Stem Cell Gene Therapy Research Articles

Transition of Care for Patients with Thalassemia.

Thalassemia is one of the most common hemoglobinopathies affecting a large number of people in India and other countries of South-East Asia. For patients with most severe form of the disease- Transfusion Dependent Thalassemia (TDT), stem cell transplantation or gene therapy are only curative treatment which are not available to most of the patients because of lack of experts, financial constraints and lack of suitable donors. In such situations, most cases are managed with regular blood transfusion and iron chelation therapy. With this treatment, over the years, survival of the patients has improved and 20-40% cases are entering into adulthood. In the absence of structured transition of care programs, currently most adult TDT patients are being managed by pediatricians. This article highlights the need for transition of care for TDT patients, barriers to transition and how to overcome the barriers and process of transition of care to adult care team. The importance of empowering the patients in self-management of the disease and educating the adult care team to achieve the desired outcome of transition program is highlighted.

Advances in therapies for neurological lysosomal storage disorders.

Lysosomal Storage Disorders (LSDs) are a diverse group of inherited, monogenic diseases caused by functional defects in specific lysosomal proteins. The lysosome is a cellular organelle that plays a critical role in catabolism of waste products and recycling of macromolecules in the body. Disruption to the normal function of the lysosome can result in the toxic accumulation of storage products, often leading to irreparable cellular damage and organ dysfunction followed by premature death. The majority of LSDs have no curative treatment, with many clinical subtypes presenting in early infancy and childhood. Over two-thirds of LSDs present with progressive neurodegeneration, often in combination with other debilitating peripheral symptoms. Consequently, there is a pressing unmet clinical need to develop new therapeutic interventions to treat these conditions. The blood-brain barrier is a crucial hurdle that needs to be overcome in order to effectively treat the central nervous system (CNS), adding considerable complexity to therapeutic design and delivery. Enzyme replacement therapy (ERT) treatments aimed at either direct injection into the brain, or using blood-brain barrier constructs are discussed, alongside more conventional substrate reduction and other drug-related therapies. Other promising strategies developed in recent years, include gene therapy technologies specifically tailored for more effectively targeting treatment to the CNS. Here, we discuss the most recent advances in CNS-targeted treatments for neurological LSDs with a particular emphasis on gene therapy-based modalities, such as Adeno-Associated Virus and haematopoietic stem cell gene therapy approaches that encouragingly, at the time of writing are being evaluated in LSD clinical trials in increasing numbers. If safety, efficacy and improved quality of life can be demonstrated, these therapies have the potential to be the new standard of care treatments for LSD patients.

The Role of Rab GTPases in the development of genetic and malignant diseases.

Small GTPases have been shown to play an important role in several cellular functions, including cytoskeletal remodeling, cell polarity, intracellular trafficking, cell-cycle, progression and lipid transformation. The Ras-associated binding (Rab) family of GTPases constitutes the largest family of GTPases and consists of almost 70 known members of small GTPases in humans, which are known to play an important role in the regulation of intracellular membrane trafficking, membrane identity, vesicle budding, uncoating, motility and fusion of membranes. Mutations in Rab genes can cause a wide range of inherited genetic diseases, ranging from neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD) to immune dysregulation/deficiency syndromes, like Griscelli Syndrome Type II (GS-II) and hemophagocytic lymphohistiocytosis (HLH), as well as a variety of cancers. Here, we provide an extended overview of human Rabs, discussing their function and diseases related to Rabs and Rab effectors, as well as focusing on effects of (aberrant) Rab expression. We aim to underline their importance in health and the development of genetic and malignant diseases by assessing their role in cellular structure, regulation, function and biology and discuss the possible use of stem cell gene therapy, as well as targeting of Rabs in order to treat malignancies, but also to monitor recurrence of cancer and metastasis through the use of Rabs as biomarkers. Future research should shed further light on the roles of Rabs in the development of multifactorial diseases, such as diabetes and assess Rabs as a possible treatment target.

