Molecular Therapy

Heart disease affects millions of individuals and prime editing (PE) may enable curative therapies that address the underlying drivers of heart disease. Here we describe the establishment and optimization of an in vivo cardiac PE platform which mediates efficient editing in the heart with no detectable editing in the liver. We performed a proof-of-concept test on RNA binding motif protein 20 (RBM20), which if mutated, can cause dilated cardiomyopathy (DCM) in humans. Our dual-AAV based PE therapeutic rescued cardiomyopathy phenotypes in the heterozygous Rbm20R636Q mouse model. To further develop PE targeting human RBM20, we introduced a novel humanized mouse model carrying human RBM20 wildtype (WT) or R634Q mutant sequences and displaying RBM20 cardiomyopathy phenotypes. Our human RBM20 PE therapeutic efficiently corrected the pathogenic mutation and rescued phenotypes in the humanized RBM20 mouse model. Our findings demonstrate the potential of in vivo cardiac PE in treating heart disease, offer a valuable humanized DCM mouse model for developing various therapies, and present an optimized in vivo PE platform that can be adopted for targeting other organs and tissues.

The ability to efficiently place a large piece of DNA in a specific genomic location has been a goal for the gene therapy field since its inception; however, despite significant advances in gene editing technology, this had yet to be achieved. Here we describe two methods of programmable genomic integration (PGI) that overcome some of the limitations of current approaches. Using a combination of clinically validated delivery technologies (LNP, AAV), we demonstrate the ability to specifically integrate large (>2 kb) DNA sequences into endogenous introns in the liver of non-human primates (NHP). PGI was effective across multiple genomic locations and transgenes, and insertion led to expression from the endogenous promoter. PGI was highly efficient, achieving expression in >50% of liver cells after a single course of treatment, which would be curative for most monogenic recessive liver diseases. This is the first report of clinically curative level of gene insertion at endogenous loci in NHP.

Stargardt disease is a currently untreatable, inherited neurodegenerative disease that leads to macular degeneration and blindness due to loss-of-function mutations in the ABCA4 gene. We have designed a dual adeno-associated viral vector split-intein adenine base-editing strategy to correct the most common mutation in ABCA4 (c.5882G>A, p.G1961E). We optimized ABCA4 base editing in human models, including retinal organoids, iPSC-derived retinal pigment epithelial (RPE) cells, as well as adult human retinal- and RPE/choroid explants in vitro. The resulting gene therapy vectors achieved high levels of gene correction in mutation-carrying mice and in non-human primates, with an average editing of 37% of photoreceptors and 73% of RPE cells in vivo. The high editing rates in primates make way for precise and efficient gene editing in other neurodegenerative ocular diseases.

