Development of CNS tropic AAV1-like variants with reduced liver-targeting following systemic administration in mice.

Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.

Liver-specific adiponectin gene therapy suppresses microglial NLRP3-inflammasome activation for treating Alzheimer's disease.

Adiponectin (APN) is an adipokine which predominantly expresses in adipocytes with neuroprotective and anti-inflammatory effects. We have recently indicated that circulatory trimeric APN can enter the brain by crossing the blood-brain barrier (BBB) and modulate microglia-mediated neuroinflammation. Here, we found that the microglial NLR family pyrin domain containing 3 (NLRP3)-inflammasome activation was exacerbated in APN-/-5xFAD mice in age-dependent manner. The focus of this study was to develop a new and tractable therapeutic approach for treating Alzheimer's disease (AD)-related pathology in 5xFAD mice using peripheral APN gene therapy. We have generated and transduced adeno-associated virus (AAV2/8) expressing the mouse mutated APN gene (APNC39S) into the liver of 5xFAD mice that generated only low-molecular-weight trimeric APN (APNTri). Single dose of AAV2/8-APNC39S in the liver increased circulatory and cerebral APN levels indicating the overexpressed APNTri was able to cross the BBB. Overexpression of APNTri decreased both the soluble and fibrillar Aß in the brains of 5xFAD mice. AAV2/8-APNTri treatment reduced Aß-induced IL-1ß and IL-18 secretion by suppressing microglial NLRP3-inflammasome activation. The memory functions improved significantly in AAV-APNTri-treated 5xFAD mice with reduction of dystrophic neurites. These findings demonstrate that peripheral gene delivery to overexpress trimeric APN can be a potential therapy for AD.

Gene therapy for urea cycle defects: An update from historical perspectives to future prospects.

Urea cycle defects (UCDs) are severe inherited metabolic diseases with high unmet needs which present a permanent risk of hyperammonaemic decompensation and subsequent acute death or neurological sequelae, when treated with conventional dietetic and medical therapies. Liver transplantation is currently the only curative option, but has the potential to be supplanted by highly effective gene therapy interventions without the attendant need for life-long immunosuppression or limitations imposed by donor liver supply. Over the last three decades, pioneering genetic technologies have been explored to circumvent the consequences of UCDs, improve quality of life and long-term outcomes: adenoviral vectors, adeno-associated viral vectors, gene editing, genome integration and non-viral technology with messenger RNA. In this review, we present a summarised view of this historical path, which includes some seminal milestones of the gene therapy's epic. We provide an update about the state of the art of gene therapy technologies for UCDs and the current advantages and pitfalls driving future directions for research and development.

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy.

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

Gain-of-function factor H-related 5 protein impairs glomerular complement regulation resulting in kidney damage.

Genetic variation within the factor H-related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

Gene Therapy for Paediatric Homozygous Familial Hypercholesterolaemia.

The clinical outcome for children and adolescents with homozygous familial hypercholesterolaemia (HoFH) can be devastating, and treatment options are limited in the presence of a null variant. In HoFH, atherosclerotic risk accumulates from birth. Gene therapy is an appealing treatment option as restoration of low-density lipoprotein receptor (LDLR) gene function could provide a cure for HoFH. A clinical trial using a recombinant adeno-associated vector (rAAV) to deliver LDLR DNA to adult patients with HoFH was recently completed; results have not yet been reported. However, this treatment strategy may face challenges when translating to the paediatric population. The paediatric liver undergoes substantial growth which is significant as rAAV vector DNA persists primarily as episomes (extra-chromosomal DNA) and are not replicated during cell division. Therefore, rAAV-based gene addition treatment administered in childhood would likely only have a transient effect. With over 2,000 unique variants in LDLR, a goal of genomic editing-based therapy development would be to treat most (if not all) mutations with a single set of reagents. For a robust, durable effect, LDLR must be repaired in the genome of hepatocytes, which could be achieved using genomic editing technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and a DNA repair strategy such as homology-independent targeted integration. This review discusses this issue in the context of the paediatric patient group with severe compound heterozygous or homozygous null variants which are associated with aggressive early-onset atherosclerosis and myocardial infarction, together with the important pre-clinical studies that use genomic editing strategies to treat HoFH in place of apheresis and liver transplantation.

In Search of Adeno-Associated Virus Vectors With Enhanced Cardiac Tropism for Gene Therapy.

Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods.

Systemic AAV8-mediated delivery of a functional copy of muscle glycogen phosphorylase (Pygm) ameliorates disease in a murine model of McArdle disease.

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1-3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor ß and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

The implementation of newborn screening for spinal muscular atrophy: the Australian experience.

