Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders.

The TATA-box binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in 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 suffered from the lack of a genetic mouse model of TAF1 disease to explore mammalian mechanism and treatments. Here, we generated and validated a conditional cre-lox allele, and the first ubiquitous Taf1 knock-out mouse. We discovered that Taf1 deletion in males was embryonically lethal, which may explain why no human null-variants have been identified. In the brains of Taf1 heterozygous females, no differences were found in gross structure, overall expression, and protein localization, 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 a small subset of neurons has been negatively impacted by Taf1 loss. Finally, this new mouse may be a future platform for the development of TAF1 disease therapeutics.

OnTarget: in silico design of MiniPromoters for targeted delivery of expression.

MiniPromoters, or compact promoters, are short DNA sequences that can drive expression in specific cells and tissues. While broadly useful, they are of high relevance to gene therapy due to their role in enabling precise control of where a therapeutic gene will be expressed. Here, we present OnTarget (http://ontarget.cmmt.ubc.ca), a webserver that streamlines the MiniPromoter design process. Users only need to specify a gene of interest or custom genomic coordinates on which to focus the identification of promoters and enhancers, and can also provide relevant cell-type-specific genomic evidence (e.g. accessible chromatin regions, histone modifications, etc.). OnTarget combines the provided data with internal data to identify candidate promoters and enhancers and design MiniPromoters. To illustrate the utility of OnTarget, we designed and characterized two MiniPromoters targeting different cell populations relevant to Parkinson Disease.

ABE8e Corrects Pax6-Aniridic Variant in Humanized Mouse ESCs and via LNPs in Ex Vivo Cortical Neurons.

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.

rAAV-PHP.B escapes the mouse eye and causes lethality whereas rAAV9 can transduce aniridic corneal limbal stem cells without lethality.

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.

Intracerebroventricular Administration of AAV9-PHP.B SYN1-EmGFP Induces Widespread Transgene Expression in the Mouse and Monkey Central Nervous System.

Viral vectors made from adeno-associated virus (AAV) have emerged as preferred tools in basic and translational neuroscience research to introduce or modify genetic material in cells of interest. The use of viral vectors is particularly attractive in nontransgenic species, such as nonhuman primates. Injection of AAV solutions into the cerebrospinal fluid is an effective method to achieve a broad distribution of a transgene in the central nervous system. In this study, we conducted injections of AAV9-PHP.B, a recently described AAV capsid mutant, in the lateral ventricle of mice and rhesus macaques. To enhance the expression of the transgene (the tag protein emerald green fluorescent protein [EmGFP]), we used a gene promoter that confers high neuron-specific expression of the transgene, the human synapsin 1 (SYN1) promoter. The efficacy of the viral vector was first tested in mice. Our results show that intracerebroventricular injections of AAV9-PHP.B SYN1-EmGFP-woodchuck hepatitis virus posttranscriptional regulatory element resulted in neuronal EmGFP expression throughout the mice and monkey brains. We have provided a thorough characterization of the brain regions expressing EmGFP in both species. EmGFP was observed in neuronal cell bodies over the whole cerebral cortex and in the cerebellum, as well as in some subcortical regions, including the striatum and hippocampus. We also observed densely labeled neuropil in areas known to receive projections from these regions. Double fluorescence studies demonstrated that EmGFP was expressed by several types of neurons throughout the mouse and monkey brain. Our results demonstrate that a single injection in the lateral ventricle is an efficient method to obtain transgene expression in many cortical and subcortical regions, obviating the need of multiple intraparenchymal injections to cover large brain areas. The use of intraventricular injections of AAV9-PHP.B SYN1-EmGFP could provide a powerful approach to transduce widespread areas of the brain and may contribute to further development of methods to genetically target-specific populations of neurons.

Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells.

Small and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies. In clinical gene therapy, ophthalmic trials have been leading the field, with over 50% of ocular clinical trials using promoters that restrict expression based on cell type. Here, 19 human DNA MiniPromoters were bioinformatically designed for rAAV, tested by neonatal intravenous delivery in mouse, and successful MiniPromoters went on to be tested by intravitreal, subretinal, intrastromal, and/or intravenous delivery in adult mouse. We present promoter development as an overview for each cell type, but only show results in detail for the recommended MiniPromoters: Ple265 and Ple341 (PCP2) ON bipolar, Ple349 (PDE6H) cone, Ple253 (PITX3) corneal stroma, Ple32 (CLDN5) endothelial cells of the blood-retina barrier, Ple316 (NR2E1) Müller glia, and Ple331 (PAX6) PAX6 positive. Overall, we present a resource of new, redesigned, and improved MiniPromoters for ocular gene therapy that range in size from 784 to 2484 bp, and from weaker, equal, or stronger in strength relative to the ubiquitous control promoter smCBA. All MiniPromoters will be useful for therapies involving small regulatory RNA and DNA, and proteins ranging from 517 to 1084 amino acids, representing 62.9-90.2% of human proteins.

