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Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells

Abstract

Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration. Based on their wide host range and ability to transduce quiescent cells, human immunodeficiency virus (HIV)-derived lentivirus vectors are ideally suited for this purpose. Our present studies in a murine model show that whole bone marrow cells are readily transduced after a 1-hour vector exposure in the presence of stem cell factor and CH296 fibronectin fragment. Using this rapid transduction protocol, we achieved long-term, multilineage reconstitution of murine recipients with up to 25% GFP-expressing cells in primary and secondary recipients. Our results demonstrate the unique ability of HIV-derived vectors to transduce hematopoietic stem cells in the absence of enrichment, under minimal cytokine stimulation, and following brief exposures.

Main

Gene transfer to hematopoietic repopulating cells using retrovirus vectors benefits from stem cell enrichment and ex vivo culture to accomplish efficient long-term marking. Both enrichment and transduction culture result in overall qualitative and quantitative loss of stem cells, thereby limiting gene transfer rates in vivo.1, 2, 3, 4 Further, extended ex vivo manipulation can be a critical obstacle in candidate applications with exhausted autologous stem cell pools, such as Fanconi anemia (FA), providing the added rationale to develop rapid transduction protocols.5, 6

Some of these limitations can be overcome by human immunodeficiency virus (HIV)-derived lentivirus vectors with their ability to transduce nondividing cells, thereby permitting shortened ex vivo culture durations while maintaining gene transfer to long-term repopulating cells.7 We have previously reported long-term gene transfer rates of 12–40% after VSV-G/ lentivirus vector transduction of murine whole bone marrow cells after 12-h prestimulation in murine stem cell factor (mSCF) and a single subsequent 12-h vector exposure.8 We demonstrated in those studies that single cytokine support with mSCF compared favorably with poly-cytokine support combining mSCF, Flt3-ligand, megakaryocyte growth and development factor, and interleukin-6. Additional experiments (not shown) confirmed poor cell cycle progression, high rates of apoptosis, poor viability, and poor gene marking of whole marrow cells in the absence of cytokine support.

In seeking to extend those studies, we investigated in vitro and in vivo gene transfer rates to whole bone marrow cells without prestimulation, in the presence of mSCF only and after further reduction of ex vivo vector exposure time to 1 hour.

In initial in vitro experiments we studied cytokine support, vector particle density, and minimum exposure duration requirements for efficient gene transfer to unmanipulated whole bone marrow cells. Results illustrated that gene transfer predictably improves at escalating multiplicities of infection (MOI), but without substantial differences after 1- versus 3-h vector exposures (Figure 1a). We noted that even during these brief exposures, coating culture dishes with CH296 fibronectin fragment improved gene transfer efficiency, (Figure 1b). The pronounced fibronectin effect is likely the result of colocalization of cell and vector particle, and perhaps additional antiapoptotic properties described by others.9, 10 In previous experiments (not shown), we found that gene transfer declines substantially in the absence of cytokine support. All current experiments therefore reflect transduction culture in the presence of mSCF only. We also confirmed that gene transfer is maintained to an enriched target cell population after immunomagnetic depletion of lineage committed cells, (Figure 1c) – a cell source more frequently targeted by other investigators.