PURPOSE: To evaluate the implementation of the first statewide newborn screening (NBS) program for spinal muscular atrophy (SMA) in Australia. Processes that hinder and support clinical development, translation, and sustainability of the first primary genetic screening program in Australia are appraised. METHODS: The study prospectively describes the course (timelines, health processes, and preliminary clinical outcomes) for SMA screen-positive newborns from 1 August 2018 to 31 July 2019 in New South Wales and Australian Capital Territory, Australia. RESULTS: In the first year of the program, 103,903 newborns were screened. Ten newborns screened positive for SMA. Genetic confirmation of SMA occurred in 9/10 (90%) of infants. Clinical signs of SMA evolved in 4/9 (44%) within 4 weeks of life, heralded by hypotonia and weakness initially recognized in the neck. Median time to implementing a care plan (including commencement of disease-modifying therapies) was 26.5 days (16-37 days) from birth. CONCLUSION: NBS is essential for early and equitable identification of patients with SMA. Expedient diagnosis and management are vital, as disease latency appears brief in some cases. NBS shows significant clinical utility to support early parental decision making, improve access to specialist neuromuscular expertise, and facilitate initiation of personalized therapeutic strategies.

The diagnostic utility of genome sequencing in a pediatric cohort with suspected mitochondrial disease.

PURPOSE: The utility of genome sequencing (GS) in the diagnosis of suspected pediatric mitochondrial disease (MD) was investigated. METHODS: An Australian cohort of 40 pediatric patients with clinical features suggestive of MD were classified using the modified Nijmegen mitochondrial disease severity scoring into definite (17), probable (17), and possible (6) MD groups. Trio GS was performed using DNA extracted from patient and parent blood. Data were analyzed for single-nucleotide variants, indels, mitochondrial DNA variants, and structural variants. RESULTS: A definitive MD gene molecular diagnosis was made in 15 cases and a likely MD molecular diagnosis in a further five cases. Causative mitochondrial DNA (mtDNA) variants were identified in four of these cases. Three potential novel MD genes were identified. In seven cases, causative variants were identified in known disease genes with no previous evidence of causing a primary MD. Diagnostic rates were higher in patients classified as having definite MD. CONCLUSION: GS efficiently identifies variants in MD genes of both nuclear and mitochondrial origin. A likely molecular diagnosis was identified in 67% of cases and a definitive molecular diagnosis achieved in 55% of cases. This study highlights the value of GS for a phenotypically and genetically heterogeneous disorder like MD.

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV-piggyBac Gene Therapy.

Recombinant adeno-associated viral (rAAV) vectors are highly promising vehicles for liver-targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile-onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV-mediated liver-targeted gene therapy. The Abcb4-/- mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4-/- mice with established liver disease, but not in disease-free, wild-type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase-mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile-onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life-long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV-piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation.

Great expectations: virus-mediated gene therapy in neurological disorders.

Gene therapy (GT) has tremendous potential for the treatment of neurological disorders to transform patient care. The successful application of virus-mediated GT to treat spinal muscular atrophy is a significant milestone, serving to accelerate similar progress in a spectrum of neurological conditions, with more than 50 clinical trials currently underway, across neurodevelopmental, neurodegenerative, muscular dystrophy, epilepsy, chronic pain and neoplastic diseases. This review provides an overview of the key features of virus-mediated GT, paradigms of delivery and dosing, potential risks and highlights ongoing research to optimise safe and effective delivery of vectors into the nervous system. Examples of the application of GT in various neurological diseases alongside clinical development challenges will be presented. As the development and translation of GTs gain pace, success can only ultimately be realised for patients following implementation in the health system. The challenges and controversies of daunting costs, ethics, early diagnosis and health system readiness will require innovative pricing schemes, regulatory policies, education and organisation of a skilled workforce to deliver of high-quality care in clinical practice as we prepare for advanced therapeutics in neurology.

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

AAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization.

Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.

Gene therapy clinical trials worldwide to 2017: An update.

To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our November 2017 update, we have entries on 2597 trials undertaken in 38 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and the genes that have been transferred. Details of the analyses presented, and our searchable database are available via The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in gene therapy clinical trials around the world, and discuss key trends since the previous review, namely the use of chimeric antigen receptor T cells for the treatment of cancer and advancements in genome editing technologies, which have the potential to transform the field moving forward.

Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.

BACKGROUND: Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes. METHODS: Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and ß-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes. CONCLUSIONS: These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.

Thymocyte self-renewal and oncogenic risk in immunodeficient mouse models: relevance for human gene therapy clinical trials targeting haematopoietic stem cell populations?

Emerging evidence indicates that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Here we discuss formative studies demonstrating that, in mice, early thymocytes acquire self-renewing potential when thymic progenitor supply is sub-physiological and the importance of cellular competition with this at-risk cell population to prevent lymphoid malignancy. We also consider the possibility that increased thymic residency time, established under conditions of limited cellular competition, may have contributed to oncogenesis observed in early SCID-X1 trials when combined with insertional activation of proto-oncogenes such as LMO2.