Epigenome editing technology holds great promise for treating diverse genetic disorders. While a series of advances has been made on epigenetic silencing using programmable editors, little progress has been made in leveraging epigenetic activation for therapeutic application. Here we demonstrate epigenetic activation of the LAMA1 gene for the treatment of LAMA2-CMD, a severe congenital muscle dystrophy (CMD) caused by biallelic mutations in the LAMA2 gene. LAMA1 is a sister homologue that is known to compensate for the function of LAMA2. However, supplementing LAMA1 or LAMA2 gene via viral platform is not feasible due to the large size of their coding sequences. Through a single administration of our (Adeno-associated virus) AAV vector encoding all the necessary elements for epigenetic activation, a platform termed CRISPR guide-nucleotide directed modulation (GNDM), we observed significant LAMA1 gene upregulation and phenotype improvements in DyW mice, a severe disease model of LAMA2-CMD. Notably, sustained expression of the GNDM gene and subsequent activation of the LAMA1 gene persisted beyond analyzed period of one year despite immune recognition of the GNDM protein by the host immune system. Regulatory T (Treg) cells appeared to facilitate tolerance to GNDM in the transduced muscle tissue. The muscle-tropic AAV capsid exhibited desired vector biodistribution and promising pharmacodynamics with good safety profiles in adult non-human primates (NHPs). Moreover, administration to juvenile NHPs demonstrated superior pharmacodynamics compared to adults, even at half the adult dose, suggesting safer and more effective therapeutic outcomes in mostly pediatric LAMA2-CMD patients. Our approach holds broad applicability for a range of loss-of-function genetic disorders and could offer a therapeutic breakthrough where active epigenome brings clinical benefit.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the selective deterioration of motor neurons in the central nervous system (CNS). A key driver of this pathogenesis is nuclear loss of ALS-associated protein TDP-43, leading to mis-splicing of TDP-43 targets including important neuronal genes STMN2 and UNC13A. Here, we have developed a gene therapy strategy for ALS and related TDP-43 proteinopathies, to correct mis-splicing of both STMN2 and UNC13A cryptic exons using small nuclear RNAs (snRNAs) encoded from a single vector. We identified promoter sequence elements to increase therapeutic snRNA expression by 10-fold, then further optimized the expression cassette with combinatorial snRNA targeting to rescue multiple cryptic splicing targets. The engineered snRNAs restored normal pre-mRNA processing of both STMN2 and UNC13A transcripts despite TDP-43 loss of function, rescuing stathmin-2 protein levels in iPSC derived motor neurons, restoring their axonal regeneration capacity to wild-type levels. In addition, adeno-associated virus (AAV) delivery of the snRNAs to the murine central nervous system in the constitutive cryptic splicing model Stmn2Hum{Delta}GU fully restored cortical Stmn2 pre-mRNA processing, highlighting the utility of snRNAs as a therapeutic modality in vivo. Together, this study demonstrates that snRNAs are a promising and versatile therapeutic strategy for the simultaneous correction of multiple aberrant transcripts affected by cryptic splicing in TDP-43 proteinopathies.

Mutations in HNRNPH2 cause an X-linked disorder characterized by developmental delay, intellectual disability, motor and gait disturbances, and seizures. Murine models that reproduce key clinical features of HNRNPH2-related neurodevelopmental disorder suggest that it may result from a toxic gain of function of the mutant protein or a complex loss of normal HNRNPH2 function with impaired compensation by its homolog, HNRNPH1. In this study, we tested gapmer antisense oligonucleotides (ASOs) that target murine Hnrnph2 in a non-allele-specific manner. The lead ASO reduced Hnrnph2 mRNA and protein levels while inducing compensatory upregulation of Hnrnph1 in both WT and Hnrnph2 mutant mouse brains. A single intracerebroventricular injection of the Hnrnph2 ASO into neonatal mutant Hnrnph2 mice rescued molecular and audiogenic seizure phenotypes and improved motor and cognitive functions. ASO treatment at the juvenile stage also rescued audiogenic seizures and motor deficits. In contrast, Hnrnph2 ASO administration did not improve survival, body weight, or hydrocephalus. In human iPSC-derived neurons, a human-specific HNRNPH2 research ASO reduced HNRNPH2 and upregulated HNRNPH1 mRNA levels. Mechanistically, we demonstrate that HNRNPH1 expression is regulated by alternative splicing and that HNRNPH2 modulates this process. These findings provide preclinical proof of concept for HNRNPH2 ASO therapy and offer insights into its underlying molecular mechanism. One Sentence SummaryASO-mediated Hnrnph2 knockdown induces Hnrnph1 upregulation and rescues phenotypes in a mouse model of HNRNPH2-related neurodevelopmental disorder.