Hematopoietic stem-cell gene therapy is associated with restored white matter microvascular function in cerebral adrenoleukodystrophy

Blood-brain barrier disruption marks the onset of cerebral adrenoleukodystrophy (CALD), a devastating cerebral demyelinating disease caused by loss of ABCD1 gene function. The underlying mechanism are not well understood, but evidence suggests that microvascular dysfunction is involved. We analyzed cerebral perfusion imaging in boys with CALD treated with autologous hematopoietic stem-cells transduced with the Lenti-D lentiviral vector that contains ABCD1 cDNA as part of a single group, open-label phase 2-3 safety and efficacy study (NCT01896102) and patients treated with allogeneic hematopoietic stem cell transplantation. We found widespread and sustained normalization of white matter permeability and microvascular flow. We demonstrate that ABCD1 functional bone marrow-derived cells can engraft in the cerebral vascular and perivascular space. Inverse correlation between gene dosage and lesion growth suggests that corrected cells contribute long-term to remodeling of brain microvascular function. Further studies are needed to explore the longevity of these effects.

In Vivo RNA Delivery to Hematopoietic Stem and Progenitor Cells via Targeted Lipid Nanoparticles.

Ex vivo autologous hematopoietic stem cell (HSC) gene therapy has provided new therapies for the treatment of hematological disorders. However, these therapies have several limitations owing to the manufacturing complexities and toxicity resulting from required conditioning regimens. Here, we developed a c-kit (CD117) antibody-targeted lipid nanoparticle (LNP) that, following a single intravenous injection, can deliver RNA (both siRNA and mRNA) to HSCs in vivo in rodents. This targeted delivery system does not require stem cell harvest, culture, or mobilization of HSCs to facilitate delivery. We also show that delivery of Cre recombinase mRNA at a dose of 1 mg kg-1 can facilitate gene editing to almost all (∼90%) hematopoietic stem and progenitor cells (HSPCs) in vivo, and edited cells retain their stemness and functionality to generate high levels of edited mature immune cells.

Gaucher Disease in Internal Medicine and Dentistry

Gaucher disease (GD) is a lysosomal storage pathological condition, characterized by a genetic autosomal recessive transmission. The GD cause is the mutation of GBA1 gene, located on the chromosome 1 (1q21), that induces the deficiency of the lysosomal enzyme glucocerebrosidase with consequent abnormal storage of its substrate (glucosylceramide), in macrophages. The GD incidence in the general population varies from 1:40,000 to 1:60,000 live births, but it is higher in the Ashkenazi Jewish ethnicity (1:800 live births). In the literature, five different types of GD are described: type 1, the most common clinical variant in Europe and USA (90%), affects the viscera; type 2, characterized by visceral damage and severe neurological disorders; type 3, in which the neurological manifestations are variable; cardiovascular type; and, finally, perinatal lethal type. The most affected tissues and organs are the hematopoietic system, liver, bone tissue, nervous system, lungs, cardiovascular system and kidneys. Another aspect of GD is represented by oral and dental manifestations. These can be asymptomatic or cause the spontaneous bleeding, the post oral surgery infections and the bone involvement of both arches through the Gaucher cells infiltration into the maxilla and mandibular regions. The pharmacological treatment of choice is the enzyme replacement therapy, but the new pharmacological frontiers are represented by oral substrate reduction therapy, chaperone therapy, allogeneic hematopoietic stem cell transplantation and gene therapy.

Gene Therapy for Regenerative Medicine.

The development of biological methods over the past decade has stimulated great interest in the possibility to regenerate human tissues. Advances in stem cell research, gene therapy, and tissue engineering have accelerated the technology in tissue and organ regeneration. However, despite significant progress in this area, there are still several technical issues that must be addressed, especially in the clinical use of gene therapy. The aims of gene therapy include utilising cells to produce a suitable protein, silencing over-producing proteins, and genetically modifying and repairing cell functions that may affect disease conditions. While most current gene therapy clinical trials are based on cell- and viral-mediated approaches, non-viral gene transfection agents are emerging as potentially safe and effective in the treatment of a wide variety of genetic and acquired diseases. Gene therapy based on viral vectors may induce pathogenicity and immunogenicity. Therefore, significant efforts are being invested in non-viral vectors to enhance their efficiency to a level comparable to the viral vector. Non-viral technologies consist of plasmid-based expression systems containing a gene encoding, a therapeutic protein, and synthetic gene delivery systems. One possible approach to enhance non-viral vector ability or to be an alternative to viral vectors would be to use tissue engineering technology for regenerative medicine therapy. This review provides a critical view of gene therapy with a major focus on the development of regenerative medicine technologies to control the in vivo location and function of administered genes.

