From Bone Marrow to Mobilized Peripheral Blood Stem Cells: The Circuitous Path to Clinical Gene Therapy for Fanconi Anemia

Abstract For decades, it has remained challenging to achieve long-term engraftment and correction of blood counts using gene-modified hematopoietic stem cells for Fanconi anemia. Toward this goal, our group conducted preclinical studies using a safety modified lentiviral vector encoding full-length cDNA for FANCA in normal and affected patient hematopoietic progenitor cells, and in a mutant mouse model that supported the IND for a gene therapy clinical trial for Fanconi anemia, complementation group A (NCT01331018). These studies led us to incorporate methods such as addition of N-acetylcysteine and hypoxic incubation during transduction. Because of the low stem cell numbers of Fanconi patients and initial difficulty with using plerixafor off-label for mobilization, we began our study with bone marrow as the source of stem cells. Due to concerns regarding secondary cancers, no conditioning was administered prior to infusion of gene-modified cells. The US Food and Drug Administration approved adult patients initially, but later permitted pediatric patient enrollment with a minimum age of 4 years. The primary objective of our phase I trial was safety. Secondary objectives included in vitro correction of mitomycin C (MMC) sensitivity, procurement of sufficient cell numbers, and ultimately, long-term correction of blood counts in recipients. Eligibility included absolute neutrophil count ≥0.5, hemoglobin ≥8, platelet count ≥20,000, lack of matched family donor, adequate organ function, and not meeting criteria for diagnosis of MDS. Our three enrolled patients were ages 22, 10, and 5 years. All demonstrated defects in the FANCA gene, with two patients sequenced and one patient diagnosed by complementation. Due to in-process learning and the later addition of plerixafor mobilization to the protocol, three different laboratory procedures were used to prepare the gene-modified product for each patient. Cell products were CD34+ selected bone marrow, bone marrow mononuclear cells depleted of red cells by hetastarch, and G-CSF and plerixafor mobilized cells depleted of red blood cells and cells bearing lineage markers, respectively. Transduction efficiencies were 17.7, 42.7 and 26.3% of colony forming cells (CFC) in 0 nM MMC, and 80, 100, and 100% of CFC in 10 nM MMC. Growth of hematopoietic colonies in MMC indicated functional correction of the FANCA defect. The 1st patient received 6.1×10e4, the 2nd 2.9×10e5, and the 3rd 4.3×10e6 CD34+ cells/kg. Serious adverse events included cytopenias in all patients, and hospital admission for fever due to viral upper respiratory infection in one patient. The patients remain alive at 46, 38, and 12 months after receipt of gene-modified cells. Due to worsening cytopenias, the third patient underwent hematopoietic cell transplant from an unrelated donor 10 months after infusion of gene-modified cells. To date, he has done well with transplant, and no indication that prior gene therapy impacted the outcome. The blood counts for the first 2 patients who have not undergone allogeneic transplant remain stable at 1,111 and 1,077 days post infusion compared to the first blood counts when they arrived at our center. For the 1st patient, vector was detectable in white blood cells (WBC) up to 21 days, in the 2nd up to 582 days, and the 3rd up to 81 days post infusion. Thus, in these patients, despite dramatic improvement in cell dose during the study, there was lack of persistence in detection of gene-modified WBCs beyond 1.5 years. A number of factors may have contributed, including lack of conditioning, in vitro cell manipulation including cytokine exposure, inability to transduce primitive hematopoietic stem cells, and paucity of long-term repopulating cells at the ages of the patients, suggesting earlier collection may be beneficial. This study is now closed to enrollment. Valuable information gained as a result of this study will contribute to future clinical gene therapy trials. Current work focuses on how to evaluate stem cell fitness prior to attempting gene therapy, minimizing manipulation required for gene correction and/or in vivo genetic correction and non-chemotherapy-based conditioning to facilitate engraftment. We would like to personally thank each patient and their families for participating in this study, as we could not have learned these lessons without their support. Disclosures Becker: GlycoMimetics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Trovagene: Research Funding; Rocket Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding. Adair:Miltenyi Biotec: Honoraria; RX Partners: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Kiem:Rocket Pharmaceuticals: Consultancy; Homology Medicine: Consultancy; Magenta: Consultancy.

