Advancements in programmable DNA-Binding Proteins (DBDs) that target the genome, such as zinc fingers, transcription activator-like effectors, and Cas9, have broadened drug target design beyond traditional protein substrates. Effective delivery methodologies remain a major barrier in targeting the central nervous system. Currently, adeno-associated virus is the most wellvalidated delivery system for the delivery of DBDs towards the central nervous with multiple, ongoing clinical trials. While effective in transducing neuronal cells, viral delivery systems for DBDs remain problematic due to inherent viral packaging limits or immune responses that hinder translational potential. Direct administration of DBDs or encapsulation in lipid nanoparticles may provide alternative means towards delivering gene therapies into the central nervous system. This review will evaluate the strengths and limitations of current DBD delivery strategies in vivo. Furthermore, this review will discuss the use of adult stem cells as a putative delivery vehicle for DBDs and the potential advantages that these systems have over previous methodologies.

Introduction

Brain-derived neurotrophic factor (BDNF) has been implicated in wide range of neurological diseases and injury. This neurotrophic factor is vital for neuronal health, survival, and synaptic connectivity. Many therapies focus on the restoration or enhancement of BDNF following injury or disease progression.

Areas covered

The present review will focus on the mechanisms in which BDNF exerts its beneficial functioning, current BDNF therapies, issues and potential solutions for delivery of neurotrophic factors to the central nervous system, and other disease indications that may benefit from overexpression or restoration of BDNF.

Expert opinion

Due to the role of BDNF in neuronal development, maturation, and health, BDNF is implicated in numerous neurological diseases making it a prime therapeutic agent. Numerous studies have shown the therapeutic potential of BDNF in a number of neurodegenerative disease models and in acute CNS injury, however clinical translation has fallen short due to issues in delivering this molecule. The use of MSC as a delivery platform for BDNF holds great promise for clinical advancement of neurotrophic factor restoration. The ease with which MSC can be engineered opens the door to the possibility of using this cell-based delivery system to advance a BDNF therapy to the clinic.

To improve the translational predictability of treatment strategies for Huntington's disease (HD), sensitive and analogous cognitive outcomes are needed across HD animal models and humans. Spatial memory measures are promising candidates because they are based on 'visual' or 'non-verbal' cognition, and are commonly tested in both animals and humans. Here, we consider the suitability of spatial memory for strengthening translational links between animals and humans in HD research and clinical trials. We describe findings of spatial memory impairments in human HD and mouse models, including which aspects of spatial memory are most affected and at which time points in disease progression. We also describe the neural systems that underlie spatial memory and link spatial memory impairments to HD neuropathology, focussing on striatal and hippocampal systems. We provide a critical analysis of the literature in terms of the suitability of spatial memory for bridging the translational gap between species. Finally, we discuss possible neural mechanisms that might explain the spatial memory impairments seen in HD, and their relevance to potential treatments.

Progress to date from our group and others indicate that using genetically-engineered mesenchymal stem cells (MSC) to secrete brain-derived neurotrophic factor (BDNF) supports our plan to submit an Investigational New Drug application to the Food and Drug Administration for the future planned Phase 1 safety and tolerability trial of MSC/BDNF in patients with Huntington's disease (HD). There are also potential applications of this approach beyond HD. Our biological delivery system for BDNF sets the precedent for adult stem cell therapy in the brain and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA), Alzheimer's disease, and some forms of Parkinson's disease. The MSC/BDNF product could also be considered for studies of regeneration in traumatic brain injury, spinal cord and peripheral nerve injury. This work also provides a platform for our future gene editing studies, since we will again use MSCs to deliver the needed molecules into the central nervous system.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.

Stem cell therapies have been explored as a new avenue for the treatment of neurologic disease and damage within the CNS in part due to their native ability to mimic repair mechanisms in the brain. Mesenchymal stem cells have been of particular clinical interest due to their ability to release beneficial neurotrophic factors and their ability to foster a neuroprotective microenviroment. While early stem cell transplantation therapies have been fraught with technical and political concerns as well as limited clinical benefits, mesenchymal stem cell therapies have been shown to be clinically beneficial and derivable from nonembryonic, adult sources. The focus of this review will be on emerging and extant stem cell therapies for juvenile and adult-onset Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats. Although pathogenesis has been attributed to this polyglutamine expansion, the underlying mechanisms through which the huntingtin protein functions have yet to be elucidated. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. For allele-specific targeting, transcription activator-like effectors (TALE) were designed to target single-nucleotide polymorphisms (SNP) in the mutant allele and packaged into a vector backbone containing KRAB to promote transcriptional repression of the disease-associated allele. Additional TALEs were packaged into a vector backbone containing heterodimeric FokI and were designed to be used as nucleases (TALEN) to cause a CAG-collapse in the mutant allele. Human HD fibroblasts were treated with each TALE-SNP or TALEN. Allele-expression was measured using a SNP-genotyping assay and mutant protein aggregation was quantified with Western blots for anti-ubiquitin. The TALE-SNP and TALEN significantly reduced mutant allele expression (p < 0.05) when compared to control transfections while not affecting expression of the nondisease allele. This study demonstrates the potential of allele-specific gene modification using TALE proteins, and provides a foundation for targeted treatment for individuals suffering from Huntington's or other genetically linked diseases.

Huntington's disease (HD) is a fatal autosomal-dominant neurodegenerative disease caused by a trinucleotide CAG repeat expansion of the huntingtin gene (HTT) that affects 1 in every 10 000 individuals in the United States. Our lab developed a novel immune deficient HD mouse strain, the YACNSG, from a commonly used line, the YAC128 mouse, to enable transplantation studies using engineered human cells in addition to studying the impact of the immune system on disease progression. The primary goal of this project was to characterize this novel immune deQficient HD mouse model, using behavioral assays and histology to compare this new model to the immune competent YAC128 and immune deficient mice that had engraftment of a human immune system. Flow cytometry was used to confirm that the YACNSG strain lacked immune cells, and in vivo imaging was used to assess human mesenchymal stem/stromal cell (MSC) retention compared with a commonly used immune deficient line, the NSG mouse. We found that YACNSG were able to retain human MSCs longer than the immune competent YAC128 mice. We performed behavioral assessments starting at 4 months of age and continued testing monthly until 12 months on the accelerod and in the open field. At 12 months, brains were isolated and evaluated using immunohistochemistry for striatal volume. Results from these studies suggest that the novel immune deficient YACNSG strain of mice could provide a good model for human stem-cell based therapies and that the immune system appears to play an important role in the pathology of HD.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.