Topical gene editing therapeutics using lipid nanoparticles: 'gene creams' for genetic skin diseases?

Patients living with inherited skin diseases have benefited from recent advances in DNA sequencing technologies that provide new or improved diagnostics. However, developing and delivering new treatments for the 'genodermatoses' remains challenging. The goal of creating topical preparations that can recover the inherent gene pathology remains largely aspirational. However, recent progress in two fields - the chemistry of topical delivery formulations (lipid nanoparticles) and the molecular biology of gene repair (CRISPR-Cas9, base and prime editing) - presents new opportunities to address this unmet need. In this review, we discuss how lipid nanoparticle delivery vehicles could be used to deliver gene-editing tools to formulate topical 'gene creams' suitable for the treatment of genodermatoses. We summarize the historical landscape of topical therapeutics and advances in gene editing that may herald an era of new therapies for patients with inherited skin disorders.

Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

Caenorhabditis elegans for rare disease modeling and drug discovery: strategies and strengths.

Although nearly 10% of Americans suffer from a rare disease, clinical progress in individual rare diseases is severely compromised by lack of attention and research resources compared to common diseases. It is thus imperative to investigate these diseases at their most basic level to build a foundation and provide the opportunity for understanding their mechanisms and phenotypes, as well as potential treatments. One strategy for effectively and efficiently studying rare diseases is using genetically tractable organisms to model the disease and learn about the essential cellular processes affected. Beyond investigating dysfunctional cellular processes, modeling rare diseases in simple organisms presents the opportunity to screen for pharmacological or genetic factors capable of ameliorating disease phenotypes. Among the small model organisms that excel in rare disease modeling is the nematode Caenorhabditis elegans. With a staggering breadth of research tools, C. elegans provides an ideal system in which to study human disease. Molecular and cellular processes can be easily elucidated, assayed and altered in ways that can be directly translated to humans. When paired with other model organisms and collaborative efforts with clinicians, the power of these C. elegans studies cannot be overstated. This Review highlights studies that have used C. elegans in diverse ways to understand rare diseases and aid in the development of treatments. With continuing and advancing technologies, the capabilities of this small round worm will continue to yield meaningful and clinically relevant information for human health.

CRISPR/Cas9 gene editing therapies for cystic fibrosis.

INTRODUCTION: Cystic fibrosis (CF) is a life-limiting genetic disorder affecting approximately 70,000 people worldwide. Current burden of treatment is high. While the latest pharmaceutical innovation has benefitted many, patients with certain genotypes remain excluded. Gene editing has the potential to correct the underlying cause of disease for all patients, representing a permanent cure.Areas covered: Various DNA editing-based strategies for treatment are currently being developed. Different strategies are called for based upon location of mutations (intronic vs. exonic), delivery mechanism of editing machinery, and cell type being targeted. Furthermore, the unique physiology of the CF lung presents a variety of barriers to delivery of CRISPR-Cas9 machinery.Expert opinion: The most significant obstacle to the use of CRISPR-Cas9 in vivo is the fact that the most clinically relevant and accessible CF tissue, the airway epithelium, is made up of non-dividing cells where precise editing via homology-directed repair (HDR) does not occur; rather, potentially deleterious imprecise editing via non-homologous end joining (NHEJ) dominates. Future research should focus on the development of either more precise NHEJ-based approaches, access to airway basal cells, editing approaches that do not involve introducing genomic double-strand breaks, and strategies with ex vivo edited cells.

Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata.

Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton.