An oocyte-specific Cas9-expressing mouse for germline CRISPR/Cas9-mediated genome editing.

Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.

A novel association between platelet filamin A and soluble N-ethylmaleimide sensitive factor attachment proteins regulates granule secretion.

Background and Objective: The molecular mechanisms that underpin platelet granule secretion remain poorly defined. Filamin A (FLNA) is an actin-crosslinking and signaling scaffold protein whose role in granule exocytosis has not been explored despite evidence that FLNA gene mutations confer platelet defects in humans. Methods and Results: Using platelets from platelet-specific conditional Flna-knockout mice, we showed that the loss of FLNA confers a severe defect in alpha (α)- and dense (δ)-granule exocytosis, as measured based on the release of platelet factor 4 (aka CXCL4) and adenosine triphosphate (ATP), respectively. This defect was observed following activation of both immunoreceptor tyrosine-based activation motif (ITAM) signaling by collagen-related peptide (CRP) and G protein-coupled receptor (GPCR) signaling by thrombin and the thromboxane mimetic U46619. CRP-induced spikes in intracellular calcium [Ca2+]i were impaired in FLNA-null platelets relative to controls, confirming that FLNA regulates ITAM-driven proximal signaling. In contrast, GPCR-mediated spikes in [Ca2+]i in response to thrombin and U46619 were unaffected by FLNA. Normal platelet secretion requires complexing of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptosomal-associated protein 23 (SNAP23) and syntaxin-11 (STX11). We determined that FLNA coimmunoprecipitates with both SNAP23 and STX11 upon platelet stimulation. Conclusion: FLNA regulates GPCR-driven platelet granule secretion and associates with SNAP23 and STX11 in an activation-dependent manner.

The GLP-1 agonist, exendin-4, stimulates LH secretion in female sheep.

Our previous studies showed that microinjection into the median eminence of the sheep of glucagon-like peptide- 1 (GLP-1) or its receptor agonist exendin-4 stimulates luteinising hormone (LH) secretion, but it is unknown whether the same effect may be obtained by systemic administration of the same. The present study measured the response in terms of plasma LH concentrations to intravenous (iv) infusion of exendin-4. A preliminary study showed that infusion of 2 mg exendin-4 into ewes produced a greater LH response in the follicular phase of the oestrous cycle than the luteal phase. Accordingly, the main study monitored plasma LH levels in response to either 0.5 mg or 2 mg exendin-4 or vehicle (normal saline) delivered by jugular infusion for 1 h in the follicular phase of the oestrous cycle. Blood samples were collected at 10 min intervals before, during and after infusion. Both doses of exendin-4 increased mean plasma LH concentrations and increased LH peripheral pulse amplitude. There was no effect on inter-pulse interval or timing of the preovulatory LH surge. These doses of exendin-4 did not alter plasma insulin or glucose concentrations. Quantitative PCR of the gastrointestinal tract samples from a population of ewes confirmed the expression of the preproglucagon gene (GCG). Expression increased aborally and was greatest in the rectum. It is concluded that endogenous GLP-1, most likely derived from the hindgut, may act systemically to stimulate LH secretion. The present data suggest that this effect may be obtained with levels of agonist that are lower than those functioning as an incretin.

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.

Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

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.

Targeted Expression of Retinoschisin by Retinal Bipolar Cells in XLRS Promotes Resolution of Retinoschisis Cysts Sans RS1 From Photoreceptors.

Purpose: Loss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type-specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors. Methods: Bipolar cell transgene RS1 expression was achieved using two inner retina-specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation. Results: Immunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively. Conclusions: These results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.

LNP-mediated delivery of CRISPR RNP for wide-spread in vivo genome editing in mouse cornea.

CRISPR/Cas9-based genome-editing therapies are poised to change the clinical outcome for many diseases with validated therapeutic targets awaiting an appropriate delivery system. Recent advances in lipid nanoparticle (LNP) technology make them an attractive platform for the delivery of various forms of CRISPR/Cas9, including the efficient and transient Cas9/gRNA ribonucleoprotein (RNP) complexes. In this study, we initially tested our novel LNP platform by delivering pre-complexed RNPs and template DNA to cultured mouse cortical neurons, and obtained successful ex vivo genome editing. We then directly injected LNP-packaged RNPs and DNA template into the mouse cornea to evaluate in vivo delivery. For the first time, we demonstrated wide-spread genome editing in the cornea using our LNP-RNPs. The ability of our LNPs to transfect the cornea highlights the potential of our novel delivery platform to be used in CRISPR/Cas9-based genome editing therapies of corneal diseases.