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Gene Therapy for Tolerance to Factor VIII Induced with B Cells and Bone Marrow Transduced with FVIII Domains.

Blood ◽

10.1182/blood.v106.11.3051.3051 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3051-3051

Author(s):

David W. Scott ◽

Tie Chi Lei

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

B Cells ◽

Factor Viii ◽

Tolerance Induction ◽

Class Ii ◽

Hematopoietic Stem

Abstract Tolerance induction for hemophilia inhibitor formation has recently been achieved by gene therapy using B cells transduced with a fusion protein of C2 and A2 domains of factor VIII with an IgG heavy chain. We have shown that inhibitor titers can be reduced over 90% by this approach in both naïve and highly immunized FVIII knockout mice (FVIII−/−). This process requires the presence of CD25+ regulatory cells for induction, and is stable for at least three months (Lei, T-C. and Scott, D.W. 2005. Induction of tolerance to fVIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B-cells as Ig fusion proteins. Blood, 105: 4865–4870). Herein, we wished to establish whether retroviral transduction and integration of the fusion Ig was necessary for tolerance. In addition, we also tested whether transduction of bone marrow could lead to long-term tolerance. B cells (resting and LPS blasts) from FVIII sufficient C57Bl/6 and FVIII−/− mice were pulsed with FVIII and injected into naïve FVIII−/− recipients that were subsequently challenged with therapeutic doses of FVIII in an immunogenic protocol. We reasoned that C57Bl/6 mice were FVIII sufficient and could display FVIII processed epitopes within their MHC class II. Moreover, pulsing with FVIII should lead to presentation of processed epitopes transiently in class II MHC. We found that specific tolerance to FVIII was only induced with B cells transduced with a combination of A2-IgG and C2-IgG, indicating that transient expression of FVIII epitopes and presentation by B cells is not sufficient for tolerance induction. Resting B-cells pulsed with fVIII in vitro are not tolerogenic Resting B-cells pulsed with fVIII in vitro are not tolerogenic In further studies, we examined whether tolerance could be induced by bone marrow transplantation of C2-Ig and A2-Ig transduced hematopoietic stem cells under a non-myeloablative regimen with busulfan in immunized FVIII−/− mice. This protocol could lead to long-term tolerance if pluripotential stem cells were transduced and engrafted. Our results indicate that tolerance to FVIII domains could be achieved even though the level of expression of GFP positive cells in peripheral blood of busulfan-treated mice was less than 1% (compared to >50% positive cells in lethally irradiated recipients of similarly transduced bone marrow cells). These results have implications for long-term therapy of inhibitor formation in hemophilia A. (Supported by NIH grants HL061883, AI035622 and a Lab Grant from the National Hemophilia Foundation.)

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Long-term lymphoid progenitors independently sustain naïve T and NK cell production in humans

Nature Communications ◽

10.1038/s41467-021-21834-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Natalia Izotova ◽

Christine Rivat ◽

Cristina Baricordi ◽

Elena Blanco ◽

Danilo Pellin ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Nk Cell ◽

Integration Site ◽

Hematopoietic Stem ◽

Cell Production ◽

Lymphoid Progenitors ◽

Clinical Gene Therapy ◽

Cancer Immunotherapies

AbstractOur mathematical model of integration site data in clinical gene therapy supported the existence of long-term lymphoid progenitors capable of surviving independently from hematopoietic stem cells. To date, no experimental setting has been available to validate this prediction. We here report evidence of a population of lymphoid progenitors capable of independently maintaining T and NK cell production for 15 years in humans. The gene therapy patients of this study lack vector-positive myeloid/B cells indicating absence of engineered stem cells but retain gene marking in both T and NK. Decades after treatment, we can still detect and analyse transduced naïve T cells whose production is likely maintained by a population of long-term lymphoid progenitors. By tracking insertional clonal markers overtime, we suggest that these progenitors can support both T and NK cell production. Identification of these long-term lymphoid progenitors could be utilised for the development of next generation gene- and cancer-immunotherapies.

