Transduction Of Hematopoietic Stem Cells Research Articles

Introducing a G-Makassar variant in HSCs by in vivo base editing treats sickle cell disease in mice

Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrated correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We demonstrated successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In HSC-enriched cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements of derived red blood cells. After ex vivo transduction of HSCs from a SCD mouse model and subsequent transplantation, we achieved an average of 88% editing at the target site in transplanted mice. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without showing noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. This in vivo approach requires only one non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. This technically simple approach has the potential for scalable applications in resource-limiting regions where SCD is prevalent.

Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.

Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

Advancements in Hematopoietic Stem Cell Gene Therapy: A Journey of Progress for Viral Transduction.

Hematopoietic stem cell (HSC) transduction has undergone remarkable advancements in recent years, revolutionizing the landscape of gene therapy specifically for inherited hematologic disorders. The evolution of viral vector-based transduction technologies, including retroviral and lentiviral vectors, has significantly enhanced the efficiency and specificity of gene delivery to HSCs. Additionally, the emergence of small molecules acting as transduction enhancers has addressed critical barriers in HSC transduction, unlocking new possibilities for therapeutic intervention. Furthermore, the advent of gene editing technologies, notably CRISPR-Cas9, has empowered precise genome modification in HSCs, paving the way for targeted gene correction. These striking progresses have led to the clinical approval of medicinal products based on engineered HSCs with impressive therapeutic benefits for patients. This review provides a comprehensive overview of the collective progress in HSC transduction via viral vectors for gene therapy with a specific focus on transduction enhancers, highlighting the latest key developments, challenges, and future directions towards personalized and curative treatments.

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard exvivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

Expression of a large coding sequence: Gene therapy vectors for Ataxia Telangiectasia

Ataxia telangiectasia is a monogenetic disorder caused by mutations in the ATM gene. Its encoded protein kinase ATM plays a fundamental role in DNA repair of double strand breaks (DSBs). Impaired function of this kinase leads to a multisystemic disorder including immunodeficiency, progressive cerebellar degeneration, radiation sensitivity, dilated blood vessels, premature aging and a predisposition to cancer. Since allogenic hematopoietic stem cell (HSC) transplantation improved disease outcome, gene therapy based on autologous HSCs is an alternative promising concept. However, due to the large cDNA of ATM (9.2 kb), efficient packaging of retroviral particles and sufficient transduction of HSCs remains challenging.We generated lentiviral, gammaretroviral and foamy viral vectors with a GFP.F2A.Atm fusion or a GFP transgene and systematically compared transduction efficiencies. Vector titers dropped with increasing transgene size, but despite their described limited packaging capacity, we were able to produce lentiviral and gammaretroviral particles. The reduction in titers could not be explained by impaired packaging of the viral genomes, but the main differences occurred after transduction. Finally, after transduction of Atm-deficient (ATM-KO) murine fibroblasts with the lentiviral vector expressing Atm, we could show the expression of ATM protein which phosphorylated its downstream substrates (pKap1 and p-p53).

HDAd6/35++ - A new helper-dependent adenovirus vector platform for in vivo transduction of hematopoietic stem cells

In previous studies, we achieved safe and efficient invivo hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34+ cells invitro. Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term invivo HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient invivo HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the invivo approach.

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV

We aim to develop an invivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. Invivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100μg/mL (mice) and >20μg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1μg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the invivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system with no cure yet. Here, we report genetic engineering of hematopoietic stem cells (HSCs) to express myelin oligodendrocyte glycoprotein (MOG), specifically in platelets, as a means of intervention to induce immune tolerance in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. The platelet-specific αIIb promoter was used to drive either a full-length or truncated MOG expression cassette. Platelet-MOG expression was introduced by lentivirus transduction of HSCs followed by transplantation. MOG protein was detected on the cell surface of platelets only in full-length MOG-transduced recipients, but MOG was detected in transmembrane-domain-less MOG1-157-transduced platelets intracellularly. We found that targeting MOG expression to platelets could prevent EAE development and attenuate disease severity, including the loss of bladder control in transduced recipients. Elimination of the transmembrane domains of MOG significantly enhanced the clinical efficacy in preventing the onset and development of the disease and induced CD4+Foxp3+ Treg cells in the EAE model. Together, our data demonstrated that targeting transmembrane domain-deleted MOG expression to platelets is an effective strategy to induce immune tolerance in EAE, which could be a promising approach for the treatment of patients with MS autoimmune disease.

