Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing.

After damage to the adult mammalian central nervous system (CNS), surviving neurons have limited capacity to regenerate and restore functional connectivity. Conditional genetic deletion of PTEN results in robust CNS axon regrowth, while PTEN repression with short hairpin RNA (shRNA) improves regeneration but to a lesser extent, likely due to suboptimal PTEN mRNA knockdown using this approach. Here we employed the CRISPR/dCas9 system to repress PTEN transcription in neural cells. We targeted the PTEN proximal promoter and 5' untranslated region with dCas9 fused to the repressor protein Krüppel-associated box (KRAB). dCas9-KRAB delivered in a lentiviral vector with one CRISPR guide RNA (gRNA) achieved potent and specific PTEN repression in human cell line models and neural cells derived from human iPSCs, and induced histone (H)3 methylation and deacetylation at the PTEN promoter. The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript, a construct previously used in CNS injury models. The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells, and enhanced neurite outgrowth after nerve growth factor stimulation. PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.

Neurotrophic Factors Used to Treat Spinal Cord Injury.

The application of neurotrophic factors as a therapy to improve morphological and behavioral outcomes after experimental spinal cord injury (SCI) has been the focus of many studies. These studies vary markedly in the type of neurotrophic factor that is delivered, the mode of administration, and the location, timing, and duration of the treatment. Generally, the majority of studies have had significant success if neurotrophic factors are applied in or close to the lesion site during the acute or the subacute phase after SCI. Comparatively fewer studies have administered neurotrophic factors in order to directly target the somata of injured neurons. The mode of delivery varies between acute injection of recombinant proteins, subacute or chronic delivery using a variety of strategies including osmotic minipumps, cell-mediated delivery, delivery using polymer release vehicles or supporting bridges of some sort, or the use of gene therapy to modify neurons, glial cells, or precursor/stem cells. In this brief review, we summarize the state of play of many of the therapies using these factors, most of which have been undertaken in rodent models of SCI.

Scaffolds to promote spinal cord regeneration.

Substantial research effort in the spinal cord injury (SCI) field is directed towards reduction of secondary injury changes and enhancement of tissue sparing. However, pathway repair after complete transections, large lesions, or after chronic injury may require the implantation of some form of oriented bridging structure to restore tissue continuity across a trauma zone. These matrices or scaffolds should be biocompatible and create an environment that facilitates tissue growth and vascularization, and allow axons to regenerate through and beyond the implant in order to reconnect with "normal" tissue distal to the injury. The myelination of regrown axons is another important requirement. In this chapter, we describe recent advances in biomaterial technology designed to provide a terrain for regenerating axons to grow across the site of injury and/or create an environment for endogenous repair. Many different types of scaffold are under investigation; they can be biodegradable or nondegradable, natural or synthetic. Scaffolds can be designed to incorporate immobilized signaling molecules and/or used as devices for controlled release of therapeutic agents, including growth factors. These bridging structures can also be infiltrated with specific cell types deemed suitable for spinal cord repair.

Strain-specific differences in the effects of cyclosporin A and FK506 on the survival and regeneration of axotomized retinal ganglion cells in adult rats.

The immune response can influence neuronal viability and plasticity after injury, effects differing in strains of rats with different susceptibility to autoimmune disease. We assessed the effects of i.p. injections of cyclosporin A (CsA) or FK506 on adult retinal ganglion cell (RGC) survival and axonal regeneration into peripheral nerve (PN) autografted onto the cut optic nerve of rats resistant (Fischer F344) or vulnerable (Lewis) to autoimmune disease. Circulating and tissue CsA and FK506 levels were similar in both strains. Three weeks after autologous PN transplantation the number of viable beta-III tubulin-positive RGCs was significantly greater in CsA- and FK506-treated F344 rats compared with saline-injected controls. RGC survival in Lewis rats was not significantly altered. In F344 rats, retrograde labeling of RGCs revealed that CsA or FK506 treatment significantly increased the number of RGCs that regenerated an axon into a PN autograft; however these agents had no beneficial effect on axonal regeneration in Lewis rats. PN grafts in F344 rats also contained comparatively more pan-neurofilament immunoreactive axons. In both strains, 3 weeks after transplantation CsA or FK506 treatment resulted in increased retinal macrophage numbers, but only in F344 rats was this increase significant. At this time-point PN grafts in both strains contained many macrophages and some T cells. T cell numbers in Lewis rats were significantly greater than in F344 animals. The increased RGC axonal regeneration seen in CsA- or FK506-treated F344 but not Lewis rats shows that modulation of immune responses after neurotrauma has complex and not always predictable outcomes.

Complement and myoblast transfer therapy: donor myoblast survival is enhanced following depletion of host complement C3 using cobra venom factor, but not in the absence of C5.

Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne Muscular Dystrophy and involves the injection of cultured muscle precursor cells ('myoblasts') isolated from normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT is widely accepted as being due to the immune response of the host. The role of complement as one possible mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement. Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by 3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3 increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approximately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast survival by a mechanism possibly involving activated C3 fragments.

Problems and solutions in myoblast transfer therapy.

