br CRISPR Cas Tips and Tricks

CRISPR Cas9 Tips and Tricks Dario Lupianez and Malte Spielmann (Berlin, Germany) held an engaging workshop titled CRISPR Cas9 tips and tricks. The aim was to demonstrate how this genome editing technology can be utilized in embryonic stem flt3 (ESCs) to produce genomic structural variations (SVs) in mice within ten weeks. This process would typically take a year using conventional targeting technologies, thus offering a fast alternative. SVs include deletions, inversions and duplications of genomic regions, allowing one to functionally assess gene and enhancer regions and create mouse models of human disease efficiently and with relative ease. This was exemplified by creating mouse mutants in which the Epha4 locus was manipulated to produce multiple pathogenic variations in the limb. Genomic deletions in regulatory regions resulted in brachydactyly, inversions resulted in F-syndrome (syndactyly) and duplications produced polydactyly in mice. In all cases, the mouse phenotypes recapitulated those of rare limb malformations in humans, enabling the in vivo and in vitro dissection of genetic variants seen in a clinical setting. This workshop comprised an interactive demonstration of single guide RNA (sgRNA) design, with tips on how best to maximize on-target activity with the targeting sequence while minimizing off-target activity. The pitfalls of this technology were also highlighted, including the extensive variability in targeting efficiency from one locus to another and increasing the proportion of homology-directed repair (HDR), over error-prone nonhomologous end joining (NHEJ), during precise genome-editing. Strategies to increase HDR relative to NHEJ were discussed and included the use of chemical inhibitors to target the NHEJ pathway to favor HDR. As CRISPR Cas9 technology is refined further, modeling genomic rearrangements to understand human disease will become increasingly powerful.
Antisense Therapy to Treat Spinal Muscular Atrophy Richard Finkel (Florida, USA) reported on results from a Phase II clinical trial in which antisense therapy had been used to treat infantile-onset spinal muscular atrophy (SMA). SMA is caused by a genetic defect in the SMN1 gene, leading to reduced levels of SMN, a protein required for the survival of motor neurons. This results in a variety of symptoms comprising progressive muscle weakness and feeding and breathing failure in the most severe cases. SMA is the most common genetic cause of infant death. Although SMA patients are unable to generate fully functioning SMN protein from SMN1, they all retain at least one copy of the SMN2 gene. Due to a single nucleotide mutation, the majority of SMN2 transcripts are alternatively spliced producing a truncated protein that is rapidly degraded. However, a small amount of full-length protein is produced, allowing some neurons to survive and function to some extent. One therapeutic strategy is to find splicing modulators that can promote the inclusion of Exon 7 in SMN2, usually excluded during splicing, to produce more of the fully functional SMN protein. In theory, this approach would increase the total amount of functional SMN, and thereby preserve a greater proportion of motor neurons. To test this hypothesis, companies have used a high throughput approach to test splicing modulators that are able to achieve such a result, producing functional benefit in preclinical models. In an open label Phase II study sponsored by Ionis Pharmaceuticals, 20 pre-symptomatic newborns were treated with intrathecal administration of an antisense oligonucleotide (ASO) called Nusinersen (NCT01839656). Three babies died following enrollment. However, the remaining infants continuing the trial are now >2years old, many on partial or no ventilation support, and display unprecedented improvement in motor milestones and function. These encouraging results have informed a Phase III trial in infants and children with SMA.
HPV vaccines are highly effective at preventing anogenital HPV infections and the neoplastic diseases that they cause (). Like other licensed anti-viral prophylactic vaccines, the HPV vaccines are thought to function primarily by inducing antibodies that bind the virus, thereby preventing infection (). In the case of the three licensed HPV vaccines, these antibodies are induced by antigens comprised of L1 virus-like particles (VLPs), which morphologically and immunologically resemble the outer shell of the authentic virus. Cervarix, Gardasil, and Gardasil-9 are based on VLPs of two, four, and nine HPV types, respectively. The ability of the VLPs to consistently induce robust and durable systemic virus neutralizing antibody responses after intramuscular injection almost certainly explains their high type-specific efficacy against infection in clinical trials and effectiveness in national vaccination programs. In contrast to vaccination, natural HPV infections inconsistently induce virion antibody responses, usually after a delay of several months. The responses are typically several orders of magnitude lower than after vaccination, and are detected only transiently in some individually (). The effectiveness of the antibodies induced by natural infection in preventing reinfection by the same HPV type remains controversial, although there is some evidence for protection in women, at least those with relative high titers of circulating antibodies ().