Transduction Of Stem Cells Research Articles

Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice

AbstractFanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

Retroviral Vector Integration Site Analysis in Rhesus Macaques Transplanted with Hematopoietic Stem Cells Transduced with Simian Immunodeficiency Virus Vectors.

Lentiviral vectors derived from the Simian Immunodeficiency Virus (SIV) mediate relatively efficient transduction of hematopoietic stem cells (HSCs) from rhesus macaques. While integration sites associated with onco-retroviral vectors have been extensively studied in primate transplantation experiments, much less in known about lentiviral vector integration site patterns. The existing literature is limited to one report showing that SIV vectors have a distinctive genomic integration pattern compared with onco-retroviral vectors (Hematti et al 2004). Here we report our results mapping 263 integration sites for SIV vectors in an autologous rhesus macaque transplantation model. Two SIV vectors were used that expressed either MGMT-P140K alone or MGMT-P140K together with HOXB4 from an internal MSCV promoter. Two rhesus macaques were transplanted with autologous CD34+ cells, half of which were transduced with the MGMT vector and half were transduced with MGMT-HOXB4 vector. The first animal was treated with 7 courses of temozolomide and 6-BG which has resulted in selection of transduced cells in vivo, both at the level of myeloid progenitors, and to a lesser degree, in HSCs. A total of 152 integration sites were identified from this animal based on LAM-PCR. Sequence analysis showed a favored preference for integration into transcription units, which comprised 70% of all integrations, with 64% integrations occurring within introns and 6% within exons. The highest density of SIV integration sites per Mbp were on chromosomes 17 and 19 (0.17 and 0.2 respectively). At different time points during drug treatment, multiple clones contributed to hematopoiesis and 24 clones were identified repetitively. The second animal was treated with two courses of TMZ/BG and two courses of BCNU/BG resulting in selection of transduced cells in all lineages. So far, a total of 111 integration sites have been identified in this animal and a similar general integration pattern was observed as seen in the first animal. Integration into transcription units was favored (71%) with 65% occurring within introns and 6% within exons. The three most gene-dense chromosomes 17, 19 and 22 had the highest density of SIV integration sites (0.11, 0.16 and 0.18 respectively). In this animal, 10 out 111 integration sites were identified repetitively during the drug treatments. Vector integrations near previously described oncogenes were identified in both animals (19 out 152 and 11 out of 111 integration sites for each animal respectively). However, no common integration sites (CIS) into a single oncogene were observed and no abnormal hematopoietic proliferation developed in either animal. Moreover, there were no integrations seen within the MDS/Evi locus that has been previously shown to be a CIS for onco-retroviral vectors. Our study shows that the SIV integration pattern is distinctly different from that obtained with murine oncoretroviral vectors and is consistent with the previous study. The lack of integrations within the MDS1/Evi locus represents a potential safety advantage, however further study will be necessary to determine whether the overall propensity for insertional mutagenesis and transformation is decreased. We also show that multiple clones contributed to hematopoiesis before and after MGMT-mediated selection suggesting that this approach is not necessarily associated with restrictions in clonal numbers contributing to hematopoiesis.

Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.

Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology 2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

A Phase I Dose Escalation Study of the mTOR Inhibitor Sirolimus and MEC Chemotherapy Targeting Signal Transduction in Leukemic Stem Cells for Acute Myeloid Leukemia.