Transgene distribution and immune response after ultrasound delivery of rAAV9 and PHP.B to the brain in a mouse model of amyloidosis.

Efficient disease-modifying treatments for Alzheimer disease, the most common form of dementia, have yet to be established. Gene therapy has the potential to provide the long-term production of therapeutic in the brain following a single administration. However, the blood-brain barrier poses a challenge for gene delivery to the adult brain. We investigated the transduction efficiency and immunological response following non-invasive gene-delivery strategies to the brain of a mouse model of amyloidosis. Two emerging technologies enabling gene delivery across the blood-brain barrier were used to establish the minimal vector dosage required to reach the brain: (1) focused ultrasound combined with intravenous microbubbles, which increases the permeability of the blood-brain barrier at targeted sites and (2) the recombinant adeno-associated virus (rAAV)-based capsid named rAAV-PHP.B. We found that equal intravenous dosages of rAAV9 combined with focused ultrasound, or rAAV-PHP.B, were required for brain gene delivery. In contrast to rAAV9, focused ultrasound did not decrease the rAAV-PHP.B dosage required to transduce brain cells in a mouse model of amyloidosis. The non-invasive rAAV delivery to the brain using rAAV-PHP.B or rAAV9 with focused ultrasound triggered an immune reaction including major histocompatibility complex class II expression, complement system and microglial activation, and T cell infiltration.

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 progranulin-expressing mice as a novel tool for the development of progranulin-modulating therapeutics.

The granulin protein (also known as, and hereafter referred to as, progranulin) is a secreted glycoprotein that contributes to overall brain health. Heterozygous loss-of-function mutations in the gene encoding the progranulin protein (Granulin Precursor, GRN) are a common cause of familial frontotemporal dementia (FTD). Gene therapy approaches that aim to increase progranulin expression from a single wild-type allele, an area of active investigation for the potential treatment of GRN-dependent FTD, will benefit from the availability of a mouse model that expresses a genomic copy of the human GRN gene. Here we report the development and characterization of a novel mouse model that expresses the entire human GRN gene in its native genomic context as a single copy inserted into a defined locus (Hprt) in the mouse genome. We show that human and mouse progranulin are expressed in a similar tissue-specific pattern, suggesting that the two genes are regulated by similar mechanisms. Human progranulin rescues a phenotype characteristic of progranulin-null mice, the exaggerated and early deposition of the aging pigment lipofuscin in the brain, indicating that the two proteins are functionally similar. Longitudinal behavioural and neuropathological analyses revealed no significant differences between wild-type and human progranulin-overexpressing mice up to 18 months of age, providing evidence that long-term increase of progranulin levels is well tolerated in mice. Finally, we demonstrate that human progranulin expression can be increased in the brain using an antisense oligonucleotide that inhibits a known GRN-regulating micro-RNA, demonstrating that the transgene is responsive to potential gene therapy drugs. Human progranulin-expressing mice represent a novel and valuable tool to expedite the development of progranulin-modulating therapeutics.

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.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia.

Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.

Ferroportin-mediated iron export from vascular endothelial cells in retina and brain.

Retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD) and other neurodegenerative diseases. The retina and the brain are protected from the systemic circulation by the blood retinal barrier (BRB) and blood brain barrier (BBB), respectively. Iron levels within the retina and brain need to be tightly regulated to prevent oxidative injury. The method of iron entry through the retina and brain vascular endothelial cells (r&bVECs), an essential component of the BRB and BBB, is not fully understood. However, localization of the cellular iron exporter, ferroportin (Fpn), to the abluminal membrane of these cells, leads to the hypothesis that Fpn may play an important role in the import of iron across the BRB and BBB. To test this hypothesis, a mouse model with deletion of Fpn within the VECs in both the retina and the brain was developed through tail vein injection of AAV9-Ple261(CLDN5)-icre to both experimental Fpnf/f, and control Fpn+/+ mice at P21. Mice were aged to 9 mo and changes in retinal and brain iron distribution were observed. In vivo fundus imaging and quantitative serum iron detection were used for model validation. Eyes and brains were collected for immunofluorescence. Deletion of Fpn from the retinal and brain VECs leads to ferritin-L accumulation, an indicator of elevated iron levels, in the retinal and brain VECs. This occurred despite lower serum iron levels in the experimental mice. This result suggests that Fpn normally transfers iron from retinal and brain VECs into the retina and brain. These results help to better define the method of retina and brain iron import and will increase understanding of neurodegenerative diseases involving iron accumulation.

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.

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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.