Previously, we have demonstrated that ACIS KEPTIDE, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*106) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 g/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities. In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo, significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice. HighlightsO_LIACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 & 8). Intranasal inoculation of SARS-CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6) and kidney tubular cells (Fig.8). ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8). C_LIO_LIACIS KEPTIDE completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8). C_LIO_LIACIS KEPTIDE improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult. C_LIO_LIIntranasal administration of 0.05% Beta-propiolactone ({beta}PL)-inactivated SARS-CoV2 (1 *106) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation (Supplemental videos) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality. C_LIO_LIThe peptide backbone of ACIS KEPTIDE provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8). Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8) of SARS-CoV2-treated K18-hACE2 mice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/378257v1_fig2.gif" ALT="Figure 2"> View larger version (51K): org.highwire.dtl.DTLVardef@15c9911org.highwire.dtl.DTLVardef@453819org.highwire.dtl.DTLVardef@65f8a0org.highwire.dtl.DTLVardef@a602d8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2.C_FLOATNO Effect of KEPTIDE on the Expression of ACE-2 Receptor on the Membrane of CALU-3 Human Lung Cells. Human lung epithelial cells. CALU-3 cells were grown in complete DMEM cells for 2 days until it reached 70% confluency followed by starving with serum for 2 hrs. After that, 25 M of ACIS KEPTIDE were treated for 30 mins, 1 hr, 2 hrs and 6 hrs. After each time point cells were fixed and stained for ACE-2 (Green; Rabbit anti-ACE-2 antibody; Abcam; 1:250 dilution) and KEPTIDE (blue). Thirty minutes of KEPTIDE treatment significantly stimulated the internalization of ACE-2 along with KEPTIDE. Subsequent incubation periods displayed significant down-regulation of ACE-2 receptors. Experiments were confirmed after three different experiments. C_FIG O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/378257v1_fig6.gif" ALT="Figure 6"> View larger version (98K): org.highwire.dtl.DTLVardef@104bd7forg.highwire.dtl.DTLVardef@34ecc1org.highwire.dtl.DTLVardef@a3ac74org.highwire.dtl.DTLVardef@18f0570_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 6.C_FLOATNO Protective effect of ACIS KEPTIDE on the expression of ACE-2 and the entry of SARS-CoV2 virions in lung of SARS-CoV2-insulted K18-hACE2 mice. (A-D) Dual IHC staining of ACE-2 (red) and SARS-CoV2 (brown) in bronchiolar epithelium in (A) vehicle-treated (0.05% PL-inactivated VEROE6 sup, (B)virus, (C) virus + KEPTIDE, and (D) virus + peptide-treated K18-hACE2 mice (n= 7-8). (a-h) Magnified views of outer layers and inner layers of bronchiolar epithelia of respective images enclosed in a dotted squares. Arrows were justified in the bottom of each image. Results are confirmed after three independent experiments. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/378257v1_fig8.gif" ALT="Figure 8"> View larger version (87K): org.highwire.dtl.DTLVardef@ae105aorg.highwire.dtl.DTLVardef@1b39472org.highwire.dtl.DTLVardef@d6e3e9org.highwire.dtl.DTLVardef@ce6d6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 8.C_FLOATNO Protective effect of ACIS KEPTIDE on the expression of ACE-2 and prevention of SARS-CoV2 entry in kidney of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 g/kg Bwt KEPTIDE or 50 g/kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (Results are confirmed after three independent experiments. (A-D) Dual IHC of ACE-2 (red) and SARS-CoV2 (brown) in tubular epithelium in (A) vehicle, (B) Virus, (C) Virus + KEPTIDE-, and (D) Virus + peptide-treated groups. (a) showed magnified view of kidney cortex vehicle-treated mouse with basal expression of ACE-2 (red), (b) magnified view of kidney of virus-tread animal. Upscaled expression of ACE-2(red) with degenerated Bowmans capsule and invasion of SARS CoV2 (Brown arrow). (c) Magnified view of Glomerulus of Virus + KEPTIDE-treated group. Significantly less ACE-2 expression (red arrow) and no virus-infiltration were noted. (d) Elevated expression of ACE-2 in virus +Peptide-treated group. Results were confirmed after three different experiments in 7-8 animals. C_FIG O_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC="FIGDIR/small/378257v1_fig5.gif" ALT="Figure 5"> View larger version (123K): org.highwire.dtl.DTLVardef@1072b3aorg.highwire.dtl.DTLVardef@1a70702org.highwire.dtl.DTLVardef@bd227corg.highwire.dtl.DTLVardef@12335bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 5.C_FLOATNO ACIS KEPTIDE protects acute histopathological changes in Lungs of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 g/kg Bwt KEPTIDE or 50 g /kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated media); in group 2, mice (n=7 were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (A-D) Hematoxylin Background staining of small airway alveolar parenchyma. (E-H) H & E staining of Bronchiolar epithelium, surrounding cartilaginous and alveolar parenchyma. Magnified views of bronchiolar epithelia of (Ei) Control (orange arrow indicates intact epithelial lining: orange star demonstrates preserved connective tissue), (Fii) Virus only (thin red arrow indicates the degenerated bronchiolar epithelium), (Giii) Virus + KEPTIDE (thin blue arrow indicates protected epithelium), and (Hiv) Virus + Peptide-treated (thin green arrow indicates degenerated epithelium). Results are confirmed after three different experiments in 7-8 animals. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/378257v1_fig7.gif" ALT="Figure 7"> View larger version (150K): org.highwire.dtl.DTLVardef@9d3c17org.highwire.dtl.DTLVardef@d40a51org.highwire.dtl.DTLVardef@f17652org.highwire.dtl.DTLVardef@8f3230_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 7.C_FLOATNO Effect of ACIS KEPTIDE on the protection of acute histopathological changes in Kidney of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 g/kg Bwt KEPTIDE or 50 g/kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (A-D) H & E staining of kidney cortex with detailed structures of glomeruli, tubular epithelium, and Bowmans capsule in all 4 groups. Results are confirmed after three independent experiments. C_FIG