In vitro studies have suggested that AML cells are sensitive to treatment with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. In particular, mTOR inhibition is known to enhance the sensitivity of primary AML cells and AML stem cells to etoposide based chemotherapy leading to inhibition of leukemic SRC activity in NOD/SCID mice. To determine the feasibility of applying this approach in vivo, we performed a Phase I dose escalation study of the mTOR inhibitor sirolimus (rapamycin) with a combination chemotherapy induction regimen in adults with relapsed or refractory non-M3 AML. The purpose of this trial was to determine the safety and dose limiting toxicities of sirolimus and chemotherapy in this patient population. Patients received a loading dose of oral sirolimus on day 1 followed 24 hours later by daily doses of oral sirolimus on days 2–7 plus MEC (mitoxantrone 8 mg/m2/day IV, etoposide 100 mg/m2/day IV, and cytarabine 1000 mg/m2/day IV) on days 1–5. Five sirolimus dose levels were explored by a standard 3+3 design. Sirolimus was studied at loading doses from 3–15 mg and daily doses from 1–5 mg/d. Clinical response was assessed by bone marrow biopsy upon hematologic recovery or day 42, whichever occurred first. 23 adults (14 women, 9 men) of median age 58 (range 22 to 65) with relapsed, refractory, or secondary AML were treated with sirolimus and MEC. Five subjects had antecedent hematologic disorders or prior leukemogenic chemotherapy, 18 had relapsed or refractory disease. Sirolimus was well tolerated and did not increase non-hematologic toxicity of MEC chemotherapy. Asymptomatic, reversible liver transaminase or bilirubin elevations occurred in 4 patients, two of which were > grade 2. One patient with a history of prior cytarabine cerebellar toxicity (unknown at the time of study entry) developed reversible cerebellar ataxia. Three patients died of complications related to bacterial infections during chemotherapy-induced aplasia. Dose limiting toxicity was prolonged myelosuppression at the highest planned dose level and was responsible for one treatment-related death due to infectious complications from unresolved aplasia on study day 119. For the first four dose levels the median time to ANC recovery >500/uL among evaluable patients was 27 days (range16–38). Pharmacokinetic data showed that doses of 3 mg and higher consistently achieved rapamycin levels considered therapeutic in solid organ transplantation (4–9.2 ug/L). Bone marrow studies in 2/2 evaluable patients on dose level 4 (12 mg loading dose and 4 mg per day sirolimus) showed inhibition of p70S6 kinase phosphorylation consistent with effective inhibition of mTOR at this dose level. Complete remissions occurred in four patients, all treated for first relapse. Two patients subsequently proceeded to allogeneic transplantation. These results indicate that the combination of mTOR inhibition and chemotherapy is feasible in human AML and establish an appropriate dose for phase II studies to be 12mg loading dose followed by 4 mg daily. Patient recruitment at this dose is ongoing. Confirmation of the efficacy of this regimen, which targets signal transduction in leukemic < stem cells, is planned in a randomized phase II trial at the cooperative group level.

Autologous Stem Cell Transplantation (ASCT) for AIDS-Related Lymphomas (ARL) and the Potential Role of HIV-Resistant Stem Cells.

Background: Optimal therapy of ARL relies on both effective anti-tumor chemotherapy and successful control of the underlying HIV infection. Management of HIV using HAART has been hampered by patient non-compliance with complex regimens, drug resistance and ongoing low level viral replication. Multiplexed RNA based anti-HIV gene transfer strategies to confer intrinsic cellular resistance may help circumvent these problems. Autologous stem cell transplantation for ARL is an ideal clinical platform for delivery of SiRNA transduced stem cells as combining gene transfer strategy with high dose antilymphoma therapy could provide control of both the HIV infection and the ARL.Methods: Gene transfer - anti-HIV RNA elements, including short hairpin RNA (shRNA) targeted to HIV tat/rev, a TAR-specific decoy sequence, and a ribozyme targeted to CCR5 were combined into a lentivirus vector (LV, rHIV7-shI-TAR-CCR5RZ). Using LV transduction methods, these anti-HIV RNAs were delivered into CD34+ hematopoietic progenitor cells (HPC).Results: Preclinical vector development - LV transduction allowed differentiation in liquid culture and in a SCID-hu model which produced macrophage and T cell progeny that were resistant to the HIV virus. Although HIV resistance can be induced in vitro with single anti-HIV shRNAs, no resistance was found in multiply passaged HIV in rHIV7-shI-TAR-CCR5RZ-transduced cells. In addition, cells were analyzed by microarray for mRNA and miRNA alterations and no significant changes were noted between CD34 transduced and untransduced cells. Copy number in transduced cells was 1–2/cell, and integration site analysis localized to transcriptionally active sites, usually away from terminal portions of gene sequences. This vector is proposed for use in ASCT for ARL.Update of ASCT in ARL: Between 1998 and 2006, 28 patients with high-risk ARL underwent ASCT at the City of Hope. Conditioning regimens included CBV (cyclophosphamide (Cy)100mg/kg, Carmustine150 mg/m2, VP16 60mg/kg) in 24 and FTBI/Cy/VP16 in 4. All patients engrafted, median time to ANC>500 was 11 days (range, 8–19). Regimen related toxicities included grade 3–4 hepatic toxicity n=3 and interstitial pneumonitis n=2. OI's included PCP pneumonia n=2, CMV infection (2 viremias, 1 retinitis) and 2 cases of VZV. Therapy-related MDS was seen in one patient who ultimately died of MDS while in remission from his ARL. Median HIV viral load (VL) at ASCT was 6113 gc/ml with 22 having an undetectable VL. At two years only 9 pts had an undetectable VL. Median CD4 count at ASCT was 164 (range 25–1064), this rose to 263 (95–1164) at two years post ASCT. With a median f/u of 41 months, 2yr OS is 78% (95% CI 63–87) and PFS 78% (95% CI 63–87).Conclusions: ASCT can lead to durable remission for ARL. The fluctuation in VL seen post ASCT reflects the natural history of HIV infection and limitations of current antiviral therapy. Ultimately the system of gene transfer outlined above combined with ASCT as part of our next generation clinical trial could provide the optimal chance of cure for pts with high-risk ARL by offering both effective antilymphoma and anti-HIV therapy.

Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack

Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry(-/-)) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 +/- 127 DAF molecules/cell (DAF(high)) compared with 249 +/- 94 DAF molecules/cell (DAF(low)) and 774 +/- 135 Crry molecules/cell on control mouse erythrocytes. DAF(high)-Crry(-/-) erythrocytes were significantly more resistant than either DAF(low)-Crry(-/-), DAF(-/-) -Crry(+/+) or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry(-/-) erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAF(high)-Crry(-/-) erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

Correction of sickle cell disease by homologous recombination in embryonic stem cells

AbstractPrevious studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (βS) with a normal copy of the gene (βA). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human βS-globin gene with a human βA-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematologic values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis.

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

Rapid analysis of T-cell selection in vivo using T cell–receptor retrogenic mice

Although T-cell receptor (TCR) transgenic as well as knockout and knockin mice have had a large impact on our understanding of T-cell development, signal transduction and function, the need to cross these mice delays experiments considerably. Here we provide a methodology for the rapid expression of TCRs in mice using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells of any background before adoptive transfer into RAG-1(-/-) mice. For simplicity, we refer to these as retrogenic mice. We demonstrate that these retrogenic mice are comparable to transgenic mice expressing three commonly used TCRs (OT-I, OT-II [corrected] and AND). We also show that retrogenic mice expressing male antigen-specific TCRs (HY, MataHari and Marilyn) facilitated the analysis of positive and negative selection in female and male mice, respectively. We examined various tolerance mechanisms in epitope-coupled TCR retrogenic mice. This powerful resource could expedite the identification of proteins involved in T-cell development and function.

Maintenance of Proliferative Capacity and Retroviral Transduction Efficiency of Human Fetal CD38–/CD34++Stem Cells

Methods for the efficient transduction and expansion of fetal hematopoietic stem cells could lead to novel in utero therapies for blood cell disorders and enzymatic deficiencies. Here we describe a new assay to measure rapidly the effects of cytokines on the differentiation or expansion of primitive progenitors and stem cells found among CD38(-)CD34(++) lineage() cells isolated from human midgestation liver. Importantly, conditions that otherwise supported the expansion of clonogenic progenitors reduced their proliferative capacity. A combination of megakaryocyte growth and development factor and granulocyte-macrophage colony-stimulating factor maintained proliferative potential while also yielding an intermediate level of progenitor expansion. Retroviral transduction was achieved using Moloney murine leukemia virus-based vectors. Freshly isolated candidate stem cells could be transduced at almost 17% efficiency by a 1-h exposure to virus with centrifugation to aid transduction. This was increased to a mean 35.5% transduction efficiency after 1 day of culture. Additionally, the transduction efficiency of candidate stem cells isolated from fetal placental blood was 33.0%. These findings encourage further investigation into the feasibility of ex utero gene therapy whereby fetal cells are isolated from the circulation, transduced, and expanded ex utero before being returned to the fetus.