Lentiviral Transduction of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

The nonhuman primate (NHP) animal model is an important predictive preclinical model for developing gene and cell therapies. It is also an experimental animal model used to study hematopoietic stem and progenitor cell (HSPC) biology, with the capability of serving as a step for the translation of the basic research concepts from small animals to humans. Lentiviral vectors are currently the standard gene delivery vehicles for transduction of HSPCs in the clinical setting. They have proven to be less genotoxic and more efficient than the previously used murine γ-retroviruses. Transplantation of lentiviral vector-transduced HSPCs into autologous macaques has been well developed over the past two decades. In this chapter, we provide detailed methodologies for lentiviral vector transduction of rhesus macaque HSPCs, including production and titration of lentiviral vector, purification of CD34+ HSPCs, and lentiviral vector transduction and assessment.

Intraosseous delivery of platelet-targeted factor VIII lentiviral vector in humanized NBSGW mice

AbstractWe previously showed that intraosseous (IO) delivery of factor VIII (FVIII, gene F8) lentiviral vector (LV) driven by the megakaryocyte-specific promoter Gp1bα (G-F8-LV) partially corrected the bleeding phenotype in hemophilia A (HemA) mice for up to 5 months. In this study, we further characterized and confirmed the successful transduction of self-regenerating hematopoietic stem and progenitor cells (HSPCs) in treated mice. In addition, secondary transplant of HSPCs isolated from G-F8-LV–treated mice corrected the bleeding phenotype of the recipient HemA mice, indicating the potential of long-term transgene expression following IO-LV therapy. To facilitate the translation of this technology to human applications, we evaluated the safety and efficacy of this gene transfer therapy into human HSPCs. In vitro transduction of human HSPCs by the platelet-targeted G-F8-LV confirmed megakaryocyte-specific gene expression after preferential differentiation of HSPCs to megakaryocyte lineages. Lentiviral integration analysis detected a polyclonal integration pattern in G-F8-LV–transduced human cells, profiling the clinical safety of hemophilia treatment. Most importantly, IO delivery of G-F8-LV to humanized NBSGW mice produced persistent FVIII expression in human platelets after gene therapy, and the megakaryocytes differentiated from human CD34+ HSPCs isolated from LV-treated humanized mice showed up to 10.2% FVIII expression, indicating efficient transduction of self-regenerating human HSPCs. Collectively, these results indicate the long-term safety and efficacy of the IO-LV gene therapy strategy for HemA in a humanized model, adding further evidence to the feasibility of translating this method for clinical applications.

In vivo HSC transduction in rhesus macaques with an HDAd5/3+ vector targeting desmoglein 2 and transiently overexpressing cxcr4

AbstractWe developed a new in vivo hematopoietic stem cell (HSC) gene therapy approach that involves only IV injections and does not require myeloablation/conditioning and HSC transplantation. In this approach, HSCs are mobilized from the bone marrow into the peripheral bloodstream and transduced with IV injected helper-dependent adenovirus (HDAd) vectors. A fraction of transduced HSCs returns to the bone marrow and persists there long term. Here, we report desmoglein 2 (DSG2) as a new receptor that can be used for in vivo HSC transduction. HDAd5/3+ vectors were developed that use DSG2 as a high-affinity attachment receptor, and in vivo HSC transduction and safety after IV injection of an HDAd5/3+ vector expressing green fluorescent protein (GFP) in granulocyte colony-stimulating factor/AMD3100 (plerixafor)-mobilized rhesus macaques were studied. Unlike previously used CD46-targeting HDAd5/35++ vectors, HDAd5/3+ virions were not sequestered by rhesus erythrocytes and therefore mediated ∼10-fold higher GFP marking rates in primitive HSCs (CD34+/CD45RA–/CD90+ cells) in the bone marrow at day 7 after vector injection. To further increase the return of in vivo transduced, mobilized HSCs to the bone marrow, we transiently expressed cxcr4 in mobilized HSCs from the HDAd5/3+ vector. In vivo transduction with an HDAd5/3+GFP/cxcr4 vector at a low dose of 0.4 × 1012 viral particles/kg resulted in up to 7% of GFP-positive CD34+/CD45RA–/CD90+ cells in the bone marrow. This transduction rate is a solid basis for in vivo base or prime editing in combination with natural or drug-induced expansion of edited HSCs. Furthermore, our study provides new insights into HSC biology and trafficking after mobilization in nonhuman primates.

