Elizabeth M. Simpson

Introduction: Aniridia is a rare congenital vision-loss disorder primarily caused by variants in the paired box 6 ( PAX6 ) gene. Current treatments do not maintain vision long term, nor address the underlying disease mechanism. CRISPR-mediated genome editing holds great promise for treating genetic diseases but requires demonstration of efficacy on human DNA to support translational relevance. The aim of this work was to generate and test a minimally humanized mouse model of aniridia, carrying a FLAG-tagged Pax6 and the most common recurring pathogenic aniridia-patient variant, introduced for the first time in mice. Methods: Ocular assessments (gross examination, slit lamp imaging, and fluorescent and confocal microscopy) were performed in mice homozygous for humanization and heterozygous for the variant, paralleling patients. Mice were examined immediately post-wean and at adulthood, with the former chosen as a likely time for treatment initiation. As an exploratory proof-of-principle, a lipid nanoparticle-delivered adenine base editor (ABE8e) was tested to demonstrate the utility of the model. Results: Ocular assessments revealed the expected phenotypes for an aniridia mouse model, supporting successful generation and phenotypic validation of the humanized mouse. The ABE8e treatment resulted in a modest correction (4.8% ± 0.4% SEM) of the pathogenic variant in clinically relevant ex vivo cortical neurons. Discussion: Although designed for the development of genome editing therapies, this humanized mouse is also applicable to other therapies requiring interaction with human DNA or RNA. It also enables evaluation of nonsense-suppression drugs since the readthrough proteins retain the FLAG tag. More broadly, this methodology is applicable to other pathogenic variants for aniridia, CRISPRa approaches, and other genetic diseases for which CRISPR-based therapy holds promise.

Aniridia is a rare congenital vision-loss disorder that is caused primarily by heterozygous loss-of-function variants in the PAX6 gene. There is currently no curative treatment. Gene therapy has emerged as a powerful strategy for treating inherited retinal diseases. Here, we aim to establish a systemic PAX6 gene-augmentation therapy capable of addressing retinal pathologic phenotypes in a preclinical Sey mouse model of aniridia. We evaluated the intravenous delivery of FLAG-tagged PAX6 packaged in the AAV-PHP.eB capsid. Treatment was administered on postnatal day 21 to mimic the average age at diagnosis and the likely early therapeutic window in humans. The transcript levels of the virally-delivered PAX6 were 7.1% at 1 month, and 8.9% at 5 months, of Wt endogenous levels. These transcripts produced PAX6 protein in the ganglion (GCL) and inner nuclear cell layers. Importantly, in this pilot study, a significant increase in GCL thickness at 5 months post-injection was also observed. Furthermore, this structural change in the Sey retina was preceded by a significant increase in Notch1 transcription at 1-month post-injection. Thus, we demonstrated the first successful viral-mediated augmentation of PAX6 in the retina in a preclinical mouse model of aniridia, resulting in a phenotypic change at the structural and molecular levels.

Aniridia is a rare congenital-blinding disorder caused by pathogenic variants in PAX6 for which there is no vision-saving treatment. Here we aim to deliver quantitative mouse data sufficient to provide preclinical testing of therapeutic approaches to aniridia with reliable power calculations. We histologically quantify the peripheral thickness of the cornea, retina, and their sublayers in the B6129F1-Sey mouse model of aniridia, using the largest cohort of Wt and Sey mice to date (67 mice, 134 eyes). We show a highly significant thinning of the total cornea and the corneal epithelium. However, the corneal stromal-endothelial layer thickness was unaffected by Sey. Nevertheless, the epithelium and stromal-endothelial layer thickness were highly significantly correlated in Sey mice only. We also show a highly significant thinning of the total retina, and a very-to-highly, significant thinning of the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) sublayers. In the retina, we found that the GCL layer thickness was very significantly correlated with the ONL and INL thickness, in Sey mice only. Using the data generated here, we calculated the number of mice needed to demonstrate a full or partial correction of the Sey aniridic phenotype. For example, at 80% power a 50% effect will require: for the epithelium, 21 Wt and 21 Sey mice; total cornea, 58 Wt and 58 Sey mice; and total retina, 28 Wt and 28 Sey mice. We deliver quantitative mouse data, which provides preclinical research testing of therapeutic approaches with the information needed to make reliable power calculations.

