A Point-of-Care Platform for Hematopoietic Stem Cell Gene Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4416-4416
Author(s):  
Jennifer E Adair ◽  
Kevin G Haworth ◽  
Guy Sauvageau ◽  
Shelly Heimfeld ◽  
Jonah D Hocum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Point Of Care ◽  
Great Promise ◽  
Major Advance ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract Hematopoietic stem and progenitor cell (HSPC) gene therapy holds great promise but requires highly technical and dedicated facilities. Current state-of-the-art requires ex vivo HSPC gene transfer in dedicated Good Manufacturing Practices (GMP) facility infrastructure, limiting treatment to highly developed countries. A patient point-of-care strategy would therefore make HSPC gene therapy available to patients worldwide. We developed a short, semi-automated, mostly-closed platform for ex vivo isolation and lentivirus (LV) gene modification of CD34+ HSPCs from either bone marrow or mobilized peripheral blood sources using the CliniMACS Prodigy® (Figure 1). Experiments were performed with a biosafety cabinet and personal protective equipment to simulate anticipated conditions in clinical facilities of underdeveloped countries. A total of 7 custom programs were developed for bone marrow or mobilized peripheral blood CD34+ cell isolation and LV transduction. Addition of a pyrimidoindole derivative, UM 729, permitted efficient transduction of CD34+ HSPCs in <18 hours. Complete semi-automated production took <34 hours from collection to infusion of the gene modified cell product, requiring very little operator hands-on time. Autologous, LV gene-modified CD34+ HSPCs from two nonhuman primates produced using this platform engrafted and supported multilineage hematopoietic repopulation after myeloablative total body irradiation at 1020 cGy. Total cell doses achieved were 27 x 106 and 5.4 x 106 CD34+ cells/kg body weight, respectively. We observed stable, persistent and polyclonal gene marking in peripheral blood granulocytes up to 40% and lymphocytes up to 15% within 4 months after infusion. Neither animal displayed evidence for increased toxicity, including potential contamination from the cell products. We then validated processing of human bone marrow and mobilized apheresis products from healthy adult donors. We demonstrate efficient isolation of human CD34+ HSPCs and up to 60% transduction efficiency with a clinical-grade LV currently being tested in a phase I clinical trial for treatment of HIV-associated lymphoma. Products tested met current FDA approved specifications for infusion. Xenotransplantation of these products into immunodeficient mice resulted in polyclonal engraftment over 12 weeks. These data demonstrate preclinical safety and feasibility of a patient point-of-care strategy for ex vivo LV gene transfer into HSPCs. This platform represents a major advance in global portability of LV mediated HSPC gene therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3343-3343
Author(s):  
Matteo Maria Naldini ◽  
Gabriele Casirati ◽  
Erika Zonari ◽  
Giacomo Desantis ◽  
Andrea Cammarata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Gene Therapy ◽  
Single Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Single Cell Rna Sequencing ◽  
Mobilized Peripheral Blood

Abstract Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy. We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture. By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between. After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC. Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO). To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status. As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. Disclosures No relevant conflicts of interest to declare.

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3710-3716 ◽  
Author(s):  
Peter A. Horn ◽  
Kirsten A. Keyser ◽  
Laura J. Peterson ◽  
Tobias Neff ◽  
Bobbie M. Thomasson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Lentiviral Vectors ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Lentiviral Transduction ◽  
Mobilized Peripheral Blood

Abstract The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34+ hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)– and granulocyte-colony stimulating factor (G-CSF)–primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2358-2358
Author(s):  
Ali Nowrouzi ◽  
Africa Gonzales-Murillo ◽  
Anna Paruzynski ◽  
Ariana Jacome ◽  
Paula Rio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Large Scale ◽  
Random Distribution ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
In Cis

Abstract Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (&gt;80%), partial loss of vector LTR nucleotides was detected in &gt;10% of the integrants (3–25bp). Notably, &gt;20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2108-2115 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Todd E. Meyerrose ◽  
Penny L. Ragland ◽  
Barry Grimes
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Liver ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hematopoietic Reconstitution ◽  
Late Antigen ◽  
Mobilized Peripheral Blood

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs “home” to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 × 106 FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26+ cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1+c-kit+ cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.

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