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

456. Gene Transfer to Skin Stem Cells by Early Gestational Intra_Amniotic Injection of Lentiviral Vector

Early gestational gene transfer may provide developmental opportunities for transduction of accessible stem cell populations that are not available later in development. An example with potential clinical importance is skin_associated stem cells. During development, these cells become inaccessible for gene transfer due to formation of the periderm, stratification of epidermis, and involution of skin accessory structures. To test whether gene transfer to skin stem cells could be achieved early in gestation, and whether gene expression would be maintained long_term, we injected lentiviral vector carrying the green fluorescent protein (GFP) reporter gene into the fetal murine amniotic space from the late head fold/early somite stage (E8) to E18. From E8 to E12 amniotic space injections were performed under ultrasound image guidance using a [email protected] Vevo660 system with a 40 mHz scanhead. After E12, injections were performed under direct vision using a stereoscopic microscope. We observed the skin under fluorescence stereomicroscopy at sequential time points after birth and confirmed GFP expression by immunohistochemistry.GFP expression was observed in the skin of E8 to E12 groups after birth, but no expression was observed with injections after E12. In E8 to E10 injected groups, widespread expression over the entire skin surface, including hair and nails, was seen, whereas in the E11 and E12 groups, GFP expression was limited to the hair and skin of the scalp. Expression in these distributions persisted beyond 6 months of age in all animals. Histological analysis revealed that GFP was expressed in the skin accessory structures, including the bulge region of the hair follicle, the sebaceous glands, as well as the epidermis. After epidermal injury by punch biopsy, GFP positive cells from the surrounding skin and hair follicles were recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a distinctive radial pattern consistent with stem cell transduction. FACS analysis of the skin of E9 group revealed the existence of CD34+/GFP+ cells confirming gene transfer to the bulge stem cells.These finding confirm that gene transfer can be achieved to skin associated stem cell populations, including the bulge and epidermal stem cells, by early gestational intra_amniotic injection of lentiviral vectors. This gene transfer method may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal skin directed gene therapy for specific skin disorders. Early gestational gene transfer may provide developmental opportunities for transduction of accessible stem cell populations that are not available later in development. An example with potential clinical importance is skin_associated stem cells. During development, these cells become inaccessible for gene transfer due to formation of the periderm, stratification of epidermis, and involution of skin accessory structures. To test whether gene transfer to skin stem cells could be achieved early in gestation, and whether gene expression would be maintained long_term, we injected lentiviral vector carrying the green fluorescent protein (GFP) reporter gene into the fetal murine amniotic space from the late head fold/early somite stage (E8) to E18. From E8 to E12 amniotic space injections were performed under ultrasound image guidance using a [email protected] Vevo660 system with a 40 mHz scanhead. After E12, injections were performed under direct vision using a stereoscopic microscope. We observed the skin under fluorescence stereomicroscopy at sequential time points after birth and confirmed GFP expression by immunohistochemistry. GFP expression was observed in the skin of E8 to E12 groups after birth, but no expression was observed with injections after E12. In E8 to E10 injected groups, widespread expression over the entire skin surface, including hair and nails, was seen, whereas in the E11 and E12 groups, GFP expression was limited to the hair and skin of the scalp. Expression in these distributions persisted beyond 6 months of age in all animals. Histological analysis revealed that GFP was expressed in the skin accessory structures, including the bulge region of the hair follicle, the sebaceous glands, as well as the epidermis. After epidermal injury by punch biopsy, GFP positive cells from the surrounding skin and hair follicles were recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a distinctive radial pattern consistent with stem cell transduction. FACS analysis of the skin of E9 group revealed the existence of CD34+/GFP+ cells confirming gene transfer to the bulge stem cells. These finding confirm that gene transfer can be achieved to skin associated stem cell populations, including the bulge and epidermal stem cells, by early gestational intra_amniotic injection of lentiviral vectors. This gene transfer method may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal skin directed gene therapy for specific skin disorders.

