Publications by authors named "Hans-Peter Kiem"

231 Publications

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques.

Mol Ther Methods Clin Dev 2021 Sep 24;22:304-319. Epub 2021 Jun 24.

Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. , CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion . To investigate this versus discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.
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http://dx.doi.org/10.1016/j.omtm.2021.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403686PMC
September 2021

A Gut Reaction to SIV and SHIV Infection: Lower Dysregulation of Mucosal T Cells during Acute Infection Is Associated with Greater Viral Suppression during cART.

Viruses 2021 Aug 14;13(8). Epub 2021 Aug 14.

Department of Microbiology, University of Washington, 750 Republican St., Seattle, WA 98109, USA.

Selection of a pre-clinical non-human primate (NHP) model is essential when evaluating therapeutic vaccine and treatment strategies for HIV. SIV and SHIV-infected NHPs exhibit a range of viral burdens, pathologies, and responses to combinatorial antiretroviral therapy (cART) regimens and the choice of the NHP model for AIDS could influence outcomes in studies investigating interventions. Previously, in rhesus macaques (RMs) we showed that maintenance of mucosal Th17/Treg homeostasis during SIV infection correlated with a better virological response to cART. Here, in RMs we compared viral kinetics and dysregulation of gut homeostasis, defined by T cell subset disruption, during highly pathogenic SIVΔB670 compared to SHIV-1157ipd3N4 infection. SHIV infection resulted in lower acute viremia and less disruption to gut CD4 T-cell homeostasis. Additionally, 24/24 SHIV-infected versus 10/19 SIV-infected animals had sustained viral suppression <100 copies/mL of plasma after 5 months of cART. Significantly, the more profound viral suppression during cART in a subset of SIV and all SHIV-infected RMs corresponded with less gut immune dysregulation during acute SIV/SHIV infection, defined by maintenance of the Th17/Treg ratio. These results highlight significant differences in viral control during cART and gut dysregulation in NHP AIDS models and suggest that selection of a model may impact the evaluation of candidate therapeutic interventions for HIV treatment and cure strategies.
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http://dx.doi.org/10.3390/v13081609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402906PMC
August 2021

Single-dose MGTA-145/plerixafor leads to efficient mobilization and in vivo transduction of HSCs with thalassemia correction in mice.

Blood Adv 2021 03;5(5):1239-1249

Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA.

We have developed an in vivo hemopoietic stem cell (HSC) gene therapy approach without the need for myelosuppressive conditioning and autologous HSC transplantation. It involves HSC mobilization and IV injection of a helper-dependent adenovirus HDAd5/35++ vector system. The current mobilization regimen consists of granulocyte colony-stimulating factor (G-CSF) injections over a 4-day period, followed by the administration of plerixafor/AMD3100. We tested a simpler, 2-hour, G-CSF-free mobilization regimen using truncated GRO-β (MGTA-145; a CXCR2 agonist) and plerixafor in the context of in vivo HSC transduction in mice. The MGTA-145+plerixafor combination resulted in robust mobilization of HSCs. Importantly, compared with G-CSF+plerixafor, MGTA-145+plerixafor led to significantly less leukocytosis and no elevation of serum interleukin-6 levels and was thus likely to be less toxic. With both mobilization regimens, after in vivo selection with O6-benzylguanine (O6BG)/BCNU, stable GFP marking was achieved in >90% of peripheral blood mononuclear cells. Genome-wide analysis showed random, multiclonal vector integration. In vivo HSC transduction after mobilization with MGTA-145+plerixafor in a mouse model for thalassemia resulted in >95% human γ-globin+ erythrocytes at a level of 36% of mouse β-globin. Phenotypic analyses showed a complete correction of thalassemia. The γ-globin marking percentage and level were maintained in secondary recipients, further demonstrating that MGTA145+plerixafor mobilizes long-term repopulating HSCs. Our study indicates that brief exposure to MGTA-145+plerixafor may be advantageous as a mobilization regimen for in vivo HSC gene therapy applications across diseases, including thalassemia and sickle cell disease.
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http://dx.doi.org/10.1182/bloodadvances.2020003714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948287PMC
March 2021

Targeting the membrane-proximal C2-set domain of CD33 for improved CD33-directed immunotherapy.