In Vivo Hematopoietic Stem Cell Gene Therapy for SARS-CoV2 Infection Using a Decoy Receptor.

While SARS-CoV2 vaccines have shown an unprecedented success, the ongoing emergence of new variants and necessity to adjust vaccines justify the development of alternative prophylaxis and therapy approaches. Hematopoietic stem cell (HSC) gene therapy using a secreted CoV2 decoy receptor protein (sACE2-Ig) would involve a one-time intervention resulting in long-term protection against airway infection, viremia, and extrapulmonary symptoms. We recently developed a technically simple and portable in vivo hematopoietic HSC transduction approach that involves HSC mobilization from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Considering the abundance of erythrocytes, in this study, we directed sACE2-Ig expression to erythroid cells using strong β-globin transcriptional regulatory elements. We performed in vivo HSC transduction of CD46-transgenic mice with an HDAd-sACE2-Ig vector. Serum sACE2-Ig levels reached 500–1,300 ng/mL after in vivo selection. At 22 weeks, we used genetically modified HSCs from these mice to substitute the hematopoietic system in human ACE2-transgenic mice, thus creating a model that is susceptible to SARS-CoV2 infection. Upon challenge with a lethal dose of CoV2 (WA-1), sACE2-Ig expressed from erythroid cells of test mice diminishes infection sequelae. Treated mice lost significantly less weight, had less viremia, and displayed reduced cytokine production and lung pathology. The second objective of this study was to assess the safety of in vivo HSC transduction and long-term sACE2-Ig expression in a rhesus macaque. With appropriate cytokine prophylaxis, intravenous injection of HDAd-sACE2-Ig into the mobilized animal was well tolerated. In vivo transduced HSCs preferentially localized to and survived in the spleen. sACE2-Ig expressed from erythroid cells did not affect erythropoiesis and the function of erythrocytes. While these pilot studies are promising, the antiviral efficacy of the approach has to be improved, for example, by using of decoy receptors with enhanced neutralizing capacity and/or expression of multiple antiviral effector proteins.

Retrogenic Color-Barcoding for Fate Mapping of Single Innate Lymphocytes.

Lymphocyte fate mapping using single-cell transfers has been used to study T and B cell differentiation. Recently, retrogenic color-barcoding has allowed the extension of this approach to single innate lymphocytes such as NK cells. This new and versatile technology is based on the transduction of hematopoietic stem cells (HSCs) with a collection of retroviruses encoding distinct fluorescent proteins. Through combinatorial transduction, fluorescent protein barcodes are generated, which are inherited by the progeny of HSCs after transfer. By sorting individual cells expressing unique color-barcodes from the mature lymphocyte populations derived from these HSCs, it is now possible to track the fate of innate lymphocytes in vivo.

