Postnatal transplantion to boost chimerism following intrauterine haematopoietic cell transplantation (IUHCT) in a murine model of thalassemia

British Society for Gene and Cell Therapy AnnualConference and Joint UK RegenerativeMedicine Platform MeetingUnited Kingdo

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

Characterisation of Human Fetal Liver HSC.

<p>A & B) Human fetal liver haematopoietic stem cell (hflHSC) populations contain a higher proportion of CD34+ cells than that found in umbilical cord blood (UBC) (1.8×10⁷ versus 8×10⁵; p = 0.0002 and 8.1% versus 0.4%; p = 0.0001). (C) Representative images of multilineage differentiation of hflHSC in methylcellulose culture assays.</p

More efficient transduction of fetal liver-derived than cord blood-derived HSC.

<p>(A) Human fetal liver HSC are transduced more efficiently with the A2UCOE-eGFP LV than UCB-HSC, with higher efficiencies achieved with increasing gestational age from 13 through 22 weeks. (B) Higher levels of transduction of hflHSC were achieved with the A2UCOE-eGFP LV than with the PGK-eGFP and EF1α-eGFP vectors at differing multiplicities of infection (MOI).</p

Sustained expression of A2UCOE-eGFP in <i>in vitro</i>.

<p>(A) hflHSC transduced with A2UCOE-eGFP, PGK-eGFP and EF1α-GFP LVs were maintained in culture for 24 days and analyzed at 3, 10, 17 and 24 days post-transduction. Expression from A2UCOE-eGFP was sustained over the 24 day period of culture. A rapid decline in PGK-eGFP (58% to 22%) and EF1α-eGFP (71% to 23%) expression was seen by Day 10. Average vector copy number per cell (VCN) at Day 17 of culture is given in italic text above the histogram lines. (B) A2UCOE-eGFP LV transduced cells showing maintenance of multilineage differentiation capability in methylcellulose culture assays at 17 days post-transduction. (C) Quantification of proportion of eGFP expressing colonies following differentiation of LV transduced hflHSC in semi-solid methylcellulose culture assays. Note comparable levels of eGFP-positive colonies with all three (A2UCOE, EF1α, PGK) LV types.</p

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

Major haemoglobinopathies place tremendous strain on global resources. Intrauterine haemopoietic cell (IUHCT) and gene (IUGT) therapies can potentially reduce perinatal morbidities with greater efficacy than postnatal therapy alone. We performed both procedures in the thalassaemic HbbTh3/+ murine model. Intraperitoneal delivery of coisogenic cells at E13-14 produced dose-dependent chimerism. High-dose adult bone marrow (BM) cells maintained 0.2-3.1% chimerism over ~24 weeks and treated heterozygotes demonstrated higher chimerism than wild-type pups (1.6 vs. 0.7%). Fetal liver cells produced higher chimerism compared to adult BM when transplanted at the same doses, maintaining 1.8-2.4% chimerism over ~32 weeks. We boosted transplanted mice postnatally with adult BM cells following busulfan conditioning. Engraftment was maintained at >1% only in recipients which were chimeric prior to boosting. IUHCT-treated non-chimeras and non-IUHCT mice showed micro- or no chimerism. Additional fludarabine treatment produced higher chimerism than busulfan alone. Engraftment was more effective following higher starting chimerism prior to boosting and in heterozygotes. Chimeric heterozygotes expressed 2.2-15.1% donor cells with eventual decline at 24 weeks (vs. <1% in non-chimeras) and demonstrated improved haematological indices and smaller spleens compared to untreated heterozygotes. Intravenous delivery of GLOBE lentiviral-vector expressing HBB (human β-globin) resulted in vector concentration of 0.001-0.6 copies/cell. Most haematological indices were higher in treated than untreated heterozygotes including haemoglobin and mean corpuscular volume, though still lower than in wild-types. Thus both direct IUGT and IUHCT strategies can be used to achieve haematological improvement but require further dose optimisation. IUHCT will be useful combined with postnatal transplantation to further enhance engraftment