Leukemia 2021 09 15;35(9):2496-2507. Epub 2021 Feb 15.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33/CD3 bispecific antibody (BsAb) and CD33-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 antibodies"). These antibodies internalized when bound to CD33 and, as CD33/CD3 BsAb, had potent cytolytic effects against CD33 cells. Together, our data provide the rationale for further development of CD33 antibody-based therapeutics.
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http://dx.doi.org/10.1038/s41375-021-01160-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8364569PMC
September 2021

Thresholds for post-rebound SHIV control after CCR5 gene-edited autologous hematopoietic cell transplantation.

Elife 2021 Jan 12;10. Epub 2021 Jan 12.

Vaccine and Infectious Disease Division, University of Washington, Seattle, United States.

Autologous, CCR5 gene-edited hematopoietic stem and progenitor cell (HSPC) transplantation is a promising strategy for achieving HIV remission. However, only a fraction of HSPCs can be edited ex vivo to provide protection against infection. To project the thresholds of CCR5-edition necessary for HIV remission, we developed a mathematical model that recapitulates blood T cell reconstitution and plasma simian-HIV (SHIV) dynamics from SHIV-1157ipd3N4-infected pig-tailed macaques that underwent autologous transplantation with CCR5 gene editing. The model predicts that viral control can be obtained following analytical treatment interruption (ATI) when: (1) transplanted HSPCs are at least fivefold higher than residual endogenous HSPCs after total body irradiation and (2) the fraction of protected HSPCs in the transplant achieves a threshold (76-94%) sufficient to overcome transplantation-dependent loss of SHIV immunity. Under these conditions, if ATI is withheld until transplanted gene-modified cells engraft and reconstitute to a steady state, spontaneous viral control is projected to occur.
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http://dx.doi.org/10.7554/eLife.57646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803377PMC
January 2021

Stem cell-derived CAR T cells traffic to HIV reservoirs in macaques.

JCI Insight 2021 01 11;6(1). Epub 2021 Jan 11.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.
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http://dx.doi.org/10.1172/jci.insight.141502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821595PMC
January 2021

Safe and Effective Targeting and Gene Editing in Hematopoietic Stem Cells: Strategies for Accelerating Development.

Hum Gene Ther 2021 Jan;32(1-2):31-42

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

On May 11, 2020, the National Institutes of Health (NIH) and the Bill & Melinda Gates Foundation (Gates Foundation) held an exploratory expert scientific roundtable to inform an NIH-Gates Foundation collaboration on the development of scalable, sustainable, and accessible HIV and sickle cell disease (SCD) therapies based on gene editing of hematopoietic stem cells (HSCs). A particular emphasis was on how such therapies could be developed for low-resource settings in sub-Saharan Africa. Paula Cannon, PhD, of the University of Southern California and Hans-Peter Kiem, MD, PhD, of the Fred Hutchinson Cancer Research Center served as roundtable cochairs. Welcoming remarks were provided by the leadership of NIH, NHLBI, and BMGF, who cited the importance of assessing the state of the science and charting a path toward finding safe, effective, and durable gene-based therapies for HIV and SCD. These remarks were followed by three sessions in which participants heard presentations on and discussed the therapeutic potential of modified HSCs, leveraging HSC biology and differentiation, and HSC targeting approaches. This roundtable serves as the beginning of an ongoing discussion among NIH, the Gates Foundation, research and patient communities, and the public at large. As this collaboration progresses, these communities will be engaged as we collectively navigate the complex scientific and ethical issues surrounding HSC targeting and editing. Summarized excerpts from each of the presentations are given hereunder, reflecting the individual views and perspectives of each presenter.
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http://dx.doi.org/10.1089/hum.2020.263DOI Listing
January 2021

Envelope-Specific Adaptive Immunity following Transplantation of Hematopoietic Stem Cells Modified with VSV-G Lentivirus.

Mol Ther Methods Clin Dev 2020 Dec 10;19:438-446. Epub 2020 Oct 10.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 91911, USA.