63. Long-Term Constitutive Expression of the Therapeutic Gene, IL2RG, Causes T-Cell Lymphomas in a Gene Therapy Model for XSCID

Top of pageAbstract The successful gene therapy clinical trials, where the immune function of patients with X-linked severe combined immune deficiency (XSCID) was restored, demonstrated the utility for vector mediated gene transduction of hematopoietic stem cells in the treatment of a life threatening disease. However, the occurrence of T-cell leukemias in three patients, with the suspected oncogenic event resulting from upregulation of expression of the known oncogene, LMO2, by insertional mutagenesis, has led to the re- evaluation of the safety of this procedure. To model the gene therapy adverse events and to better understand the mechanism of generation of leukemia in the patients, we expressed the therapeutic gene, IL2RG, used to treat XSCID patients, in combination with and without LMO2 in a murine model of XSCID, and followed the fates of transplanted mice up to 1.8 years post transplantation. In this study, we unexpectedly found that the therapeutic gene IL2RG alone is a major contributor to the genesis of T-cell lymphomas in mice, with a prevalence of 33%, n=15. We found that IL2RG|[minus]|/|[minus]| mice transplanted with IL2RG vector transduced IL2RG|[minus]|/|[minus]| cells or similarly transduced wild type cells developed the T-cell lymphomas. As expected, T-cell lymphomas were also detected in the positive control LMO2 transduced mice with a prevalence of 50%, n=12, with cell phenotype similar to those previously published. In contrast, none of the mock transduced mice (n=16) nor LV-GFP vector transduced mice (n=17) developed the T-cell lymphoma in this study. These findings were generated from 7 independent experiments totaling 99 mice ranging from 71 to 91 weeks posttransplant. Interestingly, the average time of onset of the T-cell lymphomas in IL2RG transduced mice was faster than the LMO2 transduced mice, 10.1 months versus 12.2 months post transplant, p=0.034. The T-cell lymphoma manifested itself as a gross thymic mass, which showed neoplastic cells with a CD3/B220 double positive phenotype, as well as larger CD4|[minus]|/CD8+ blast-like cells, which were also predominant in the spleen. Vector integration site analysis showed lymphomic cells to be of clonal origin with similar integration site profiles being present in the thymuses, bone marrow cells, and spleens of individual mice. Dual transgene expression of IL2RG and LMO2 did not significantly accelerate lymphomagenesis. Our results suggest a re-evaluation of the current view that IL2RG has minimal oncogenic properties and offer caution that current gene therapy trials for XSCID may pose increased risks to patients than previously anticipated. Furthermore, these results may explain why leukemia has been seen with such prevalence in XSCID trials when gene therapy trials for other diseases have not shown any such side effects.

341. Lentivirus Transduction of Murine Embryonic Stem Cells with Truncated Human Low-Affinity Nerve Growth Factor Permits Efficient Purification of Genetically Modified Cells without Loss of Stem Cell Characteristics

The ability to genetically modify embryonic stem cells (ESC) expands the potential of ESC to correct hereditary disorders and deliver gene products for specific therapies. Integrating gene transfer vectors, such as lentivirus vectors, have been used to deliver genes to ESC, but the transduction efficiencies are far from 100% and selection of transduced cells using drug resistance is often unpredictable and toxic. A surface marker delivered by a lentivirus vector that could be employed in a magnetic-bead purification strategy would be a powerful tool in selecting transduced from untransduced ESC. To address these issues, we hypothesized that: (1) lentivirus transfer of the truncated human low-affinity nerve growth factor (LNGFR), a non-toxic, non-tumorigenic and non- functional protein expressed on the surface of transduced cells, could be used to isolate transduced ESC; (2) LNGFR expression would not alter the characteristics of the undifferentiated ESC; and (3) LNGFR expression would not interfere with the response of ESC to differentiation signals. To assess LNGFR as a purification strategy for ESC, we utilized a lentivirus vector with a human phosphoglycerate kinase 1 (PGK) promoter to drive expression of LNGFR. Murine ESC (C57BL/6 strain) were transduced with this vector and cultured on a murine embryonic fibroblast (MEF) feeder layer. Flow cytometry analysis (FACS) 48 hr after transduction revealed LNGFR expression in 44% of the ESC and 2 % in the corresponding mouse IgG1-PE control. On day 4, LNGFR+ ESC were purified with magnetic beads using an anti-LNGFR antibody. After single purification with magnetic beads, 95% of the transduced cells were LNGFR+. Several different assessments demonstrated that LNGFR expression did not alter the characteristics of the purified undifferentiated ESC. First, LNGFR+ cells plated onto a MEF feeder layer remained undifferentiated for 2 wk and the expression pattern of SSEA1 (FACS analysis), a marker for undifferentiated ESC, was identical in mock and LNGFR+ ESC (p=0.7). Second, embryoid body formation was identical in LNGFR+ and mock transduced ESC. Third, after subcutaneous administration of purified LNGFR+ or mock transduced ESC, teratomas, tumors arising from undifferentiated ESC, developed within 2 wk. Finally, to determine whether LNGFR expression would interfere with the response of ESC to differentiation signals, differentiation to endoderm was induced by Activin A. After 5 days, FACS analysis for CXCR4, a marker for definitive endoderm, showed similar levels of expression in LNGFR+ ESC and the control group (20.4% vs 21.2%), suggesting that LNGFR expression does not interfere with differentiation to endoderm. Together, these observations demonstrate that lentivirus- mediated transfer of LNGFR is a useful tool in purifying transduced ESC without deleterious or toxic effects on maintenance or differentiation.