TEM-GBM: An Open-Label, Phase I/IIa Dose-Escalation Study Evaluating the Safety and Efficacy of Genetically Modified Tie-2 Expressing Monocytes to Deliver IFN-α within Glioblastoma Tumor Microenvironment

Background: We developed a macrophage-based treatment relying on ex vivo transduction of autologous hematopoietic stem and progenitor cells (HSPC) to express immune-payloads within the TME. Our ATMP (Temferon) targets IFN-a, an immune-modulatory molecule counteracting also neo-angiogenesis and tumor growth, to a subset of Tie2-expressing, tumor-infiltrating macrophages known as TEMs.Materials and Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed glioblastoma patients with unmethylated MGMT promoter. Key eligibility criteria include age 18-70 years, ECOG 0-1 and KPS >70%, and adequate cardiac, renal, hepatic and pulmonary function. Important exclusion criteria include the presence of active autoimmune disease or receipt of any oral or parenteral chemotherapy or immunotherapy within 2 years of screening. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and transduced ex vivo with a 3 rd generation lentiviral vector encoding for IFN-a2. Transgene expression is confined to TEMs by the Tie2 promoter and post-transcriptional regulation by microRNA-126 thus achieving tumor specificity. The study evaluates safety and biological activity of Temferon in 7 cohorts of three patients each, where escalating doses of Temferon are co-administered with a fixed CD34+ cell dose of non-manipulated supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or + Busulfan).The primary endpoints for this study are: •Engraftment of Temferon over the first 90 Days•The proportion of patients achieving hematologic recovery by Day +30 from ASCT•Short-term tolerability of Temferon; stable blood counts and absence of cytopenias, absence of significant organ toxicities (> grade 2); absence of Replication Competent LentivirusThe figure below reports the TEM-GBM study design.Results: As of 28th June 2021, 18 patients have been enrolled; 15 received Temferon (D+0) with follow-up of 30 - 697 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil and platelet engraftment occurred at D+13 and D+12 for patients in cohort 1-3 and D+16 and D+15 for patients assigned to cohort 4 and 5, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted subsequently, albeit at lower levels (up to 18 months). Very low concentrations of IFNa were detected in the plasma (average 7.8 pg/ml at D+30; baseline < LLOQ) and in the cerebrospinal fluid (average 1.6 pg/ml at D+30; baseline < LLOQ), suggesting tight regulation of transgene expression. Seven deaths occurred: six at D+241, +322, +340, +402, +478, +646 after Temferon administration due to disease progression, and one at D+60 due to complications following the conditioning regimen. Nine patients had progressive disease (PD; range D-12 to +239). SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation and poor performance status compatible with autologous stem cell transplantation, concomitant medications and PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present and increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion (as defined by MRI) had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA and TCR sequencing is ongoing.Conclusion: These interim results show that Temferon is generally well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon's potential to activate the immune system and reprogram the tumor microenvironment (TME), as predicted by preclinical studies. [Display omitted] DisclosuresNaldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Persistent Control of SIV Infection in Rhesus Macaques By Expressing a Highly Potent SIV Decoy Receptor after In Vivo HSC Transduction

Despite the success achieved by antiretroviral HIV-1 therapies, long-term and repeated drug administration is associated with toxicity, virus evasion and high cost. We aim to develop a gene therapy approach for persistent control against HIV-1 infection by delivering the transgene expressing a decoy receptor (eCD4-Ig) to hematopoietic stem cells (HSCs) by in vivo transduction. In this approach, HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with intravenously injected virus vectors. We use an integrating, helper-dependent adenovirus (HDAd5/35++) vector system that targets human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase and transgene marking in peripheral blood cells can be increased by in vivo selection. The efficacy and safety of our in vivo HSC transduction/selection strategy has been previously demonstrated for the treatment of b-hemoglobinopathies, hemophilia A, and cancer in murine disease models. In non-human primates, we showed efficient transgene (g-globin) expression in peripheral red blood cells using this strategy. eCD4-Ig functions like a neutralizing antibody and shows broad neutralization spectrum against HIV-1, HIV-2 and SIV isolates. We have designed and produced an HDAd5/35++ vector expressing rhesus eCD4-Ig (rh-eCD4-Ig) from a constitutive and highly active EF1a promoter. In CD46-transgenic mice, over 50µg/mL eCD4-Ig in serum was measured after in vivo transduction and selection, with no obvious adverse events observed. Neutralization assay with serum samples showed that the produced eCD4-Ig effectively inhibited HIV-1 and SIV infection. We then performed studies in a rhesus macaque. After in vivo HSC transduction/selection of a rhesus macaque, eCD4-Ig serum levels were stable at 20-30 mg/ml. In vitro SIVmac239 neutralization assays using week 13 serum and recombinant eCD4-Ig protein determined that the IC50 of eCD4-Ig in rhesus serum is 1.0 mg/ml. This implies that the serum eCD4-Ig concentration at the time of SIVmac239 challenge was ~25-fold higher than the IC50. High-level eCD4-Ig expression had no clinical or hematological side effects. The first SIVmac239 challenge (20pg) was given on June 22 nd. Increasing rechallenge doses are currently injected monthly. So far, the viral load measured by quantitative RT-PCR is below detection limit. The animal is without symptoms and has normal lymphocyte/subset counts. Our study demonstrates an in vivo HSC transduction approach for potential long-term control of HIV-1 infection. DisclosuresKiem: VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company; Homology Medicines: Consultancy. Lieber: Ensoma: Research Funding.

