 8 aberrations that were found to be present on a BM specimen obtained pre-GTx (Blood 2007; 109:503). Our second patient enrolled in January 2007. He received 5x10^6 CD34+ cells/kg that showed 40–200 units (U) of ADA activity (normal range 58–128). Over 6 months, this patient showed a slow increase in ALC (up to 750/mcL) and lymphocyte function. PBMC ADA activity has been up to 50U. The deoxyadenosine metabolite (dAXP) level has decreased to <10%, levels typically seen after allogeneic BM transplant. A third patient was treated in May 2007 and received 2x10^6 CD34+ cells/kg with ADA activity of 7-87U. Over three months, lymphocyte function has increased over pretreatment level. PBMC ADA activity has been up to ∼24U and the dAXP levels have decreased to <10%. Both patients have been off PEG-ADA since two weeks pretransplant and had mild post-GTx courses, with no documented infection, and no need for blood product transfusions. AST and ALT rose to < 5 x the upper limit of normal (ULN) in the second patient, and to < 3xULN in the third patient, and then resolved, an effect attributed to PEG-ADA withdrawal. Without myeloid growth factors, times to neutrophil count >500/mcL were 36 and 45 days, respectively. In >200 combined days of observation, there has been only one temperature above 38 °C, which resolved with acetaminophen. These data are consistent with the positive results of GTx for ADA-SCID obtained in Milan and London and show that PEG-ADA withdrawal and reduced conditioning improve the outcome of GTx for this disease. Longer follow-up should allow us to study engraftment of cells containing vector-specific sequences and conclude if either vector contributes more to recovery of lymphoid immunity.

Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

AbstractEfficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)–expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K–expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

Characterization of an immunodeficient mouse model of mucopolysaccharidosis type I suitable for preclinical testing of human stem cell and gene therapy

Mucopolysaccharidosis type I (MPS-I or Hurler syndrome) is an inherited deficiency of the lysosomal glycosaminoglycan (GAG)-degrading enzyme α- l-iduronidase (IDUA) in which GAG accumulation causes progressive multi-system dysfunction and death. Early allogeneic hematopoietic stem cell transplantation (HSCT) ameliorates clinical features and extends life but is not available to all patients, and inadequately corrects its most devastating features including mental retardation and skeletal deformities. To test novel therapies, we characterized an immunodeficient MPS-I mouse model less likely to develop immune reactions to transplanted human or gene-corrected cells or secreted IDUA. In the liver, spleen, heart, lung, kidney and brain of NOD/SCID/MPS-I mice IDUA was undetectable, and reduced to half in heterozygotes. MPS-I mice developed marked GAG accumulation (3–38-fold) in these organs. Neuropathological examination showed GM 3 ganglioside accumulation in the striatum, cerebral peduncles, cerebellum and ventral brainstem of MPS-I mice. Urinary GAG excretion (6.5-fold higher in MPS-I mice) provided a non-invasive and reliable method suitable for serially following the biochemical efficacy of therapeutic interventions. We identified and validated using rigorous biostatistical methods, a highly reproducible method for evaluating sensorimotor function and motor skills development. This Rotarod test revealed marked abnormalities in sensorimotor integration involving the cerebellum, striatum, proprioceptive pathways, motor cortex, and in acquisition of motor coordination. NOD/SCID/MPS-I mice exhibit many of the clinical, skeletal, pathological and behavioral abnormalities of human MPS-I, and provide an extremely suitable animal model for assessing the systemic and neurological effects of human stem cell transplantation and gene therapeutic approaches, using the above techniques to measure efficacy.

Integrating Ideas on Insertional Mutagenesis by Gene Transfer Vectors

The 3rd Stem Cell Clonality and Genotoxicity Retreat was held 12–13 December 2006, directly following the 48th annual meeting of the American Hematological Society held in Orlando, Florida. This series of retreats was organized by David Williams, Chris Baum, and Christof von Kalle of Cincinnati Children's Hospital. The impetus was the incidence of insertional leukemogenesis arising in the French gene therapy trial treating X-linked severe combined immunodeficiency disease (X-SCID) in 2002. Prior to 2002, the risk of insertional mutagenesis from gene transfer vectors was a theoretical one that had not been followed up with rigorous proactive studies. Of course, this all changed after the adverse events in the French trial and a case of insertional mutagenesis discovered in an animal study earlier the same year by Chris Baum's group. In the following years, evidence was found for clonal dominance in hematopoietic stem cells transduced with retroviral vectors in both mice and macaques, and in patients with chronic granulomatous disease (CGD) treated with gene-corrected hematopoietic cells.