Current approaches for hematopoietic stem cell gene therapy typically involve lentiviral gene transfer in tandem with a conditioning regimen to aid stem cell engraftment. Although many pseudotyped envelopes have the capacity to be immunogenic due to their viral origins, thus far immune responses against the most common envelope, vesicular stomatitis virus glycoprotein G (VSV-G), have not been reported in hematopoietic stem cell gene therapy trials. Herein, we report on two Fanconi anemia patients who underwent autologous transplantation of a lineage-depleted, gene-modified hematopoietic stem cell product without conditioning. We observed the induction of robust VSV-G-specific immunity, consistent with low/undetectable gene marking in both patients. Upon further interrogation, adaptive immune mechanisms directed against VSV-G were detected following transplantation in both patients, including increased VSV-G-specific T cell responses, anti-VSV-G immunoglobulin G (IgG), and cytotoxic responses that can specifically kill VSV-G-expressing target cell lines. A proportion of healthy controls also displayed preexisting VSV-G-specific CD4 and CD8 T cell responses, as well as VSV-G-specific IgG. Taken together, these data show that VSV-G-pseudotyped lentiviral vectors have the ability to elicit interfering adaptive immune responses in the context of certain hematopoietic stem cell transplantation settings.
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http://dx.doi.org/10.1016/j.omtm.2020.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683283PMC
December 2020

AMD3100 redosing fails to repeatedly mobilize hematopoietic stem cells in the nonhuman primate and humanized mouse.

Exp Hematol 2021 01 1;93:52-60.e1. Epub 2020 Dec 1.

Stem Cell and Gene Therapy Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

AMD3100 (plerixafor) is a vital component of many clinical and preclinical transplant protocols, facilitating harvest of hematopoietic stem and progenitor cells through mobilization into the peripheral blood circulation. Repeat mobilization with AMD3100 is also necessary for many patients with suboptimal first stem cell collection or those requiring repeat transplantation. In this study we investigated the mobilization efficacy of repeated AMD3100 dosages in the nonhuman primate and humanized mouse models. In nonhuman primates, we observed effective mobilization after the first AMD3100 administration but a significantly poorer response in CD34 and hematopoietic stem cell-enriched CD90 cells with subsequent doses of the drug. A similar loss of efficacy with repeated administration was noted in immunodeficient mice engrafted with human CD34 cells, in whom the total human white cell population, and particularly human hematopoietic stem and progenitor cells, mobilized significantly less effectively following a second AMD3100 administration when compared with the first dose. Together, our results are expected to inform future mobilization protocols for the purposes of peripheral blood hematopoietic stem cell extraction or for applications in which hematopoietic stem cells must be made accessible for in vivo-delivered gene targeting agents.
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http://dx.doi.org/10.1016/j.exphem.2020.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8340682PMC
January 2021

Isolation of a Highly Purified HSC-enriched CD34CD90CD45RA Cell Subset for Allogeneic Transplantation in the Nonhuman Primate Large-animal Model.

Transplant Direct 2020 Aug 15;6(8):e579. Epub 2020 Jul 15.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side effects in nontarget cells. We have previously shown that purified CD34CD90CD45RA cells are enriched for multipotent HSCs with long-term multilineage engraftment potential, which can reconstitute the entire hematopoietic system in an autologous nonhuman primate transplant model. Here, we tested the feasibility of transplantation with purified CD34CD90CD45RA cells in the allogeneic setting in a nonhuman primate model.

Methods: To evaluate the feasibility of this approach, CD34CD90CD45RA cells from 2 fully major histocompatibility complex-matched, full sibling rhesus macaques were sort-purified, quality controlled, and transplanted. Engraftment and donor chimerism were evaluated in the peripheral blood and bone marrow of both animals.

Results: Despite limited survival due to infectious complications, we show that the large-scale sort-purification and transplantation of CD34CD90CD45RA cells is technically feasible and leads to rapid engraftment of cells in bone marrow in the allogeneic setting and absence of cotransferred T cells.

Conclusions: We show that purification of an HSC-enriched CD34 subset can serve as a potential stem cell source for allo-HCTs. Most importantly, the combination of allo-HCT and HSC gene therapy has the potential to treat a wide array of hematologic and nonhematologic disorders.
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http://dx.doi.org/10.1097/TXD.0000000000001029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581184PMC
August 2020

Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors.

Hum Gene Ther 2021 Jan 2;32(1-2):96-112. Epub 2020 Nov 2.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4 T cells , are being explored for delivery of anti-HIV therapeutic modalities . However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.
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http://dx.doi.org/10.1089/hum.2020.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020555PMC
January 2021

Factors associated with outcomes after a second CD19-targeted CAR T-cell infusion for refractory B-cell malignancies.