Platelet-Targeted Gene Transfer Induces Immune Tolerance in a Primed Model

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) is effective in inducing immune tolerance not only to the coagulation factor VIII and IX, but also to the non-coagulant protein ovalbumin (OVA) in a model without pre-existing immunity (the non-primed model). In the present study, we explored whether platelet-specific gene transfer could induce immune tolerance in the primed model. To address this question, we first used C57BL6 wild type (WT) mouse models. Both donors and recipients were immunized with OVA to induce anti-OVA antibody development. OVA expression was introduced by 2bOVA lentivirus (LV) transduction of hematopoietic stem cells (HSCs) from WT/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with a sub-lethal 6.6Gy total body irradiation (TBI). 2bGFP was used as a control vector in parallel. Animals were analyzed after at least 4 weeks of bone marrow (BM) reconstitution. The viability of OVA genetically modified HSCs was confirmed by PCR in all 2bOVA-transduced recipients. After full bone marrow constitution, which takes about 12 weeks, the engraftment in the 2bOVA group (87.4±4.4%, n=5) was similar to that in the 2bGFP group (87.8±3.9%, n=5) as determined by flow cytometry. The OVA protein level was 6.7±2.8 ng/108platelets (n=5) in 2bOVA transduced recipients. Anti-OVA antibody titers declined with time in both groups. Six months after transplantation, recipients were rechallenged with OVA. All of the anti-OVA titers in 2bGFP-transduced recipients (n=5) increased dramatically, while only one in the 2bOVA group slightly increased. Normalized anti-OVA antibody titers in the 2bOVA group (90.8±152.7) were significantly lower than those in the 2bGFP group (1278±2101) after OVA rechallenge, demonstrating that platelet-specific OVA gene delivery to HSCs can suppress anti-OVA immune responses in OVA-primed WT mice.To explore how immune suppression is established after platelet-specific gene transfer in the OVA-primed model, we transduced HSCs from OVA-specific TCR transgenic mice (OTII/CD45.2, in which greater than 97% CD4 T+ cells are OVA-specific) with 2bOVALV and transplanted into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy TBI. Untransduced transplanted mice were used as a control. After BM reconstitution, the OVA protein level was 32.3±6.3 ng/108 platelets (n=5) in the 2bOVA-transduced group. Anti-OVA antibody titers in both groups declined with time. When all the recipients were rechallenged with OVA, 2 of 3 anti-OVA titers in the untransduced group increased, while none of the titers in the 2bOVA group increased. Normalized anti-OVA antibody titers in the 2bOVA group (4.1±4.9) were significantly lower than those in the un-transduced group (152.3±214.5), demonstrating that platelet-specific OVA gene delivery to HSCs can also suppress the anti-OVA immune response in the OVA-primed WT mice that received 2bOVA-transduced HSCs from OTII mice (the OTII/WT model). After BM reconstitution, the engraftments in the 2 groups were similar (81.4±2.6%, and 80.9±2.5%, respectively), but donor-derived CD45.2+CD4+ (OTII/CD4) T cells in the 2bOVA (0.2±0.1%) group were consistently significantly lower than in the untransduced group (4.2±1.1%) in peripheral blood during the entire study course. Also, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA group compared to the untransduced group. Interestingly, there were no differences in either percentage or total cell number of CD45.2+CD4+ T cells in the thymus between the 2 groups, indicating that central tolerance may not play a role in the immune tolerance induced after platelet-targeted gene therapy. Meanwhile, the frequency and total number of endogenous CD4+ T cells were similar in the 2 groups. Of note, the percentages of donor-derived regulatory T (Treg) cells in the 2bOVA group were significantly higher than those in the untransduced group in peripheral blood, spleen, and lymph nodes, while there were no significant differences in the total numbers of donor-derived Treg cells between the 2 groups.Taken together, our studies demonstrate that platelet-specific gene therapy can induce immune tolerance in a primed system through two distinct pathways, peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. DisclosuresNo relevant conflicts of interest to declare.

