Niraja Dighe

Stable Human FIX Expression After 0.9G Intrauterine Gene Transfer of Self-complementary Adeno-associated Viral Vector 5 and 8 in Macaques

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.

The Fetal Mouse Is a Sensitive Genotoxicity Model That Exposes Lentiviral-associated Mutagenesis Resulting in Liver Oncogenesis

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Long-term reproducible expression in human fetal liver hematopoietic stem cells with a UCOE-based lentiviral vector.

Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg -/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.

A comparison of intrauterine hemopoietic cell transplantation and lentiviral gene transfer for the correction of severe β-thalassemia in a HbbTh3/+ murine model

Highlights • The HbbTh3/+ mouse is a good model of severe thalassemia for in utero therapy.• In utero and postnatal transplantation with immunosuppression resulted in better chimerism.• In utero gene therapy produced partial hematological correction but not full rescue.• Both strategies need further optimization to overcome the hostile microenvironment.

251. Intrauterine Haemopoietic Stem Cell Therapy Followed By Postnatal Cell Re-Infusion To Enhance Engraftment in a Murine Thalassaemia Model

Intrauterine haemopoietic stem cell therapy (IUHSCT) can be considered for treating haemoglobinopathies and certain metabolic diseases in early pregnancy, for the ability to easily escalate cell dose for higher engraftment and to target the pre-immune fetus. The potential therapeutic benefits are currently unrealised due to sub-therapeutic cell engraftment from host cell competition for haemopoietic niches or neutralising immune response.We investigated the strategy of IUHSCT followed by post-natal donor cell re-infusion to achieve clinically-relevant long-term engraftment in an HbbTh3-/+ mouse model of thalassaemia. Congenic IUHSCT was performed with murine fetal liver-derived mononuclear cells (MNC, 20% Lin-), followed by selective postnatal donor cell re-infusion to microchimeric pups. Adult bone marrow MNC were used when fetal liver was unavailable. Both injected pups and donor cells were derived from C57BL6 strains, with the former expressing surface antigen CD45.2. Donor cells expressed CD45.1 or GFP and were CD26-inhibited with Diprotin A prior to transplantation. Each pup received 2 or 5E+6 GFP or CD45.1 cells in utero at E14-17. Surviving pups were examined serially for chimerism after weaning. Chimeric mice (>1% engraftment) were treated with IV busulphan and received serial postnatal transplantation of the same strain of donor cells. Increasing cell doses ranging from 5E+6, and 1E+7 to 4E+7 cells per mouse were administered at six to 20 weeks postnatal.Initial donor cell engraftment exhibited a dose-response relationship following IUHSCT. Median chimerism in pups receiving 2E+6 cells in utero was 2% (range 1-6%) and 3% (3-5%) at three and eight postnatal weeks respectively. In comparison, recipients of 5E+6 cells showed 6% chimerism at three weeks (2-7%, p=0.1). Postnatal reinfusions maintained chimerism only transiently. The first dose of 5E+6 cells/pup maintained chimerism at 2-5% for the following eight weeks, and this steadily declined to 1% over the next four weeks. The second postnatal dose (1E+7 cells) had no effect as chimerism decreased to 0-2% by 20 weeks. Donor cell levels increased to 2-6% following the third dose (3E+7 cells) before becoming undetectable levels by 32 weeks. Following IUHSCT with 5E+6 cells, postnatal reinfusion (4E+7 cells) achieved stable chimerism of 4% (range 3-5%) for the following 12 weeks, eventually declining to 2% by 16 weeks. Unchallenged chimeric pups showed a steady decline in engraftment to 2%. Sub-therapeutic chimerism predicts engraftment failure despite postnatal boost, possibly reflecting a non-tolerant environment despite fetal exposure to donor cells. However the main problem remains an inability to sustain a relevant chimerism long-term. Immune-modulation of recipients may have to be considered to improve outcomes.

Erratum: "Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice" (Molecular Therapy (2005) vol. 12 (763-771) 10.1016/j.ymthe.2005.07.358)

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

Corrigendum to “Oncogenesis Following Delivery of a Nonprimate Lentiviral Gene Therapy Vector to Fetal and Neonatal Mice”

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

827. Oncogenesis Following Delivery of Lentiviral Vectors to Fetal and Neonatal Mice

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases due to the ability of these vectors to integrate in a stable manner into the patients’ chromosomes. Since three cases of T-cell leukaemia have been identified after retrovirus gene therapy for X-linked severe combined immune deficiency as being associated with the integrating vector used for gene therapy the need for animal models to test for vector safety has become of paramount importance. Our previous work has shown that a high frequency of hepatocellular carcinomas has occurred following in utero and neonatal injection with certain lentivirus vectors. It has been hypothesized that the woodchuck post regulatory element (WPRE) carried by the vectors used in this study could be implicated in the tumour development process. Our recent study using novel vectors with mutations in the WPRE shows that mice treated with these vectors still develop liver tumours. In this report we discuss these findings and preliminary data to support an alternative cause for tumorigenesis. We also discuss the fetal and neonatal system as a novel and sensitive in vivo model to test the effects and safety of integrating vectors under consideration for clinical applications.