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Abstract Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations in vitro. NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ T cells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.

Integrating vectors are associated with alterations in cellular function related to disruption of normal gene function. This has been associated with clonal expansion of cells and, in some instances, cancer. These events have been associated with replication-defective vectors and suggest that the inadvertent exposure to a replication-competent virus arising during vector manufacture would significantly increase the risk of treatment-related adverse events. These risks have led regulatory agencies to require specific monitoring for replication-competent viruses, both prior to and after treatment of patients with gene therapy products. Monitoring the risk of cell expansion and malignancy is also required. In this review, we discuss the rational potential approaches and challenges to meeting the US FDA expectations listed in current guidance documents.

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2′-5′-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

Adenosine deaminase (ADA) deficiency is an inborn error of metabolism affecting multiple systems and causing severe combined immunodeficiency. We tested intravenous administration of recombinant adeno-associated virus (AAV) 2/8-ADA vector in ADA-deficient neonate and adult mice or as part of a bimodal approach comprised of rAAV treatment at birth followed by infusion of lentiviral vector (LV)-modified lineage-depleted bone marrow cells at 8 weeks. ADA−/− mice treated with rAAV and enzyme replacement therapy (ERT) for 30 days were rescued from the lethal pulmonary insufficiency, surviving out to 180 days without further treatment. rAAV vector copy number (VCN) was highest in liver, lung, and heart and was associated with near-normal ADA activity and thymocyte development. In the bimodal approach, rAAV-mediated ADA expression supported survival during the 4 weeks before infusion of the LV-modified bone marrow cells and during the engraftment period. Conditioning prior to infusion may have resulted in the replacement of rAAV marked cells in marrow and liver, with LV VCN 100- to 1,000-fold higher in hematopoietic tissue compared with rAAV VCN, and was associated with immune cell reconstitution. In conclusion, a bimodal approach may be an alternative for patients without reliable access to ERT before receiving a stem cell transplant or gene therapy.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

Adenosine deaminase (ADA)-deficient mice and healthy rhesus monkeys were studied to determine the impact of age at treatment, vector dosage, dosing schedule, repeat administration, biodistribution, and immunogenicity after systemic delivery of lentiviral vectors (LVs). In Ada−/− mice, neonatal treatment resulted in broad vector marking across all tissues analyzed, whereas adult treatment resulted in marking restricted to the liver, spleen, and bone marrow. Intravenous administration to infant rhesus monkeys also resulted in dose-dependent marking in the liver, spleen, and bone marrow. Using an ELISA to monitor anti-vector antibody development, Ada−/− neonatal mice did not produce an antibody response, whereas Ada−/− adult mice produced a strong antibody response to vector administration. In mice and monkeys with repeat administration of LV, a strong anti-vector antibody response was shown in response to the second LV administration, which resulted in LV inactivation. Three separate doses administered to immune competent mice resulted in acute toxicity. Pegylation of the vesicular stomatitis virus G protein (VSV-G)-enveloped LVs showed a less robust anti-vector response but did not prevent the inactivation of the second LV administration. These studies identify important factors to consider related to age and timing of administration when implementing systemic delivery of LVs as a potential therapeutic agent. Keywords: lentiviral vector, gene therapy, rhesus monkeys, in vivo, immune response, ADA-deficiency, ERT, repeat administration

Abstract: To address the global burden of sickle cell disease and the need for novel therapies, the American Society of Hematology partnered with the US Food and Drug Administration to engage the work of 7 panels of clinicians, investigators, and patients to develop consensus recommendations for clinical trial end points. The panels conducted their work through literature reviews, assessment of available evidence, and expert judgment focusing on end points related to patient-reported outcome, pain (non–patient-reported outcomes), the brain, end-organ considerations, biomarkers, measurement of cure, and low-resource settings. This article presents the findings and recommendations of the end-organ considerations, measurement of cure, and low-resource settings panels as well as relevant findings and recommendations from the biomarkers panel.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve in humans. Spike protein mutations increase transmission and potentially evade antibodies raised against the original sequence used in current vaccines. Our evaluation of serum neutralizing activity in both persons soon after SARS-CoV-2 infection (in April 2020 or earlier) or vaccination without prior infection confirmed that common spike mutations can reduce antibody antiviral activity. However, when the persons with prior infection were subsequently vaccinated, their antibodies attained an apparent biologic ceiling of neutralizing potency against all tested variants, equivalent to the original spike sequence. These findings indicate that additional antigenic exposure further improves antibody efficacy against variants. IMPORTANCE As SARS-CoV-2 evolves to become better suited for circulating in humans, mutations have occurred in the spike protein it uses for attaching to cells it infects. Protective antibodies from prior infection or vaccination target the spike protein to interfere with its function. These mutations can reduce the efficacy of antibodies generated against the original spike sequence, raising concerns for reinfections and vaccine failures, because current vaccines contain the original sequence. In this study, we tested antibodies from people infected early in the pandemic (before spike variants started circulating) or people who were vaccinated without prior infection. We confirmed that some mutations reduce the ability of antibodies to neutralize the spike protein, whether the antibodies were from past infection or vaccination. Upon retesting the previously infected persons after vaccination, their antibodies gained the same ability to neutralize mutated spike as the original spike, suggesting that the combination of infection and vaccination drove the production of enhanced antibodies to reach a maximal level of potency. Whether this can be accomplished by vaccination alone remains to be determined, but the results suggest that booster vaccinations may help improve efficacy against spike variants through improving not only antibody quantity, but also quality.

Summary: Cell-based immunotherapy has become the new-generation cancer medicine, and “off-the-shelf” cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.

The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. We generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2-expressing cells with high specificity and contain minimal off-target transduction of ACE2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein, we generated an expression plasmid encoding an optimized protein that produces S-pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000-fold higher than the unmodified S protein and 4 to 10-fold more specific than the widely used delta-19 S-pseudotyped lentiviral vectors. S-pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S-pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE2-expressing cells.

Lentiviral vector (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. Clinical trials are currently underway to treat sickle cell disease using LVs expressing designed anti-sickling globin genes. However, because of the large size and complexity of the human β-globin gene, LV products often have low titers and transduction efficiency, requiring large amounts to treat a single patient. Furthermore, transduction of patient HSPCs often fails to achieve a sufficiently high vector copy number (VCN) and transgene expression for clinical benefit. We therefore investigated the combination of two compounds (PGE2 and poloxamer synperonic F108) to enhance transduction of HSPCs with a clinical-scale preparation of Lenti/G-AS3-FB. Here, we found that transduction enhancers increased the in vitro VCN of bulk myeloid cultures ∼10-fold while using a 10-fold lower LV dose. This was accompanied by an increased percentage of transduced colony-forming units. Importantly, analysis of immune-deficient NSG xenografts revealed that the combination of PGE2/synperonic F108 increased LV gene transfer in a primitive HSC population, with no effects on lineage distribution or engraftment. The use of transduction enhancers may greatly improve efficacy for LV-based HSPC gene therapy. Keywords: HSCs, lentivirus, gene therapy, sickle cell disease

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%–80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12–14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3. Keywords: gene therapy, sickle cell disease, lentiviral vector, integration, genotoxicity, hematopoietic stem-progenitor cells, stem cell clonality, xenograft, NSG mice

Summary: SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990–2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription (“SIN” vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs.

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management.

Abstract Background Mutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction. Methods Here we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples. Results Comparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure. Conclusions This multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544.

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

Abstract Background Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. Methods We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Results Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. Conclusions This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images.