Duchenne muscular dystrophy is a severe X-linked neuromuscular disease that affects approximately 1/3500 live male births in every human population, and is caused by a mutation in the gene that encodes the muscle protein dystrophin. The characterization and cloning of the dystrophin gene in 1987 was a major breakthrough and it was considered that simple replacement of the dystrophin gene would ameliorate the severe and progressive skeletal muscle wasting characteristic of Duchenne muscular dystrophy. After 20 years, attempts at replacing the dystrophin gene either experimentally or clinically have met with little success, but there have been many significant advances in understanding the factors that limit the delivery of a normal dystrophin gene into dystrophic host muscle. This review addresses the host immune response and donor myoblast changes underlying some of the major problems associated with myoblast-mediated dystrophin replacement, presents potential solutions, and outlines other novel therapeutic approaches.

Why do cultured transplanted myoblasts die in vivo? DNA quantification shows enhanced survival of donor male myoblasts in host mice depleted of CD4+ and CD8+ cells or Nk1.1+ cells.

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.

Immunobiology and the future of myoblast transfer therapy.

Myoblast transfer therapy (MTT) is a cell-mediated gene transfer method aimed at the restoration of normal dystrophin expression in Duchenne muscular dystrophy (DMD). Initial clinical MTT trials were conducted amid much controversy, as they were based on very few animal studies. Unfortunately, the trials were of little therapeutic benefit. As a result, there has been a renaissance of interest in experimental studies in animal models. In MTT, myoblasts are obtained by muscle biopsy from normal, i.e., dystrophin-positive, donors, expanded in culture, and injected directly into the muscles of dystrophic recipients. The major requirement for successful MTT is the survival of injected donor myoblasts in the host environment. However, a vast majority of donor cells fail to survive for more than 1 h after injection, and very few last beyond the first week. This review on the immunological aspects of MTT focuses in particular on the roles of specific components of the host immune response, the effects of tissue culture on donor cells, and strategies under development to circumvent the problem of donor myoblast death after injection in vivo.

Development and characterization of new flavivirus-resistant mouse strains bearing Flv(r)-like and Flv(mr) alleles from wild or wild-derived mice.

A single genetic locus, flavivirus resistance (Flv), controls virus titres and severity of flavivirus infection in mouse brain. It has been mapped to mouse chromosome 5 and shown to include different allelic forms. While the majority of laboratory mouse strains are susceptible to flaviviruses and carry the Flv(s) allele, wild mice and laboratory mouse strains recently derived from them are resistant and carry flavivirus-resistance alleles including Flv(r)-like and Flv(mr) alleles. Although there is a mouse model of flavivirus resistance conferred by the Flv(r) allele, other resistance alleles have not been adequately studied due to a lack of appropriate animal models. In this paper we describe the development of new flavivirus-resistant mouse strains, C3H.M.domesticus-Flv(r) and C3H.MOLD-Flv(mr), which carry the novel resistance alleles Flv(r)-like and Flv(mr) on the genetic background of flavivirus susceptible C3H/HeJ mice. The new strains were created by 10 to 11 generations of backcrossing followed by brother-sister matings resulting in a generation of homozygous founder stocks. Genome analysis of the newly developed mouse strains has revealed chromosomal regions of approximately 9 and 11 cM, respectively, encompassing Flv on chromosome 5, which are derived from resistant donor mice. These segments are much smaller than the segment of approximately 31 cM described in the congenic resistant mouse strain C3H.PRI-Flv(r) (also known as C3H/RV). The new congenic mouse strains, which were created to carry the Flv(r)-like and Flv(mr) alleles on the standardized genetic background of susceptible mice, represent new animal models of flavivirus resistance conferred by these novel resistance alleles.

Characterization of defective viral RNA produced during persistent infection of Vero cells with Murray Valley encephalitis virus.

Defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal RNA viruses. Due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. In many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. In this paper, we describe for the first time the presence of truncated or defective interfering viral RNAs produced in Vero cells persistently infected with the flavivirus Murray Valley encephalitis virus. While these RNAs have not been detected in acutely infected Vero cells, their appearance coincided with the establishment of persistent infection. We also show for the first time that the defective viral RNAs replicate well in both cell culture and cell-free virus replication systems, indicating that they may interfere with the replication of parental virus at the level of viral RNA synthesis. Significantly, structural analyses of these RNA species including nucleotide sequencing have revealed that they carry similar nucleotide deletions encompassing the genes coding for the prM and E proteins and various gene segments coding for the N terminus of the NS1 protein. These deletions are in frame, allowing the synthesis of truncated NS1 proteins to occur in persistently infected cells. This may have further implications for the interference with the parental virus at the level of viral RNA synthesis in addition to a major one at the level of virion assembly and release.

Differential transcription efficiency of two Ig VH promoters in vitro.

Each Ig variable region gene segment is transcribed from its own unique promoter. While all of these promoters share common consensus elements that contribute to the B cell-specific expression of these genes, the DNA sequence of each promoter is distinct. In this study, we have directly compared the transcription efficiencies of two murine heavy chain (VH) promoters in a murine B cell in vitro transcription system. We found that the promoters differed in both transcription efficiency and the ability to bind specific protein complexes. While some of the transcription differences may be attributed to differences in basal promoter elements, the spacing between the octamer and the heptamer consensus elements was found to be important. Others have reported a direct correlation between transcription efficiency and the probability that individual variable region gene segments will rearrange. Our studies may be of direct importance to those interested in identifying B cell-specific transcription factors and may ultimately help to explain differences in the expression of some VH gene segments.