An Improved Lentiviral Fluorescent Genetic Barcoding Approach Distinguishes Hematopoietic Stem Cell Properties in Multiplexed In Vivo Experiments.

Hematopoietic stem cells (HSCs) represent a rare cell population of particular interest for biomedical research and regenerative medicine. Various marker combinations enable the isolation of HSCs, but fail to reach purity in transplantation assays. To reduce animal consumption, we developed a multiplexing system based on lentiviral fluorescent genetic barcoding (FGB) to enable the parallel characterization of multiple HSC samples within single animals. While previous FGB-mediated HSC multiplexing experiments achieved high in vitro gene marking rates, in vivo persistence of transduced cells remained suboptimal. Thus, we aimed to optimize vector design and gene transfer protocols to demonstrate the applicability of FGB for functional characterization of two highly similar HSC populations in a reduced number of mice. We developed a set of six new lentiviral FGB vectors, utilizing individual and combinatorial expression of Azami Green, mCherry, and YFP derivatives. Gene transfer rates were optimized by overnight transduction of prestimulated HSCs with titrated vector doses. Populations for competitive transplantation experiments were identified by immunophenotyping murine HSCs. This identified an LSK-SLAM- (Lin-Sca-1+cKit+CD48-CD150+EPCR-) cell subpopulation that lacks EPCR expression and exhibits prospectively reduced self-renewal potential compared with prototypical ESLAM (CD45+EPCR+CD48-CD150+) HSCs. We monitored 30 data points per HSC-subpopulation in two independent experiments (each n = 5) after cotransplantation of three uniquely color-coded ESLAM and LSK-SLAM- samples per recipient. While the first experiment was hampered by data fluctuations, increasing cell numbers and exchange of the internal promoter in the second experiment led to 74.4% chimerism, with 87.1% of fluorescent cells derived from ESLAM HSCs. Furthermore, ESLAM-derived cells produced 88.1% of myeloid cells, which is indicative of their origin from long-term repopulating HSCs. This work verifies the importance of EPCR for long-term repopulating HSCs and demonstrates the applicability of our optimized FGB-driven multiplexing approach for the efficient characterization of blood cell populations in biomedical research.

Effect of supraphysiological alpha-L-iduronidase (IDUA) expression on skeletal manifestations in mucopolysaccharidosis type I (MPS I) mice following ex vivo lentiviral vector transduction of hematopoietic stem cells

Effect of supraphysiological alpha-L-iduronidase (IDUA) expression on skeletal manifestations in mucopolysaccharidosis type I (MPS I) mice following ex vivo lentiviral vector transduction of hematopoietic stem cells

Ex vivo lentiviral transduction of hematopoietic stem cells in mucopolysaccharidosis type II (MPS II) mice achieves high levels of systemic iduronate-2-sulfatase (IDS) enzyme activity and normalization of glycosaminoglycans (GAGs)

Ex vivo lentiviral transduction of hematopoietic stem cells in mucopolysaccharidosis type II (MPS II) mice achieves high levels of systemic iduronate-2-sulfatase (IDS) enzyme activity and normalization of glycosaminoglycans (GAGs)

A “Shot in the Arm” for Sickle Cell Disease

A “Shot in the Arm” for Sickle Cell Disease