Figure 1
figure1

Brief vector exposures result in efficient transduction of whole bone marrow and enriched cell populations. (a) Transduction efficiencies are not substantially different after 1-h (open squares) versus 3-h (closed circles) exposures. (b) Transduction in the presence of the CH296 fragment and murine stem cell factor (mSCF, 50 ng/ml) (Takara Shuzo, Japan) (open squares) improves transduction efficiency after a 1-h exposure. (c) Comparative gene transfer rates to whole bone marrow, lineage depleted (lin−), and lineage enriched (lin+) cell populations after a 1-h vector exposure at different multiplicities of infection. Whole bone marrow cells harvested from 8–12-week-old C57BL/6J donor animals were depleted of red blood cells, washed and immediately transduced on fibronectin fragment CH296 in Iscoves medium in the presence of protamine sulfate, mSCF (50 ng/ml) (Peprotech, Rocky Hill, NJ), 10% fetal bovine serum (FBS, Hyclone, Logan UT, USA), and 1% penicillin/streptomycin (GIBCO). Immunomagnetic enrichment (Miltenyi Biotec, Auburn, CA, USA) was conducted using a murine lineage antibody pool (Miltenyi) with enrichment purities exceeding 90% in all cases. We used a central polypurine tract (cPPT) containing self-inactivating lentiviral backbone with an internal phosphoglycerate kinase (PGK) promoter, enhanced green fluorescent protein (EGFP) expression cassette, and a woodchuck hepatitis virus post-transcriptional regulatory element (wPRE) (RRLsincPPThPGKGFPwPRE, kindly provided by L. Naldini, Torino, Italy). This second generation vector was produced by transient co-transfection of 293T cells with transfer vector, gag-pol construct (pCMV.DR8.74—containing Rev sequence), and VSV-G envelope expression construct (pMD.G) followed by ultracentrifugation for volume concentration as described earlier.8 Vector (titer between 1.5 and 2 × 108 TU/ml concentrated vector supernatant) was added for 1 or 3 h after which cells were washed twice before return to culture media and continued culture at 37°C. Analyses of GFP expression by flow-cytometry were performed 72 h after transduction. Error bars denote standard deviations from average based on two (three for panel c) independent experiments.

In spite of the encouraging in vitro results illustrated in Figure 1, and in the light of the heterogeneity of the targeted whole bone marrow cell population, we next investigated the extent of gene transfer to long-term repopulating cells in vivo under these conditions. In repopulation experiments in myeloablated murine recipients, animals received cells transduced under the same limiting conditions, namely (a) neither in vivo nor in vitro stem cell enrichment prior to transduction, (b) a single 1-hour exposure, and (c) cytokine support with mSCF only. Following injection, all animals showed ready hematopoietic reconstitution (data not shown) and demonstrated average GFP marking of 31% (range: 17–41%) in peripheral blood 20 weeks after transplantation, (Figure 2). Half of the initial cohort of animals (n=5) served as donors for secondary transplantation. Gene marking in secondary recipients 32 weeks after reconstitution (n=15, three recipient animals per donor) persisted at 25% on average (range: 5–66%), (Figure 3).

Figure 2
figure2

GFP-marking among peripheral blood leukocytes in primary recipients at indicated time points after transplantation (flow-cytometric analysis of GFP expression). Bone marrow cells from 8–12-week-old mice (C57BL/6J) were processed by ACK hemolysis and passed through a 70 μm cell strainer. Transduction in the presence of mSCF (50 ng/ml) over 1 h was performed at an MOI of 30, as described above. Following transduction, cells were pooled, washed twice, and equal numbers (2 × 106) per animal were subsequently injected via the tail vein of lethally irradiated (1050 cGy) recipients. Myeloablated secondary recipients received 5 × 106 whole bone marrow cells each, harvested from primary recipients at killing. SCT, Stem Cell Transplantation. Cohort size for primary recipients: n=10, average cohort marking is indicated by crossbar. Open symbols and star denote animals used as donors (n=5) for subsequent secondary SCT (Note: open triangle symbol illustrating marking of animal A is obscured by that of others). Identical symbols in the panels of Figure 3 identify recipients of marrow from a specific donor animal.

Figure 3
figure3

GFP-marking among peripheral blood leukocytes in secondary recipients at indicated time points after transplantation assayed by flow-cytometric analysis of GFP expression. For secondary SCT, donor animals did not receive cytokine or 5-fluoruracil treatment. Unstimulated marrow from different donor animals was not pooled. Open symbols and stars in the panels identify recipients of marrow from a specific donor animal (animals A–E). Cohort size for secondary recipients: n=3.

Immunophenotyping analysis of peripheral blood obtained from primary animals at 20 weeks after transplantation confirmed the transduction of granulocytes, B-, and T-lymphocytes, (Figure 4a). As seen in our previous study, using this expression cassette (PGK-EGFP wpre), our results demonstrated that long-term stability of proviral expression, or rather GFP fluorescence intensity, is maintained in primary (Figure 4b) and secondary (Figure 4c) recipients. Taken together, the reconstitution of secondary recipients and multilineage transgene marking indicate the transduction of long-term repopulating cells after a single 1-hour exposure to HIV-lentivector. Furthermore, our results confirmed favorable lentivirus vector expression characteristics described by others, as well as our own group.8, 11, 12