Prime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept for in vivo prime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed an in vivo prime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at the Dnmt1 locus using AAV doses of 2.5x1013 vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2 mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5x1013 vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 {micro}mol/l to below the therapeutic threshold of 600 {micro}mol/l. Our study demonstrates the feasibility of in vivo gene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.

Neural stem cells play a vital role in maintaining tissue stability and extending lifespan. Transplanting these cells to treat neurodegenerative diseases faces challenges like cellular aging, low viability, and immune rejection. We have effectively reprogrammed human fibroblasts into induced neural stem cells (iNSCs) via a single-factor miR-302a strategy, which converted skin fibroblasts into human-induced neural stem cells (hiNSCs) within 2-3 days. These cells showed delayed aging and increased resistance to oxidative stress compared to wild-type cells. Implanting them into the hippocampus of senescence-accelerated mice improved cognitive performance in severe Alzheimers, prolonged lifespan by 34%, increased fatigue resistance, and improved hair regeneration and reproductive capacity. Our findings suggest that miR-302a-hiNSCs can improve functional recovery in Alzheimers and promote healthy aging.

Point mutations in the KCNJ13 gene cause an autosomal recessive, childhood blindness, Leber congenital amaurosis (LCA16) due to a loss-of-function Kir7.1 channel. In the present study, we investigated the etiology of LCA16 caused by a KCNJ13 missense mutation (c.431T>C, p.Leu144Pro) and explored the activity of two cytosine base editors mRNAs (CBEs, BE4max-WTCas9, and evoCDA-SpCas9-NG) as a proof-of-concept therapeutic option. We observed the KCNJ13-related retinopathy phenotype in patients harboring L144P mutation. Our in-silico prediction and in vitro validation demonstrated that L144P mutation affects the channel function. We observed high on-target efficiency in the CBEs treated L144P mutant gene expressing HEK-293 cells. Strikingly, our evaluation of base editing efficacy using electrophysiology showed negligible channel function. We found that the editing bystander Cs in the protospacer region led to a missense change (L143F) in evoCDA edited cells and only silent changes in BE4max edited cells. Upon investigation of the effect of the synonymous codon, our extended analysis revealed distortion of mRNA structure, altered half-life, and/or low abundance of the cognate tRNA. We propose that KCNJ13-L144P mutation or other genes that share similar genetic complexity may be challenging to correct with the current generation of CRISPR base editors, and a combinational therapy using CRISPR base editors with a tighter editing window and requisite cognate-tRNA supplementation could be an alternative therapeutic approach to restore Kir7.1 channel function in LCA16 patients. Other options for hard-to-rescue alleles could employ homology-directed repair using CRISPR/Cas9 nucleases, Prime editing, and AAV-mediated gene augmentation.