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

Blood ◽

10.1182/blood.v112.11.2358.2358 ◽

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 (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >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.

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Hybrid AAV6/2-Mediated Human β-Globin Expression in β-Thalassemia Patient Hematopoietic Stem Cells Transplanted Into Irradiated BALB/c Nude Mouse

Blood ◽

10.1182/blood.v118.21.4715.4715 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4715-4715

Author(s):

Mengqun Tan ◽

Zhenqing Liu ◽

Juan Zhang ◽

Zhiyan Li ◽

Liujiang Song ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Globin Gene ◽

Blood Transfusions ◽

Rt Pcr ◽

Thalassemia Patient ◽

Hematopoietic Stem ◽

Human Hscs

Abstract Abstract 4715 β -Thalassemia is one of the most common worldwide monogenic human diseases,caused by molecular defects in the human β -globin gene cluster leading to decrease or absence of β-globin. Loss of β -globin chains causes ineffective production of oxygen-carrying hemoglobin and therefore results in severe anemia. The treatment for β -Thalassemia major usually includes lifelong blood transfusions but chronic blood transfusion often causes iron overload, and accumulated iron produces tissue damage in multiple organs, so that iron chelating treatment is also needed. Bone marrow transplantation is another effective therapy, which can eliminate a patient's dependence on blood transfusions, however, it is difficult to find a matching donor for most patients; therefore it is only available for a minority of patients. Gene therapy is one potential novel therapy for treatment of inherited monogenic disorders. The long–term therapeutic strategy for this disease is to replace the defective β-globin gene via introduction of a functional gene into hematopoietic stem cells (HSCs). Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. AAV can infect both dividing and non-dividing cells and wild AAV integrates preferentially at a specific site on human chromosome 19. In the absence of helper virus, recombinant AAV will stably integrate into the host cell genome, mediating long-term and stable expression of the transgene. In this study, we used a hybrid rAAV6/2 vector carrying the human β-globin gene to transduce HSCs from a β -Thalassemia patient, followed by transplantation into irradiated BALB/c nude mice. One month post-transplantation, Hb was prepared from peripheral blood and analyzed by Western Blot and HPLC respectively. RNA and DNA were isolated from bone marrow cells (BMCs) from recipient mice transplanted with mock-infected or hybrid rAAV–globin-infected cells and analyzed by RT-PCR and PCR respectively. The results showed: 1. Human β-actin and β-globin transcripts were detected by RT-PCR in BMCs from all recipient mice, indicating that human HSCs were successfully transplanted in these mice and that the human β-globin gene was transcriptionally active in the donor cells. 2. The level of human hemoglobin expressed in peripheral red blood cells of recipient mice as measured by HPLC (ratio of β/α) was increased to 0.3 from 0.05 of pre-transplantation levels. Expression of human β-globin was also confirmed in recipient mice by Western Blot; a 2–3-fold increase compared with that of controls. Our results indicate that human HSCs from a β-Thalassemia patient can be efficiently transduced by a hybrid rAAV6/2-β-globin vector followed by expression of normal human β-globin protein. This study provides a proof-of-concept that rAAV6/2-mediated gene transfer into human HSCs might be a potential approach for gene therapy of β-Thalassemia. Disclosures: No relevant conflicts of interest to declare.

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Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

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Presence of mixed colony-forming cells in long-term hamster bone marrow suspension cultures: response to pokeweed spleen conditioned medium

Blood ◽

10.1182/blood.v60.2.495.bloodjournal602495 ◽

1982 ◽

Vol 60 (2) ◽

pp. 495-502

Author(s):

CE Eastment ◽

FW Ruscetti ◽

E Denholm ◽

I Katznelson ◽

E Arnold ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conditioned Medium ◽

In Vitro Study ◽

Suspension Cultures ◽

Rapid Expansion ◽

Pokeweed Mitogen ◽

Hematopoietic Stem

In contrast to the murine system, long-term hamster bone marrow suspension cultures maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen-stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies assayed in soft-agar, These mixed colonies, which contained four cell lineages--granulocytic, erythroid, megakaryocytic, and macrophage--could be generated from cells grown in suspension for over 6 mo. Addition of SCM also induces an initial rapid expansion of the myeloid compartment, and this expansion results in 70% of the cells being terminally differentiated granulocytes. In contrast, addition of SCM to hamster bone marrow cultures containing both adherent cells and hematopoietic stem cells produced no change in the number of mixed colonies generated in the culture. This system allows the in vitro study of the process of stem cell proliferation and differentiation and also provides a means to examine the relationship of adherent and supernatant bone marrow populations.