An innovative hematopoietic stem cell gene therapy approach benefits CLN1 disease in the mouse model

Hematopoietic stem and progenitor cells (HSPCs) can establish a long‐lasting microglia‐like progeny in the central nervous system of properly myeloablated hosts. We exploited this approach to treat the severe CLN1 neurodegenerative disorder, which is the most aggressive form of neuronal ceroid lipofuscinoses due to palmitoyl‐protein thioesterase‐1 (PPT1) deficiency. We here provide the first evidence that (i) transplantation of wild‐type HSPCs exerts partial but long‐lasting mitigation of CLN1 symptoms; (ii) transplantation of HSPCs over‐expressing hPPT1 by lentiviral gene transfer enhances the therapeutic benefit of HSPCs transplant, with first demonstration of such a dose–effect benefit for a purely neurodegenerative condition like CLN1 disease; (iii) transplantation of hPPT1 over‐expressing HSPCs by a novel intracerebroventricular (ICV) approach is sufficient to transiently ameliorate CLN1‐symptoms in the absence of hematopoietic tissue engraftment of the transduced cells; and (iv) combinatorial transplantation of transduced HSPCs intravenously and ICV results in a robust therapeutic benefit, particularly on symptomatic animals. Overall, these findings provide first evidence of efficacy and feasibility of this novel approach to treat CLN1 disease and possibly other neurodegenerative conditions, paving the way for its future clinical application.

Sertoli cell-only syndrome: advances, challenges, and perspectives in genetics and mechanisms.

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children.

Manifestations of B-cell expansion with NF-κB and T-cell anergy disease overlapping with hemophagocytic lymphohistiocytosis.

B cell expansion with NF-κB and T cell anergy (BENTA) is a disease genetically linked with heterozygous gain-of-function (GOF) mutations in the CARD11 gene with an autosomal dominant expression. Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous group of disorders characterized by systemic inflammation and hypercytokinaemia. Some BENTA patients share similar clinical manifestations as HLH in various aspects, including feverand splenomegaly. In this study, we reported a 15-month-old boy diagnosed as BENTA meeting with diagnostic criteria of HLH. The complications were resolved by antibiotics for controlling severe infection, together with the reduced format of dexamethasone and etoposide for subsiding HLH activities. While the patient was not subjected to disease recurrence and maintained free of infection, a persistent lymphocytosis derived mainly from the expansion of polyclonal B cells was ascertained. Flow cytometry analysis indicated that the subdued degranulation of NK cells prior to treatment had been restored as the HLH-related complications waned. With largely reduced number and ratios in CD4 and CD8 T cells, their proliferation and Vβ diversity remained in normal ranges. In vitro stimulation experiment revealed a functional abbreviation of T cells as the percentage of IFNγ-releasing CD3+CD4+ T cells augmented while the percentage reduced in CD3+CD4- T cells. Whole exome sequencing revealed a de novo G123D missense mutation in the CARD11 gene. This new case of BENTA showcased a scenario of predominant HLH activities accompanied by a severe infection normally associated with BENTA. In addition, a brief treatment quenching HLH complication in cooperation with antibiotics for infection control was not able to solve the underlined T cell abnormality as well as B cell expansion caused by CARD11 mutation. A haematopoietic stem cell transplantation or gene therapy is still a pursuit to remedy this inborn error of immunity.

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.

Protocol for a high titer of BaEV-Rless pseudotyped lentiviral vector: Focus on syncytium formation and detachment

The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly-L-lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders.

A systematic review comparing allogeneic hematopoietic stem cell transplant to gene therapy in sickle cell disease