Intravenously Injected Human Fibroblasts Home to Skin Wounds, Deliver Type VII Collagen, and Promote Wound Healing

Patients with dystrophic epidermolysis bullosa (DEB) have incurable skin fragility, blistering, and multiple skin wounds because of mutations in the gene that encodes for type VII collagen (C7), which holds together the epidermal and dermal layers of human skin. The intradermal injection of gene-corrected DEB fibroblasts, recombinant C7 protein, or lentiviral vectors expressing C7 is a potential therapy for DEB. Nevertheless, severe DEB causes widespread wounds and treatment would require multiple injections. An alternative strategy might be to inject genetically engineered cells into the patient's circulation that home to the skin wounds and deposit the transgene product. In this study, we demonstrated that intravenously (IV) injected, molecularly engineered DEB fibroblasts (overexpressing human C7) homed to murine skin wounds and continuously delivered C7 at the wound site, where it incorporated into the skin's basement membrane zone and formed anchoring fibril structures. Wounds made on murine or grafted human skin demonstrated accelerated healing when the animals were IV injected with gene-corrected DEB fibroblasts. Our data demonstrate that abundant C7 promotes wound healing. This is also the first evidence that IV injected, molecularly engineered skin fibroblasts can deliver C7 to skin wounds. This strategy could be useful for treating DEB patients.

Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

BackgroundMucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder.MethodsThe murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis.ResultsTransduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment.ConclusionLentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA.

Stem Cell Collection and Gene Transfer in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34(+) stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34(+) cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34(+) cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Polyclonality of Retroviral Insertion Sites Following Successful Retroviral-Mediated Gene Transfer of CD18 in Canine Leukocyte Adhesion Deficiency.

Canine leukocyte adhesion deficiency (CLAD) represents a large-animal model for the human disease leukocyte adhesion deficiency, type 1 (LAD-1). Both CLAD and LAD are characterized by life-threatening bacterial infections and are due to mutations in the leukocyte integrin CD18. We recently reported successful gene therapy in six dogs with CLAD following infusion of autologous CD34+ bone marrow hematopoietic stem cells transduced with the retroviral vector PG13/MSCV-cCD18. The level of CD18+ gene corrected leukocytes ranged from 1.3% to 8.4% at one year post-transplant in the six CLAD dogs. We continue to monitor four of the six dogs for long-term follow-up. To date no gene transfer-related adverse events have been observed in any of the dogs. However, verification of polyclonality of insertion sites in the gene-corrected cells and characterization of the genomic sites of retroviral integration remain critical safety issues. To this end, we have demonstrated the polyclonality of contributing retrovirus-transduced cells in all six dogs using linear-amplification mediated PCR (LAM-PCR) with DNA obtained from peripheral blood leukocytes up to 2 years following infusion of gene-corrected cells. Multiple clones were shown to contribute to hematopoiesis; no predominant clones emerged over time. Retroviral insertion sites in the six dogs were identified using ligation-mediated PCR (LM-PCR) followed by sequencing of the genomic sequence adjacent to the integrated viral 3′ LTR. Mapping of insertion sites was facilitated by the recent completion of the annotated dog genome sequence. To date, we have identified 341 unique retroviral insertion sites. Analysis of these insertion sites indicated that 46% of the sites are located within gene-coding regions of the genome (introns or exons). Another 35% of the sites were located within 50 kb of the transcription start site of a gene-coding sequence. These observations match previous reports of preferential integration of MLV retroviral vectors near coding sequences. The insertion sites were well-distributed across all chromosomal locations, with no evidence for insertional hotspots or preferential growth of clones containing common insertion sites: only six pairs of insertion sites were located within 5 kb of each other. Further analysis was performed on a sub-group of 294 genes that had insertion sites located either in them or within 50 kb of the transcription start site. 19 of these genes (6.5%) were present in either or both of two cancer gene databases (Sanger Institute Cancer Gene Census Table, Mouse Retrovirus Tagged Cancer Gene Database). Functional annotation of the 294 genes revealed some groups of interest: 17% of the genes were involved in phosphorylation, 4% were proto-oncogenes, 12% encoded zinc-finger proteins, 23% were involved in signal transduction, 2% encoded transforming proteins, 5% were involved in cell proliferation, 8% were involved in transcriptional regulation and 4% encoded protein kinases. We are continuing to sequence and characterize a large number of retroviral insertion sites from the gene-corrected CLAD dogs. Analysis of these insertion sites should provide considerable insight into the risk of genotoxicity through insertional mutagenesis in a disease-specific large-animal model of a human hematopoietic disease.