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An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Blood ◽

10.1182/blood.v74.8.2755.bloodjournal7482755 ◽

1989 ◽

Vol 74 (8) ◽

pp. 2755-2763 ◽

Cited By ~ 2

Author(s):

RE Ploemacher ◽

JP van der Sluijs ◽

JS Voerman ◽

NH Brons

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem ◽

Dilution Assay ◽

Limiting Dilution Assay ◽

Colony Forming Units ◽

Limiting Dilution

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.

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Stable Polyclonal Murine Long-Term Hematopoiesis without Clonal Exhaustion of MGMT P140K Expressing Murine Hematopoietic Stem Cells after Extended Reduced Intensity Selection.

Blood ◽

10.1182/blood.v108.11.3271.3271 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3271-3271

Author(s):

Claudia R. Ball ◽

Manfred Schmidt ◽

Ingo H. Pilz ◽

Fessler Sylvia ◽

David A. Williams ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Hematopoietic Stem ◽

Reduced Intensity ◽

Selection Of

Abstract Gene therapy is a promising approach for the therapy of hereditary diseases, but after the occurrence of adverse side effects in a SCID-X1 gene therapy trial increased biological safety has become a major goal of gene therapy. A reduction of the number of transplanted cells could help achieve this goal by reducing the statistical likelihood of insertional mutagenesis simply by simply reducing the number of transplanted cells carrying potentially untoward insertion sites. As we have previously shown, incorporation of the selectable marker gene MGMT P140K into a retroviral vector allows a reduced intensity and toxicity in vivo selection of low numbers of genetically modified hematopoietic cells by chemotherapy with O6-benzylguanine (O6BG) and nitrosourea drugs such as 1,3-bis-2 chloroethyl-1-nitrosourea (BCNU). However, it is still not known whether extended selection over longer periods of time influences the long-term proliferation and differentiation capacity of murine haematopoietic stem cells. To address this question, serial transplantations of murine MGMT-P140K-expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were performed. After ex vivo gene transfer of a MGMT/IRES/eGFP-encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and serially transplanted. First, 2nd and 3rd generation recipient mice were subsequently treated every four weeks in order to amplify treatment effects on the long-term clonal behaviour of modified hematopoietic stem cells. Lineage contribution of transduced hematopoiesis was monitored by FACS over a total of 17 rounds of selection and clonality was monitored by LAM-PCR over a total of 16 rounds of selection. In primary mice, the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without inducing persistent peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential in the transduced compared to control cells in 1st and 2nd generation animals. LAM PCR analysis of peripheral blood revealed stable oligo- to polyclonal hematopoiesis in 1st, 2nd and 3rd generation mice. Evidence of predominant clones or clonal exhaustion was not observed despite of up to 16 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice which had received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis. This is molecular proof that extensive self-renewal of transplantable stem cells had occurred in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K-expressing hematopoietic stem cells by repetitive chemotherapy does not affect differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies intending to employ amplification of a limited number of genetically modified clones in clinical gene therapy.

