Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene (HTT) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and guide RNAs (gRNAs) efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased in the liver by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion strategies in HD and potentially other repeat expansion disorders.

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene ( HTT ) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and gRNAs efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion base editing strategies in HD and potentially other repeat expansion disorders.

PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.

Huntington's disease (HD) is caused by an expanded CAG repeat in huntingtin (HTT). Since HD is dominant and loss of HTT leads to neurological abnormalities, safe therapeutic strategies require selective inactivation of mutant HTT. Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant HTT. Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to identify target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant HTT and prevented the transcription of mutant HTT mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNA-seq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these three PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population.

Prostate-specific membrane antigen targeted gold nanoparticles for prostate cancer radiotherapy: does size matter for targeted particles?

Since the introduction of PSA testing, significantly more men have been diagnosed and treated for prostate cancer. Localized prostate cancer typically is treated with prostatectomy, however there is still a high risk of recurrence after surgery, and adjuvant radiation has been shown to mitigate disease progression. X-ray therapy is frequently used as an adjuvant to treat prostate cancer, but is an imperfect tool. In this report we describe the development of a targeted-radiosensitizing nanoparticle that significantly improves X-ray therapy. Taking advantage of the demonstrated radiosensitizing activity of gold nanoparticles (AuNPs) we developed targeted AuNPs and varied both surface ligand density and AuNP size to develop an optimized AuNP for X-ray radiotherapy. We conjugated a prostate-specific membrane antigen (PSMA) targeting ligand, PSMA-1, to AuNPs and found that the targeting ligand dramatically improved gold uptake by PSMA-expressing PC3pip cells compared with PC3flu cells lacking the PSMA receptors. Further, enhancement of radiotherapy was significantly more pronounced by internalization of smaller PSMA targeted-AuNPs. Our studies provide a foundation for design of size-selected AuNPs for targeted radiotherapy and, for the first time, systematically investigate both the effect of ligand and AuNP size on the cell uptake, tumor targeting and radiotherapy efficacy.

Targeted Gold Nanocluster-Enhanced Radiotherapy of Prostate Cancer.

For over a hundred years, X-rays have been a main component of the radiotherapeutic approaches to treat cancer. Yet, to date, no radiosensitizer has been developed to selectively target prostate cancer. Gold has excellent X-ray absorptivity and is used as a radiotherapy enhancing material. In this work, ultrasmall Au25 nanoclusters (NCs) are developed for selective prostate cancer targeting, radiotherapy enhancement, and rapid clearance from the body. Targeted-Au25 NCs are rapidly and selectively taken up by prostate cancer in vitro and in vivo and also have fast renal clearance. When combined with X-ray irradiation of the targeted cancer tissues, radiotherapy is significantly enhanced. The selective targeting and rapid clearance of the nanoclusters may allow reductions in radiation dose, decreasing exposure to healthy tissue and making them highly attractive for clinical translation.

Association between TDP-43 and mitochondria in inclusion body myositis.

Inclusion body myositis (IBM) is the most common cause of primary myopathy in individuals aged 50 years and over, and is pathologically characterized by protein aggregates of p62 and mislocalized cytoplasmic TDP-43, as well as mitochondrial abnormalities in affected muscle fibers. Our recent studies have shown the accumulation of TDP-43 in mitochondria in neurons from patients with amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), and revealed mitochondria as critical mediators of TDP-43 neurotoxicity. In this study, we investigated the association between mitochondria and TDP-43 in biopsied skeletal muscle samples from IBM patients. We found that IBM pathological markers TDP-43, phosphorylated TDP-43, and p62 all coexisted with intensively stained key subunits of mitochondrial oxidative phosphorylation complexes I-V in the same skeletal muscle fibers of patients with IBM. Further immunoblot analysis showed increased levels of TDP-43, truncated TDP-43, phosphorylated TDP-43, and p62, but decreased levels of key subunits of mitochondrial oxidative phosphorylation complexes I and III in IBM patients compared to aged matched control subjects. This is the first demonstration of the close association of TDP-43 accumulation with mitochondria in degenerating muscle fibers in IBM and this association may contribute to the development of mitochondrial dysfunction and pathological protein aggregates.

Mitofusin 2 Regulates Axonal Transport of Calpastatin to Prevent Neuromuscular Synaptic Elimination in Skeletal Muscles.

Skeletal muscles undergo atrophy in response to diseases and aging. Here we report that mitofusin 2 (Mfn2) acts as a dominant suppressor of neuromuscular synaptic loss to preserve skeletal muscles. Mfn2 is reduced in spinal cords of transgenic SOD1G93A and aged mice. Through preserving neuromuscular synapses, increasing neuronal Mfn2 prevents skeletal muscle wasting in both SOD1G93A and aged mice, whereas deletion of neuronal Mfn2 produces neuromuscular synaptic dysfunction and skeletal muscle atrophy. Neuromuscular synaptic loss after sciatic nerve transection can also be alleviated by Mfn2. Mfn2 coexists with calpastatin largely in mitochondria-associated membranes (MAMs) to regulate its axonal transport. Genetic inactivation of calpastatin abolishes Mfn2-mediated protection of neuromuscular synapses. Our results suggest that, as a potential key component of a novel and heretofore unrecognized mechanism of cytoplasmic protein transport, Mfn2 may play a general role in preserving neuromuscular synapses and serve as a common therapeutic target for skeletal muscle atrophy.