ABSTRACTIntroductionAllogeneic hematopoietic stem cell transplant (HSCT) and gene therapy (GT) are two potentially curative approaches for sickle cell disease (SCD), but they have never been compared in clinical trials.ObjectiveTo compare the safety and efficacy of HSCT and GT to assist clinicians and patients in making informed treatment decisions.MethodsPhase I-III clinical trials and case reports/series were included. Regimens included HSCT from all stem cell sources, lentiviral gene therapy, and gene editing, with any conditioning regimen. We searched Medline and EMBASE databases as of 1st June 2020 for studies reporting HSCT and GT outcomes in SCD. The Newcastle-Ottawa scale was used to assess the risk of bias. Descriptive statistics and post-hoc imputation for standard deviations of mean change in FEV1 and FVC were performed.ResultsIn total, 56 studies (HSCT, n = 53; GT, n = 3) representing 1,198 patients met inclusion criteria (HSCT, n = 1,158; GT, n = 40). Length of follow-up was 3,881.5 and 58.7 patient-years for HSCT and GT, respectively. Overall quality of evidence was low, with no randomized controlled trials identified. Two-year overall survival for HSCT was 91%; mortality was 2.5% for GT. Acute chest syndrome and vaso-occlusive episodes were reduced post-HSCT and GT. Meta-analysis was not possible due to lack of comparator and heterogeneity in outcome measures reporting. Very few studies reported post-transplant end-organ function. Six secondary malignancies (5 post-HSCT, 1 post-GT) were reported.DiscussionReporting of SCD-related complications and patient-important outcomes is lacking for both strategies. We advocate for standardized reporting to better compare outcomes within and between treatment groups.

The Guard1 clinical trial - A first in-human, phase 1/2 study evaluating AVR-RD-02, a hematopoietic stem cell (HSC) gene therapy for Gaucher disease: Preliminary safety, pharmacodynamic and clinical efficacy results from the subjects observed for up to 24 months post-infusion

The Guard1 clinical trial - A first in-human, phase 1/2 study evaluating AVR-RD-02, a hematopoietic stem cell (HSC) gene therapy for Gaucher disease: Preliminary safety, pharmacodynamic and clinical efficacy results from the subjects observed for up to 24 months post-infusion

Current and new therapies for mucopolysaccharidoses.

The mucopolysaccharidoses (MPSs) are a subset of lysosomal storage diseases caused by deficiencies in the enzymes required to metabolize glycosaminoglycans (GAGs), a group of extracellular heteropolysaccharides that play diverse roles in human physiology. As a result, GAGs accumulate in multiple tissues, and affected patients typically develop progressive, multi-systemic symptoms in early childhood. Over the last 30 years, the treatments available for the MPSs have evolved tremendously. There are now multiple therapies that delay the progression of these debilitating disorders, although their effectiveness varies according to MPS sub-type. In this review, we discuss the basic principle underlying MPS treatment (enzymatic "cross correction"), and we review the three general modalities currently available: hematopoietic stem cell transplantation, enzymatic replacement, and gene therapy. For each treatment type, we discuss its effectiveness across the MPS subtypes, its inherent risks, and future directions. Long term, we suspect that treatment for the MPSs will continue to evolve, and through a combination of early diagnosis and effective management, these patients will continue to live longer lives with improved outcomes for quality of life.

Validation of an assay to measure iduronate-2-sulfatase activity in cerebrospinal fluid to assess the efficacy of a hematopoietic stem cell (HSC) gene therapy

Validation of an assay to measure iduronate-2-sulfatase activity in cerebrospinal fluid to assess the efficacy of a hematopoietic stem cell (HSC) gene therapy

Sustained improvement of clinical CNS and somatic features of Gaucher disease type 3 after haematopoietic stem cell (HSC) gene therapy: A first-in-world report

Sustained improvement of clinical CNS and somatic features of Gaucher disease type 3 after haematopoietic stem cell (HSC) gene therapy: A first-in-world report

Phase 1/2 clinical trial of autologous hematopoietic stem and progenitor cell (HSPC) gene therapy for cystinosis

Phase 1/2 clinical trial of autologous hematopoietic stem and progenitor cell (HSPC) gene therapy for cystinosis

Atidarsagene autotemcel, a European post-regulatory approval model for delivery of autologous hematopoietic stem cell gene therapy products via a network of qualified treatment centers (QTCs)

Atidarsagene autotemcel, a European post-regulatory approval model for delivery of autologous hematopoietic stem cell gene therapy products via a network of qualified treatment centers (QTCs)

Long-term clinical outcomes of atidarsagene autotemcel (autologous hematopoietic stem cell gene therapy [HSC-GT] for metachromatic leukodystrophy) with up to 11 years follow-up

Long-term clinical outcomes of atidarsagene autotemcel (autologous hematopoietic stem cell gene therapy [HSC-GT] for metachromatic leukodystrophy) with up to 11 years follow-up