Charcot-Marie-Tooth disease type 2A (CMT2A) is the most common axonal CMT and is associated with an early onset and severe motor-dominant phenotype. CMT2A is mainly caused by dominant mutations in the MFN2 gene, encoding Mitofusin-2, a GTPase located in the outer membrane of the mitochondria and endoplasmic reticulum (ER). Mutations in MFN2 are known to affect mitochondrial dynamics. We previously demonstrated that the mutated MFN2Arg94Gln further disrupts contacts between the ER and the mitochondria, leading to progressive axonal degeneration. There is no effective therapeutic approach to slow or reverse the progression of CMT2A, and treatments currently under development primarily focus on restoring mitochondrial function. Here, we provide proof-of-concept that neuronal overexpression of wild-type MFN2 (MFN2WT) provides therapeutic benefit in transgenic CMT2A mice carrying the mutated MFN2Arg94Gln. Intrathecal delivery of an AAV9 vector expressing MFN2WT effectively targets motor and sensory neurons, restoring ER-mitochondria contacts and mitochondrial morphology, thereby preserving both neuromuscular junction integrity and motor function. Strikingly, therapeutic efficacy is also achieved following vector injection after the onset of symptoms, rescuing the molecular hallmarks of CMT2A pathology and reversing locomotor. Notably, AAV administration was well tolerated, with no evidence neither of hepatotoxicity nor dorsal root ganglion inflammation. These results establish that boosting MFN2s levels using gene therapy is a promising therapeutic avenue for CMT2A.

TAR DNA-binding protein 43kDa (TDP-43) dysfunction is an early pathogenic mechanism that underlies amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder that lacks disease modifying therapies. We previously developed a mouse model in which TDP-43 is selectively deleted from motor neurons (ChAT-Cre;Tardbpf/f) that mimics the early stages of ALS. Here, we demonstrate that intravenous delivery of a blood-brain-barrier (BBB) permeable AAV capsid expressing our rationally designed splicing repressor CTR (AAV-PHP.eB-CTR) in symptomatic ChAT-Cre;Tardbpf/f mice markedly slowed disease progression and prevented paralysis. Systemic delivery of AAV-PHP.eB-CTR led to transduction of [~]80% of spinal motor neurons, repression of TDP-43-associated cryptic exons within motor neurons expressing CTR, and attenuation of motor neuron loss. Notably, the addition of the TARDBP 3UTR autoregulatory element to CTR maintained its expression within a physiological range. In control littermates that received AAV-PHP.eB-CTR and were monitored for >20 months, grip strength and body weight remained normal, and no histopathological abnormalities were observed, underscoring a favorable safety profile for this gene therapy. These results provide preclinical proof-of-concept that BBB-crossing AAV delivery of CTR can rescue motor neuron disease through the restoration of TDP-43 function, offering a promising mechanism-based therapeutic strategy for ALS.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C*G-to-T*A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2, reverting the exon 7 mutation via an A*T-to-G*C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.