Blood 2021 01;137(3):323-335

Clinical Research Division and.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet, CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (acute lymphoblastic leukemia [ALL], n = 14; chronic lymphocytic leukemia [CLL], n = 9; non-Hodgkin lymphoma [NHL], n = 21) who received CART2 on a phase 1/2 trial (NCT01865617) at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 cytokine release syndrome, 9%; grade ≥3 neurotoxicity, 11%). After CART2, complete response (CR) was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before the first CAR T-cell infusion (CART1) and an increase in the CART2 dose compared with CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received cyclophosphamide and fludarabine (Cy-Flu) lymphodepletion before CART1 and a higher CART2 compared with CART1 cell dose. The identification of 2 modifiable pretreatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies.
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http://dx.doi.org/10.1182/blood.2020006770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819764PMC
January 2021

Purification of Human CD34CD90 HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy.

Mol Ther Methods Clin Dev 2020 Sep 15;18:679-691. Epub 2020 Jul 15.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34CD90 cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133 cells or CD38 subsets of CD34 cells from human blood products. We directly compared these strategies to the isolation of CD90 cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90 cell selection results in about 30-fold fewer target cells in comparison to CD133 or CD38 CD34 hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential . Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90 fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90 cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.
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http://dx.doi.org/10.1016/j.omtm.2020.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424231PMC
September 2020

CAR T-cell therapy for cancer and HIV through novel approaches to HIV-associated haematological malignancies.

Lancet Haematol 2020 Sep 10;7(9):e690-e696. Epub 2020 Aug 10.

Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, Division of Medical Oncology, University of Washington, Seattle, WA, USA. Electronic address:

People living with HIV are a global population with increased cancer risk but their access to modern immunotherapies for cancer treatment has been limited by socioeconomic factors and inadequate research to support safety and efficacy in this population. These immunotherapies include immune checkpoint inhibitors and advances in cellular immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy. Despite the field of cancer immunotherapy rapidly expanding with ongoing clinical trials, people with HIV are often excluded from such trials. In 2019, post-approval evaluation of anti-CD19 CAR T-cell therapy in people with HIV and aggressive B-cell lymphoma showed the feasibility of CAR T-cell therapy for cancer in this excluded group. Along with expanded treatment options for people with HIV is the ability to assess the effects of immunotherapy on the latent HIV reservoir, with certain immunotherapies showing the ability to alleviate this burden. This Series paper addresses the increased cancer burden in people with HIV, the increasing evidence for the safety and efficacy of immunotherapies in the context of HIV and cancer, and opportunities for novel applications of CAR-T therapy for the treatment of both haematological malignancies and HIV.
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http://dx.doi.org/10.1016/S2352-3026(20)30142-3DOI Listing
September 2020

Gene Therapy for Canine SCID-X1 Using Cocal-Pseudotyped Lentiviral Vector.

Hum Gene Ther 2021 Jan 23;32(1-2):113-127. Epub 2020 Sep 23.

Stem Cell and Gene Therapy Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Hematopoietic stem and progenitor cell (HSPC)-based gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3 T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.
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http://dx.doi.org/10.1089/hum.2020.127DOI Listing
January 2021

Call for Papers: Expanding the Scale and Scope of Therapeutic Gene Editing.

Mol Ther 2020 08 16;28(8):1743. Epub 2020 Jul 16.

Department of Pediatrics, Gene and Cell Therapy Program, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.

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http://dx.doi.org/10.1016/j.ymthe.2020.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403465PMC
August 2020

Robust expansion of HIV CAR T cells following antigen boosting in ART-suppressed nonhuman primates.

Blood 2020 10;136(15):1722-1734

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA.

Chimeric antigen receptor (CAR) T cells targeting CD19+ hematologic malignancies have rapidly emerged as a promising, novel therapy. In contrast, results from the few CAR T-cell studies for infectious diseases such as HIV-1 have been less convincing. These challenges are likely due to the low level of antigen present in antiretroviral therapy (ART)-suppressed patients in contrast to those with hematologic malignancies. Using our well-established nonhuman primate model of ART-suppressed HIV-1 infection, we tested strategies to overcome these limitations and challenges. We first optimized CAR T-cell production to maintain central memory subsets, consistent with current clinical paradigms. We hypothesized that additional exogenous antigen might be required in an ART-suppressed setting to aid expansion and persistence of CAR T cells. Thus, we studied 4 simian/HIV-infected, ART-suppressed rhesus macaques infused with virus-specific CD4CAR T cells, followed by supplemental infusion of cell-associated HIV-1 envelope (Env). Env boosting led to significant and unprecedented expansion of virus-specific CAR+ T cells in vivo; after ART treatment interruption, viral rebound was significantly delayed compared with controls (P = .014). In 2 animals with declining CAR T cells, rhesusized anti-programmed cell death protein 1 (PD-1) antibody was administered to reverse PD-1-dependent immune exhaustion. Immune checkpoint blockade triggered expansion of exhausted CAR T cells and concordantly lowered viral loads to undetectable levels. These results show that supplemental cell-associated antigen enables robust expansion of CAR T cells in an antigen-sparse environment. To our knowledge, this is the first study to show expansion of virus-specific CAR T cells in infected, suppressed hosts, and delay/control of viral recrudescence.
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http://dx.doi.org/10.1182/blood.2020006372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544543PMC
October 2020

CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside.

Br J Haematol 2021 01 7;192(1):33-49. Epub 2020 Jun 7.

Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Genome editing therapies represent a significant advancement in next-generation, precision medicine for the management of haematological diseases, and CRISPR/Cas9 has to date been the most successful implementation platform. From discovery in bacteria and archaea over three decades ago, through intensive basic research and pre-clinical development phases involving the modification of therapeutically relevant cell types, CRISPR/Cas9 genome editing is now being investigated in ongoing clinic trials. Despite the widespread enthusiasm brought by this new technology, significant challenges remain before genome editing can be routinely recommended and implemented in the clinic. These include risks of genotoxicity resulting from off-target DNA cleavage or chromosomal rearrangement, and suboptimal efficacy of homology-directed repair editing strategies, which thus limit therapeutic options. Practical hurdles such as high costs and inaccessibility to patients outside specialised centres must also be addressed. Future improvements in this rapidly developing field should circumvent current limitations with novel editing platforms and with the simplification of clinical protocols using in vivo delivery of editing reagents.
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http://dx.doi.org/10.1111/bjh.16807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118330PMC
January 2021

Assessment and streamlined preparation of low-cytotoxicity lentiviral vectors for mobilized human hematopoietic stem cell transduction.

Exp Hematol 2020 06 27;86:28-42.e3. Epub 2020 May 27.

Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH. Electronic address:

As important vectors for ectopic protein expression, gene silencing, and progenitor cell barcoding, lentiviruses continue to emerge as versatile research and clinical tools. For studies employing cell types that are relatively resistant to transduction, high-titer lentivirus preparations with low cytotoxicity are required. During lentivirus production, carryover plasmid DNA endotoxins, transfection reagents, damaged packaging cells, and virus concentration procedures are potential sources of cytotoxicity. As an often unevaluated property of lentivirus preparations, cytotoxicity can unwittingly skew estimates of functional titers and complicate interpretations of transduced cell phenotypes. By employing hematopoietic UT7epo cells cultured in erythropoietin (EPO) below maximal dosing, we first define a sensitive flow cytometric bioassay for critically assessing the cytotoxicity (and titers) of lentivirus preparations. Bioassay of custom preparations of research-grade lentiviruses from six commercial sources unexpectedly revealed substantial cytotoxicity (with certain preparations additionally registering titers several log below designated values). To overcome such limiting properties, we further report on unique, efficient workflows for reproducibly preparing and processing high-titer, low-cytotoxicity (HTLC) lentiviruses at research scale. These HTLC lentiviruses reliably transduce peripheral blood hematopoietic stem/progenitor cells (PB-HSPCs) at frequencies ≥40%, with low cytotoxicity. In addition, by employing cyclosporin H (to inhibit IFITM3), PB-HSPCs can be transduced at heightened efficiency with nominal cytotoxicity. Overall, this work provides straightforward approaches to (1) critical assessment of the cytotoxicity of lentivirus preparations; (2) reproducible generation (and concentration) of high-quality lentiviruses via a streamlined workflow; and (3) transduction of PB-HSPCs at benchmark levels with nominal cytotoxicity.
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http://dx.doi.org/10.1016/j.exphem.2020.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372909PMC
June 2020

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

Cell 2020 05 27;181(5):1016-1035.e19. Epub 2020 Apr 27.

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
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http://dx.doi.org/10.1016/j.cell.2020.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252096PMC
May 2020

DNA Barcoding in Nonhuman Primates Reveals Important Limitations in Retrovirus Integration Site Analysis.