129. Adenovirus Serotype 35 Vector-Mediated Transduction in Mouse Hematopoietic Stem Cells Isolated from Human CD46-Transgenic Mice

Because of their capacity for self-renewal and multilineage differentiation into all types of mature blood cells, hematopoietic stem cells (HSCs) are highly attractive, not only as targets of gene therapy but also as cell sources for regeneration medicine. Hence, efficient transduction into HSCs holds promise for treating a number of diseases that result from abnormal blood cell function, and it would provide a powerful approach for understanding their basic biology and for manipulating their properties in vitro and in vivo.

826. Transduction of Human Hematopoietic Stem Cells by RD114-TR-Pseudotyped Lentiviral Vectors

HIV-1-derived lentiviral vectors are efficiently pseudotyped by a chimeric envelope (RD114-TR) encoding the extracellular and transmembrane domains of the FLV RD114 glycoprotein fused to cytoplasmic tail (TR) of the MLV 4070A amphotropic glycoprotein. RD114-TR pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are of particular interest for both ex vivo and in vivo gene therapy applications. We carried out a comparative analysis of VSV-G and RD114-TR-pseudotyped lentiviral vectors in transducing human cord blood-derived CD34+ hematopoietic stem/progenitor cells. Transduction efficiency was comparatively analysed in CD34+ cells in liquid culture, in the progeny of CD34+ clonogenic progenitors in semi-solid culture, and in the progeny of CD34+ repopulating stem cells after xeno-transplantation in NOD-SCID mice. In all cases, RD114-TR-pseudotyped vectors transduced hematopoietic cells at lower m.o.i., resulting in lower toxicity and more efficient stable transduction at comparable vector copy number per genome. Potential changes in CD34+ cells transcription profile and phenotype upon transduction with RD114-TR or VSV-G-pseudotyped vectors was investigated by Affymetrix Gene Chips microarray analysis. We found no significant difference in gene expression patterns between mock-RD114-TR and VSV-G-transduced cells. Our study show that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors.

92. Gene Transfer to Ocular Stem Cells by Early Gestational Intra-Amniotic Injection of Lentiviral Vector

Ocular gene transfer has generally been approached by direct intraocular injection. In this study, we hypothesized that an opportunity exists during early gestation when specific ocular stem cell populations are accessible for gene transfer. These include the stem cell populations that maintain the cornea, lens, and retina throughout life. To test this hypothesis, we injected lentiviral vector encoding the green fluorescent protein (GFP) reporter gene into the murine amniotic space from the late head fold/early somite stage postcoital day 8 (E8) to E18 and performed sequential analysis of GFP expression in ocular tissues. Depending on the timing of vector exposure, significant GFP expression was observed in all ectoderm-derived tissues in the eye. With injection at early gestational time points, GFP expression persisted long term, with evidence of high efficiency stem cell transduction in the cornea, lens, and retina. The observed patterns and duration of gene expression confirm the accessibility of ocular stem cell populations for lentiviral vector-based gene transfer at specific developmental time points in early gestation. This model may be useful for the investigation of mechanisms of genetic and/or developmental ocular disease and for the development of prenatal gene therapy for specific ocular disorders.