Figure 4
figure4

Proviral GFP expression in multiple leukocyte lineages of a single animal (a), over time in bulk leukocytes of primary (b), and secondary (c) recipients. (a) Multilineage GFP expression in a representative primary recipient 6 months from transplantation. Four additional animals showed similar differential marking in all analyzed lineages. Peripheral blood white blood cells were stained with PE-labeled monoclonal anti-mouse antibodies against CD90.1 (T-lymphocyte), B 220 (B-lymphocyte), and Gr-1 (granulocyte) epitopes (all antibodies purchased from PharMingen, San Diego, CA, USA), and analyzed on a Becton DickinsonFACSCalibur® cytometer using Cellquest® software. Numbers in quadrants refer to the percentage of cells in the respective dot-plot quadrants. Analysis after exclusion of cells staining with propidium iodide. (b) Median fluorescence intensity (MFI) in peripheral blood leukocytes of primary recipients at indicated time points after SCT. (c) MFI in peripheral blood leukocytes of secondary recipients at indicated time points after SCT. Note: the first time point represents the MFI in peripheral blood leukocytes of the donor animal for those secondary recipients on the day of killing. Error bars denote standard deviation in MFI values among the three secondary recipients of marrow cells from an identical donor.

Along with others, we previously demonstrated the close correlation of vector concentration (or MOI) at the time of transduction and the copy number in target cell progeny.13, 14, 15 The use of lentivectors in particular has been shown to result in multicopy insertion, which, in turn, may increase the risk for insertional leukemogenesis, and may interfere with studies involving clonal tracking of stem cell kinetics.3, 16 We note that a reduction in vector exposure time from 12 to 1 h in the current experimental system (MOI 30) also resulted in a decrease in target cell copy number (data not shown), an observation confirmed by others.17 To determine how vector particle density (or MOI) and rapid transduction sequence in these experiments affect average proviral copy numbers in progeny, we performed real-time PCR. Our results analyzing DNA extracted from bone marrow cells of primary recipients obtained at 20 or 40 weeks after transplantation showed an average copy number of 1.5 copies per cell in recipient marrow cells (Table 1). This is consistent with data by Mostoslavsky et al.,17 employing a similar strategy of minimal manipulation and rapid transduction.

Table 1 Average proviral copy number as determined by real-time PCR in DNA extracted from bone marrow cells of nine primary recipient animals

A recent study by Blomer et al.18 reports the presence of infectious VSV-G pseudotype lentiviral particles on cells injected into animals after a 5-h ex vivo transduction (at MOI 7), and persisting at low levels even after extensive washing. We have not evaluated to what extent injected cells in our model serve as a ‘shuttle’ for unincorporated viral particles, but it is worthwhile to emphasize the differences between their protocol and the one proposed here. Specifically, this concerns the choice of target cell (cardiomyocytes in the study by Blomer et al.), and the intravenous route of cell administration in our experimental design. The latter has been shown to result in serum complement inactivation of VSV-G pseudotyped vector particles.19 Nevertheless, further studies are required to confirm the scope and implication of any potential viral particle persistence after transduction culture.

Our present study confirms that HIV-derived lentivirus vectors are ideally suited for the transduction of murine long-term repopulating cells under these restrictive conditions. Rapid 1-hour transduction may improve in vivo gene marking by a number of mechanisms, including reduced target cell differentiation, and by limiting toxic effects of VSV-G protein on the target cell population.20 Our strategy may prove particularly useful in situations where the target stem cell quantity is greatly limited and cells are of poor ex vivo viability, such as FA. In support of such an approach, recent studies in a murine model of FA demonstrated the development of myeloproliferative abnormalities in recipients of Fancc−/− stem cells. Those abnormalities appeared to arise specifically from cells that had undergone prolonged transduction culture, but remained untransduced, and, consequently, genetically uncorrected.21 Finally, this protocol represents an ideal platform for subsequent in vivo selection to achieve complete phenotype correction and high-level therapeutic chimerism required for some applications. Clearly, studies in large animal models and targeting human CD34-enriched progenitor cell populations are needed to confirm the feasibility of our strategy.

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Correspondence to P Kurre.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt).

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Kurre, P., Anandakumar, P. & Kiem, HP. Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells. Gene Ther 13, 369–373 (2006). https://doi.org/10.1038/sj.gt.3302659

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Keywords

  • lentivirus
  • hematopoietic stem cells

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