How do genes that escape from X-chromosome inactivation contribute to Turner syndrome?

X-chromosome inactivation generally results in dosage equivalence for expression of X-linked genes between 46,XY males and 46,XX females. The 20-30% of genes that escape silencing are thus candidates for having a role in the phenotype of Turner syndrome. Understanding which genes escape from silencing, and how they avoid this chromosome-wide inactivation is therefore an important step toward understanding Turner Syndrome. We have examined the mechanism of escape using a previously reported knock-in of a BAC containing the human escape gene RPS4X in mouse. We now demonstrate that escape from inactivation for RPS4X is already established by embryonic Day 9.5, and that both silencing and escape are faithfully maintained across the lifespan. No overt abnormalities were observed for transgenic mice up to 1 year of age despite robust transcription of the human RPS4X gene with no detectable downregulation of the mouse homolog. However, there was no significant increase in protein levels, suggesting translational compensation in the mouse. Finally, while many of the protein-coding genes have been assessed for their inactivation status, less is known about the X-linked RNA genes, and we propose that for many microRNA genes their inactivation status can be predicted as they are intronic to genes for which the inactivation status is known.

Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele.

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.

Correction: Epistasis between Pax6Sey and genetic background reinforces the value of defined hybrid mouse models for therapeutic trials.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly.

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina.

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)-based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Leber's hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.

Epistasis between Pax6Sey and genetic background reinforces the value of defined hybrid mouse models for therapeutic trials.

The small eye (Sey) mouse is a model of PAX6-aniridia syndrome (aniridia). Aniridia, a congenital ocular disorder caused by heterozygous loss-of-function mutations in PAX6, needs new vision saving therapies. However, high phenotypic variability in Sey mice makes development of such therapies challenging. We hypothesize that genetic background is a major source of undesirable variability in Sey mice. Here we performed a systematic quantitative examination of anatomical, histological, and molecular phenotypes on the inbred C57BL/6J, hybrid B6129F1, and inbred 129S1/SvImJ backgrounds. The Sey allele significantly reduced eye weight, corneal thickness, PAX6 mRNA and protein levels, and elevated blood glucose levels. Surprisingly, Pax6Sey/Sey brains had significantly elevated Pax6 transcripts compared to Pax6+/+ embryos. Genetic background significantly influenced 12/24 measurements, with inbred strains introducing severe ocular and blood sugar phenotypes not observed in hybrid mice. Additionally, significant interactions (epistasis) between Pax6 genotype and genetic background were detected in measurements of eye weight, cornea epithelial thickness and cell count, retinal mRNA levels, and blood glucose levels. The number of epistatic interactions was reduced in hybrid mice. In conclusion, severe phenotypes in the unnatural inbred strains reinforce the value of more naturalistic F1 hybrid mice for the development of therapies for aniridia and other disorders.

Human cis-acting elements regulating escape from X-chromosome inactivation function in mouse.

A long-standing question concerning X-chromosome inactivation (XCI) has been how some genes avoid the otherwise stable chromosome-wide heterochromatinization of the inactive X chromosome. As 20% or more of human X-linked genes escape from inactivation, such genes are an important contributor to sex differences in gene expression. Although both human and mouse have genes that escape from XCI, more genes escape in humans than mice, with human escape genes often clustering in larger domains than the single escape genes of mouse. Mouse models offer a well-characterized and readily manipulated system in which to study XCI, but given the differences in genes that escape it is unclear whether the mechanism of escape gene regulation is conserved. To address conservation of the process and the potential to identify elements by modelling human escape gene regulation using mouse, we integrated a human and a mouse BAC each containing an escape gene and flanking subject genes at the mouse X-linked Hprt gene. Escape-level expression and corresponding low promoter DNA methylation of human genes RPS4X and CITED1 demonstrated that the mouse system is capable of recognizing human elements and therefore can be used as a model for further refinement of critical elements necessary for escape from XCI in humans.

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

rAAV-compatible MiniPromoters for restricted expression in the brain and eye.

BACKGROUND: Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. METHODS: For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. RESULTS: The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. CONCLUSIONS: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice.

BACKGROUND: The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. RESULTS: In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. CONCLUSIONS: We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.