Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Abstract

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells.Evidence for transduction ofpluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XID) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.

Retrovirus molecular conjugates: a versatile and efficient gene transfer vector system for primitive human hematopoietic progenitor cells

“…Integration analysis for retroviral sequences was performed using inverse PCR using a previously published protocol with slight modification. 22,23 Genomic DNA extracted from individual colonies was digested with Taq I (NEB Beverly, MA) for 2 h at 651C following RNAse treatment (Invitrogen). The DNA digest was then incubated overnight with T4 ligase at 161C (NEB, Beverly, MA).…”

“…22,23 This specific PCR reaction amplifies integrated proviral sequences with their host genome junction fragments. The size of the amplification product depends on the s ite of retroviral integration into the host genome and varies with the location of specific enzymatic restriction sites occurring within the host genome relative to the provirus.…”

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

“…38,39 DNA from 48 provirus-positive colonies at the first peak of increase in transduced cells (1 and 2 months posttransplantation; Figure 2) and from 54 provirus-positive colonies at the second peak (10 and 11 months posttransplantation) in the animal S1 were analyzed. Thirtyfive colonies yielded distinct bands on inverse PCR (Figure 4a), and amplified DNA segments were sequenced.…”

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors

“…For PCR analysis, colonies were lysed in 200 l proteinase K buffer (0.01 m Tris pH 7.4, 0.15 m NaCl, 0.01 m EDTA pH 8, 0.05% SDS) with 30 g proteinase K (Sigma) for 2 h at 56°C as described. 53 The sample was extracted once with 200 l phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 2 g glycogen (Gibco BRL), 18 l 10 m NH 4 OAc, and 500 l EtOH. Following resuspension, the DNA was amplified with the Neo-specific primers 5′-CCCTCACTCCTTCTCTAGG-3′ and 5′-TTGTGCCC AGTCATAGCCGAA-3′, 49 for 50 cycles of 94°C 30 s, 55°C 60 s, and 72°C 90 s.…”