Staufen1 (STAU1) is a multifunctional RNA binding protein that controls mRNA degradation and subcellular localization. STAU1 interacts with the ATXN2 protein, that is polyglutamine expanded in spinocerebellar ataxia type 2 (SCA2). We previously showed that STAU1 is elevated and aggregated in cells from SCA2 patients, cells from amyotrophic lateral sclerosis (ALS) patients, and in SCA2 and ALS mouse models. We also found that reduction of STAU1 abundance in vivo by genetic interaction improved motor behavior in an SCA2 mouse model, normalized the levels of several SCA2-related proteins, and reduced aggregation of polyglutamine-expanded ATXN2. Here we developed antisense oligonucleotides (ASOs) lowering STAU1 expression toward developing a therapeutic that may be effective for treating SCA2 and ALS. We performed a screen of 118 20mer phosphorothioate 2-O-methoxyethyl (MOE) ASO gapmers targeting across the STAU1 mRNA coding region for lowering STAU1 expression in HEK-293 cells. ASO hits lowering STAU1 by >45 % were rescreened in SCA2 patient fibroblasts, and 10 of these were tested for lowering STAU1 abundance in vivo in a new BAC-STAU1 mouse model. This identified efficacious ASOs targeting human STAU1 in vivo that normalized autophagy marker proteins, including ASO-45 that also targets mouse Stau1. When delivered by intracerebroventricular (ICV) injection, ASO-45 normalized autophagy markers and abnormal mRNA abundances in cerebella of ATXN2-Q127 SCA2 mice, as well as ChAT, NeuN and cleaved caspase-3 in spinal cord of Thy1- TDP-43 transgenic mice. Targeting STAU1 may be an effective strategy for treating ALS and SCA2 as well as other disorders characterized by its overabundance.

AbstractHuntingtons disease (HD) arises from the toxic gain of function caused by a CAG expansion in the coding region of the HTT gene. HD is increasingly appreciated to emerge from multiple pathogenic processes, including somatic instability in mutant HTTs (mHTT) CAG repeat tract, which leads to diverse deleterious consequences. These include the alternative processing of HTT pre-mRNA to generate the HTT1a transcript that encodes the very toxic, mHTT isoform referred to as HTT1a. We set out to compare the efficacy and safety of allele-selective lowering of mHTT compared to non-allele-selective lowering using antisense oligonucleotides (ASOs) in heterozygous HttQ111 (Q111) mice. We developed a mutant specific ASO (MutASO) targeting Htt intron-1 that selectively reduced mutant full-length HTT, as well as HTT1a, in the brains of Q111 mice. Compared to the rescue provided by a pan-allele-targeting ASO (PanASO) that lowers wild-type HTT and full-length mHTT (sparing HTT1a), the MutASO essentially eliminated aggregate formation, and provided marked protection from transcriptional dysregulation in HD knock-in mice. Thus, by targeting the ASO to the region upstream of the cryptic polyadenylation sites required to generate the HTT1a transcript, our allele-selective MutASO potently reduced HTT1a protein levels. Here, our findings advocate that HTT1a may have a disproportionate impact on aggregate formation and transcriptional dysregulation and that lowering the levels of HTT1a could provide benefit when designing HTT-lowering based therapeutic strategies for HD.

Amyotrophic lateral sclerosis 4 (ALS4) is an autosomal dominant motor neuron disease that is molecularly characterized by reduced R-loop levels and caused by pathogenic variants in senataxin (SETX). SETX encodes an RNA/DNA helicase that resolves three-stranded nucleic acid structures called R-loops. Currently, there are no disease-modifying therapies available for ALS4. Given that SETX is haplosufficient, removing the product of the mutated allele presents a potential therapeutic strategy. We designed a series of siRNAs to selectively target the RNA transcript from the ALS4 allele containing the c.1166T>C mutation (p.Leu389Ser). Transfection of HEK293 cells with siRNA and plasmids encoding either wild-type or mutant (Leu389Ser) epitope tagged SETX revealed that three siRNAs specifically reduced mutant SETX protein levels without affecting the wild-type SETX protein. In ALS4 primary fibroblasts, siRNA treatment silenced the endogenous mutant SETX allele, while sparing the wild-type allele, and restored R-loop levels in patient cells. Our findings demonstrate that mutant SETX, differing from wild-type by a single nucleotide, can be effectively and specifically silenced by RNA interference, highlighting the potential of allele-specific siRNA as a therapeutic approach for ALS4.