Weaver syndrome is a rare neurodevelopmental disorder that encompasses macrocephaly, tall stature, obesity, brain anomalies, intellectual disability, and increased susceptibility to cancer. This dominant monogenic disorder is caused by germline variants in enhancer of zeste 2 polycomb repressive complex 2 subunit ( EZH2 ), a key epigenetic writer. Unfortunately, there are no effective treatments for Weaver syndrome. However, preclinical results support the potential for therapeutic gains, despite the prenatal onset. Thus, for the first time, we tested whether CRISPR/Cas9 gene-editing strategies may be able to “correct” a Weaver syndrome variant at the DNA level. We initiated these preclinical studies by humanizing the region surrounding the most-common recurring patient-variant location in mouse embryonic stem cells (ESCs). Humanization ensures that DNA-binding strategies will be directly translatable to human cells and patients. We then introduced into ESCs the humanized region, but now carrying the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys, and characterized the enzymatic properties of this missense variant. Our data showed a significant and dramatic reduction in EZH2-enzymatic activity, supporting previous cell-free studies of this variant as well as in vitro and in vivo mouse work by other teams. Intriguingly, this most-common variant does not create a complete loss-of-function, but rather is a hypomorphic allele. Together with prior reports describing hypomorphic effects of missense EZH2 variants, these results demonstrate that the etiology of Weaver syndrome does not require complete loss of EZH2 enzymatic activity. Toward therapy, we tested four CRISPR gene-editing strategies. We demonstrated that Streptococcus pyogenes Cas9 ( Sp Cas9) showed the highest variant correction (70.5%), but unfortunately also the highest alteration of the nonvariant allele (21.1–26.2%), an important consideration for gene-editing treatment of a dominant syndrome. However, Staphylococcus aureus Cas9 ( Sa Cas9) gave a variant correction (52.5%) that was not significantly different than Sp Cas9, and encouragingly the lowest alteration of the nonvariant allele (2.0%). Thus, the therapeutic strategy using the small Sa Cas9 enzyme, a size that allows flexibility in therapeutic delivery, was the most optimal for targeting the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys.

The TATA box-binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of the basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in human males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked dystonia–parkinsonism, a neurodegenerative disorder. However, this field has lacked a genetic mouse model of TAF1 disease to explore its mechanism in mammals and treatments. Here, we generated and validated a conditional cre-lox allele and the first ubiquitous Taf1 knockout mouse. We discovered that Taf1 deletion in male mice was embryonically lethal, which may explain why no null variants have been identified in humans. In the brains of Taf1 heterozygous female mice, no differences were found in gross structure, overall expression and protein localisation, suggesting extreme skewed X inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting that a small subset of neurons was negatively impacted by Taf1 loss. Finally, this new mouse model may be a future platform for the development of TAF1 disease therapeutics.

Introduction Aniridia is a rare congenital vision-loss disease caused by heterozygous variants in the PAX6 gene. There is no vision-saving therapy, but one exciting approach is to use CRISPR/Cas9 to permanently correct the causal genomic variants. Preclinical studies to develop such a therapy in animal models face the challenge of showing efficacy when binding human DNA. Thus, we hypothesized that a CRISPR gene therapy can be developed and optimized in humanized mouse embryonic stem cells (ESCs) that will be able to distinguish between an aniridia patient variant and nonvariant chromosome and lay the foundation for human therapy. Methods To answer the challenge of binding human DNA, we proposed the “CRISPR Humanized Minimally Mouse Models” (CHuMMMs) strategy. Thus, we minimally humanized Pax6 exon 9, the location of the most common aniridia variant c.718C > T. We generated and characterized a nonvariant CHuMMMs mouse, and a CHuMMMs cell-based disease model, in which we tested five CRISPR enzymes for therapeutic efficacy. We then delivered the therapy via lipid nanoparticles (LNPs) to alter a second variant in ex vivo cortical primary neurons. Results We successfully established a nonvariant CHuMMMs mouse and three novel CHuMMMs aniridia cell lines. We showed that humanization did not disrupt Pax6 function in vivo, as the mouse showed no ocular phenotype. We developed and optimized a CRISPR therapeutic strategy for aniridia in the in vitro system, and found that the base editor, ABE8e, had the highest correction of the patient variant at 76.8%. In the ex vivo system, the LNP-encapsulated ABE8e ribonucleoprotein (RNP) complex altered the second patient variant and rescued 24.8% Pax6 protein expression. Conclusion We demonstrated the usefulness of the CHuMMMs approach, and showed the first genomic editing by ABE8e encapsulated as an LNP-RNP. Furthermore, we laid the foundation for translation of the proposed CRISPR therapy to preclinical mouse studies and eventually patients with aniridia.

Recently safety concerns have been raised in connection with high doses of recombinant adeno-associated viruses (rAAV). Therefore, we undertook a series of experiments to test viral capsid (rAAV9 and rAAV-PHP.B), dose, and route of administration (intrastromal, intravitreal, and intravenous) focused on aniridia, a congenital blindness that currently has no cure. The success of gene therapy for aniridia may depend on the presence of functional limbal stem cells (LSCs) in the damaged aniridic corneas and whether rAAV can transduce them. Both these concerns were unknown, and thus were also addressed by our studies. For the first time, we report ataxia and lethality after intravitreal or intrastromal rAAV-PHP.B virus injections. We demonstrated virus escape from the eye and transduction of non-ocular tissues by rAAV9 and rAAV-PHP.B capsids. We have also shown that intrastromal and intravitreal delivery of rAAV9 can transduce functional LSCs, as well as all four PAX6-expressing retinal cell types in aniridic eye, respectively. Overall, lack of adverse events and successful transduction of LSCs and retinal cells makes it clear that rAAV9 is the capsid of choice for future aniridia gene therapy. Our finding of rAAV lethality after intraocular injections will be impactful for other researchers developing rAAV-based gene therapies.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 10¹¹ GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.