Mol Ther Methods Clin Dev 2020 Jun 30;17:796-809. Epub 2020 Mar 30.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

tracking of retrovirus-tagged blood stem and progenitor cells is used to study hematopoiesis. Two techniques are used most frequently: sequencing the locus of retrovirus insertion, termed integration site analysis, or retrovirus DNA barcode sequencing. Of these, integration site analysis is currently the only available technique for monitoring clonal pools in patients treated with retrovirus-modified blood cells. A key question is how these two techniques compare in their ability to detect and quantify clonal contributions. In this study, we assessed both methods simultaneously in a clinically relevant nonhuman primate model of autologous, myeloablative transplantation. Our data demonstrate that both methods track abundant clones; however, DNA barcode sequencing is at least 5-fold more efficient than integration site analysis. Using computational simulation to identify the sources of low efficiency, we identify sampling depth as the major factor. We show that the sampling required for integration site analysis to achieve minimal coverage of the true clonal pool is likely prohibitive, especially in cases of low gene-modified cell engraftment. We also show that early subsampling of different blood cell lineages adds value to clone tracking information in terms of safety and hematopoietic biology. Our analysis demonstrates DNA barcode sequencing as a useful guide to maximize integration site analysis interpretation in gene therapy patients.
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http://dx.doi.org/10.1016/j.omtm.2020.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184234PMC
June 2020

The evolution of viral integration site analysis.

Blood 2020 04;135(15):1192-1193

Fred Hutchinson Cancer Research Center.

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http://dx.doi.org/10.1182/blood.2020005115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146016PMC
April 2020

HLA-Haploidentical Hematopoietic Cell Transplantation for Treatment of Nonmalignant Diseases Using Nonmyeloablative Conditioning and Post-Transplant Cyclophosphamide.

Biol Blood Marrow Transplant 2020 07 28;26(7):1332-1341. Epub 2020 Mar 28.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington; Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington; Division of Hematology and Oncology, Seattle Children's Hospital, Seattle, Washington. Electronic address:

Allogeneic hematopoietic cell transplant (HCT) is often the only curative therapy for patients with nonmalignant diseases; however, many patients do not have an HLA-matched donor. Historically, poor survival has been seen after HLA-haploidentical HCT because of poor immune reconstitution, increased infections, graft-versus-host disease (GVHD), and graft failure. Encouraging results have been reported using a nonmyeloablative T cell-replete HLA-haploidentical transplant approach in patients with hematologic malignancies. Here we report the outcomes of 23 patients with various nonmalignant diseases using a similar approach. Patients received HLA-haploidentical bone marrow (n = 17) or granulocyte colony-stimulating factor-mobilized peripheral blood stem cell (n = 6) grafts after conditioning with cyclophosphamide 50 mg/kg, fludarabine 150 mg/m, and 2 or 4 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclophosphamide, mycophenolate mofetil, tacrolimus, ± sirolimus. Median patient age at HCT was 10.8 years. Day 100 transplant-related mortality (TRM) was 0%. Two patients died at later time points, 1 from intracranial hemorrhage/disseminated fungal infection in the setting of graft failure and 1 from infection/GVHD. The estimated probabilities of grades II to IV and III to IV acute GVHD at day 100 and 2-year National Institutes of Health consensus chronic GVHD were 78%, 26%, and 42%, respectively. With a median follow-up of 2.5 years, the 2-year overall and event-free rates of survival were 91% and 78%, respectively. These results are encouraging and demonstrate favorable disease-specific lineage engraftment with low TRM in patients with nonmalignant diseases using nonmyeloablative conditioning followed by T cell-replete HLA-haploidentical grafts. However, additional strategies are needed for GVHD prevention to make this a viable treatment approach for patients with nonmalignant diseases.
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http://dx.doi.org/10.1016/j.bbmt.2020.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306430PMC
July 2020

Effective Multi-lineage Engraftment in a Mouse Model of Fanconi Anemia Using Non-genotoxic Antibody-Based Conditioning.

Mol Ther Methods Clin Dev 2020 Jun 8;17:455-464. Epub 2020 Feb 8.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow, facilitating donor cell engraftment. Although effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk for secondary malignancies. Antibody conjugates targeting hematopoietic cells are an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity. Specifically, this platform would be useful in diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita, Schwachman-Diamond syndrome, and Fanconi anemia (FA). Our approach utilizes antibody-drug conjugates (ADC) as an alternative conditioning regimen in an FA mouse model of autologous transplantation. Antibodies targeting either CD45 or CD117 were conjugated to saporin (SAP), a ribosomal toxin. knockout mice were conditioned with either CD45-SAP or CD117-SAP prior to receiving whole marrow from a heterozygous healthy donor. Bone marrow and peripheral blood analysis revealed equivalent levels of donor engraftment, with minimal toxicity in ADC-treated groups as compared with cyclophosphamide-treated controls. Our findings suggest ADCs may be an effective conditioning strategy in stem cell transplantation not only for diseases where traditional chemotherapy is not tolerated, but also more broadly for the field of blood and marrow transplantation.
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http://dx.doi.org/10.1016/j.omtm.2020.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096734PMC
June 2020

Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy.

Nat Commun 2020 01 10;11(1):219. Epub 2020 Jan 10.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, 98109, USA.

Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. However, clonal kinetics and transcriptional programs that regulate the fate of CAR-T cells after infusion remain poorly understood. Here we perform TCRB sequencing, integration site analysis, and single-cell RNA sequencing (scRNA-seq) to profile CD8 CAR-T cells from infusion products (IPs) and blood of patients undergoing CD19 CAR-T immunotherapy. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool after infusion. Although integration site does not appear to be a key driver of clonal kinetics, scRNA-seq demonstrates that clones that expand after infusion mainly originate from infused clusters with higher expression of cytotoxicity and proliferation genes. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion.
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http://dx.doi.org/10.1038/s41467-019-13880-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954177PMC
January 2020

In-Vivo Gene Therapy with Foamy Virus Vectors.

Viruses 2019 11 23;11(12). Epub 2019 Nov 23.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Foamy viruses (FVs) are nonpathogenic retroviruses that infect various animals including bovines, felines, nonhuman primates (NHPs), and can be transmitted to humans through zoonotic infection. Due to their non-pathogenic nature, broad tissue tropism and relatively safe integration profile, FVs have been engineered as novel vectors (foamy virus vector, FVV) for stable gene transfer into different cells and tissues. FVVs have emerged as an alternative platform to contemporary viral vectors (e.g., adeno associated and lentiviral vectors) for experimental and therapeutic gene therapy of a variety of monogenetic diseases. Some of the important features of FVVs include the ability to efficiently transduce hematopoietic stem and progenitor cells (HSPCs) from humans, NHPs, canines and rodents. We have successfully used FVV for proof of concept studies to demonstrate safety and efficacy following in-vivo delivery in large animal models. In this review, we will comprehensively discuss FVV based in-vivo gene therapy approaches established in the X-linked severe combined immunodeficiency (SCID-X1) canine model.
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http://dx.doi.org/10.3390/v11121091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950547PMC
November 2019

Mouse models in hematopoietic stem cell gene therapy and genome editing.

Biochem Pharmacol 2020 04 6;174:113692. Epub 2019 Nov 6.

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA.

Gene therapy has become an important treatment option for a variety of hematological diseases. The biggest advances have been made with CAR T cells and many of those studies are now FDA approved as a routine treatment for some hematologic malignancies. Hematopoietic stem cell (HSC) gene therapy is not far behind with treatment approvals granted for beta-hemoglobinopathies and adenosine deaminase severe combined immune deficiency (ADA-SCID), and additional approbations currently being sought. With the current pace of research, the significant investment of biotech companies, and the continuously growing toolbox of viral as well as non-viral gene delivery methods, the development of new ex vivo and in vivo gene therapy approaches is at an all-time high. Research in the field of gene therapy has been ongoing for more than 4 decades with big success stories as well as devastating drawbacks along the way. In particular, the damaging effect of uncontrolled viral vector integration observed in the initial gene therapy applications in the 90s led to a more comprehensive upfront safety assessment of treatment strategies. Since the late 90s, an important read-out to comprehensively assess the quality and safety of cell products has come forward with the mouse xenograft model. Here, we review the use of mouse models across the different stages of basic, pre-clinical and translational research towards the clinical application of HSC-mediated gene therapy and editing approaches.
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http://dx.doi.org/10.1016/j.bcp.2019.113692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050335PMC
April 2020

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Blood 2019 10;134(16):1298-1311

The Scripps Research Institute, La Jolla, CA.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.
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http://dx.doi.org/10.1182/blood.2019000040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888140PMC
October 2019

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Sci Transl Med 2019 07;11(503)

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34CD90CD45RA, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.
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http://dx.doi.org/10.1126/scitranslmed.aaw3768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8407476PMC
July 2019
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