Usher syndrome type 1F (USH1F), resulting from mutations in the protocadherin-15 (PCDH15) gene, is characterized by congenital lack of hearing and balance, and progressive blindness in the form of retinitis pigmentosa. In this study, we explore a novel approach for USH1F gene therapy, exceeding the single AAV packaging limit by employing a dual adeno-associated virus (AAV) strategy to deliver the full-length PCDH15 coding sequence. We demonstrate the efficacy of this strategy in mouse USH1F models, effectively restoring hearing and balance in these mice. Importantly, our approach also proves successful in expressing PCDH15 in clinically relevant retinal models, including human retinal organoids and non-human primate retina, showing efficient targeting of photoreceptors and proper protein expression in the calyceal processes. This research represents a major step toward advancing gene therapy for USH1F and the multiple challenges of hearing, balance, and vision impairment.

Tauopathies, including Alzheimers disease, are neurodegenerative disorders characterized by the accumulation of microtubule-associated protein tau, which is closely linked to cognitive decline. Reduction of tau is a potential and promising strategy for addressing tau-linked brain disorders. We report the development of a therapeutic approach using adeno-associated virus mediated delivery of an artificial microRNA targeting human tau. In a tauopathy mouse model, we demonstrate that a one-time intra-cisterna magna administration of vector resulted in reduced total tau, decreased pathological tau seeds, fewer tau inclusions, and amelioration of tau-related neuropathology. Notably, intervention at late disease stages, after onset of tau deposition and neurodegeneration, improved quality of life and extended survival. We further demonstrated the durability of therapeutic benefit and defined the minimally effective dose in tauopathy mice. These findings provide preclinical support for the advancement of a vectorized tau-lowering strategy as a disease-modifying approach for tauopathies and enable progression towards an investigational new drug application.

Targeted AAV vectors are needed for safe and efficient delivery to and transduction of specific tissue target(s) in patients. Effective intravitreal delivery for retina gene therapy is not feasible with wildtype AAV. We employed directed evolution in nonhuman primates (NHP) to discover an AAV variant (R100) for intravitreal treatment of multiple target cells in the primate retina. R100 demonstrated superior transduction of human retinal cells compared to wildtype AAV. Furthermore, three R100-based gene therapeutics demonstrated safety, delivery, and durable pan-retinal expression of intracellular or secreted transgenes throughout the NHP retina following intravitreal administration. Finally, efficacy of R100-mediated delivery of therapeutic transgenes was demonstrated in patient-derived retinal cells (monogenic diseases) and in an NHP model of pathogenic retinal angiogenesis.

PROM1 is widely expressed across various tissues. However, its pathogenic mutations are exclusively associated with inherited retinal dystrophy (IRD). The mechanisms underlying this retina-specific vulnerability remain poorly understood, and no effective treatment currently exists for PROM1-IRD. Here, we utilized urine cells, hiPSCs, hiPSC-RPE cells, retinal organoids (ROs) and Prom1-/- mice to address these challenges. During photoreceptor development in ROs, PROM1 co-localized with ciliary marker ARL13B and outer segment (OS) marker PRPH2. It exhibited photoreceptor-specific mRNA splicing isoforms and unique N-glycosylation. In IRD patient-specific models with the PROM1 c.619G>T (p.E207X) homozygous mutation, we observed nonsense-mediated mRNA decay and altered splicing, leading to complete loss of PROM1 protein and OS-like structure disruption, faithfully recapitulating PROM1-IRD pathology. To rescue these defects, we engineered a photoreceptor-specific AAV7m8-CRXp-hPROM1, which successfully restored PROM1 expression and OS-like structures in patient-derived ROs. Therapeutic efficacy was further validated in Prom1-/- mice, where subretinal delivery of AAV8-CRXp-hPROM1 led to photoreceptor-specific expression of human PROM1, significantly preserving OS morphology and improving visual function. These findings not only provide the first solid preclinical evidence supporting gene therapy for PROM1-IRD, but also reveal photoreceptor-specific vulnerability to PROM1 mutations, offering a novel conceptual framework for investigating and treating related IRDs.