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Gene Therapy for Fanconi Anemia, Complementation Group a: Updated Results from Ongoing Global Clinical Studies of RP-L102

Blood ◽

10.1182/blood-2020-137106 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-14

Author(s):

Agnieszka Czechowicz ◽

Rajni Agarwal ◽

Julián Sevilla ◽

Paula Río ◽

Susana Navarro ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Fanconi Anemia ◽

Research Funding ◽

Phase 1 ◽

Phase 2 ◽

Medicinal Products ◽

Publicly Traded ◽

Hematopoietic Stem

Background: Fanconi anemia (FA) is a rare inherited disorder of defective cellular deoxyribonucleic acid (DNA) repair, associated with developmental abnormalities and characterized by progressive bone marrow failure (BMF) and a predisposition to hematologic malignancies and solid tumors. Approximately 60-70% of all cases result from mutations in the Fanconi Anemia Complementation Group A (FANCA) gene (FA-A). 80% of FA patients develop BMF within the first decade of life. Although allogeneic hematopoietic stem cell transplant (allo-HSCT) is a potentially curative treatment for BMF, its utilization and efficacy are limited by availability of suitable human leukocyte antigen (HLA)-matched donors, risk of graft-versus-host disease (GVHD) and transplant-related toxicities. Ex-vivo lentiviral mediated gene therapy of autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) has been shown to confer a survival advantage to gene-modified HSPCs in preclinical studies and, most recently, in the investigator initiated Phase 1/2 FANCOLEN-I clinical trial conducted in Madrid, Spain. Based on the highly favorable safety profile and promising preliminary efficacy data, global studies using "Process B" optimization including transduction enhancers, commercial-grade vector, and modified cell processing are underway. Herein, we report updated results from the US Phase 1 clinical trial and preliminary data from the global Phase 2 study in US and EU. Design and Methods: Subjects with a confirmed FANCA gene mutation aged 1 year or older, with no HLA-matched sibling donor and at least 30 CD34+ cells/µL in bone marrow (BM) were eligible for enrollment. Peripheral blood (PB) mononuclear cells were collected via leucocytapheresis on two consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs were enriched, transduced with a lentiviral vector (PGK-FANCA-WPRE) and infused fresh (not cryopreserved) without any antecedent conditioning. Patients are being followed for 3 years post-infusion for safety assessments (replication competent lentivirus (RCL), insertion site analysis (ISA)) and to ascertain evidence of efficacy (increasing PB vector copy number (VCN) and BM mitomycin-C (MMC) resistance), along with stabilization/correction of cytopenias. Results: As of August 2020, 2 subjects (aged 5 and 6 years) have received RP-L102 infusion on the Phase 1 study with over 12 months of follow up. Preliminary evidence of gene marking in PB post-RP-L102 infusion at various timepoints has been observed in both subjects. Increased bone marrow (BM) mitomycin-C (MMC) resistance post treatment has also been identified in at least 1 subject. Subject L102-001-1001 has had blood count stabilization over the 12 months following gene therapy administration. Subject L102-001-1002's course has been complicated by influenza B infection with concomitant decreases in blood counts requiring red blood cell transfusions. Transfusion requirements have decreased following resolution of infection. Since November 2019, 5 additional subjects have been enrolled onto the global Phase 2 study and received investigational infusion. Updated preliminary safety and efficacy data including PB VCN, blood counts and BM MMC resistance will be available at the time of presentation for subjects with over 12 months of follow up; drug product (DP) information (VCN and CD34+ cell dose) will be available for all treated subjects. Conclusions: DP has been successfully manufactured in the Phase I (N=2) and Phase 2 (N=5) to meet the required specificationsSafety profile of RP-L102 continues to be highly favorable.Evidence of engraftment has been seen in at least 1 subject with follow up of at least 12 months as indicated by PB genetic markings and increasing BM CFC MMC resistance; 12+ months of follow-up may be required to observe the proliferative advantage of transduced HSPCs. Disclosures Czechowicz: Rocket Pharmaceuticals, Inc.: Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Río:Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: PR has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Wagner:BlueRock: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding.

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Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer

Anemia ◽

10.1155/2010/947816 ◽

2010 ◽

Vol 2010 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Ouassila Habi ◽

Johanne Girard ◽

Valérie Bourdages ◽

Marie-Chantal Delisle ◽

Madeleine Carreau

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Ex Vivo ◽

Direct Injection ◽

Negative Effects ◽

Hematopoietic Stem

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs ofFancC−/−mice. Using this approach, we show thatFancC−/−HSCs were efficiently corrected. Intrafemoral gene transfer of theFancCgene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

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