Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci.

Gene replacement therapies in genetic medicine primarily rely on adeno-associated viral (AAV) vectors for transgene expression. However, episomal expression can decline over time due to epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to the ease of access, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two high-expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.

Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy.

Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.

Longitudinal examination of perfusion and angiogenesis markers in primary colorectal tumors shows distinct signatures for metronomic and maximum-tolerated dose strategies.

Metronomic chemotherapy (MET) has been developed to address the shortcomings of maximum-tolerated chemotherapy (MTD) in regard to toxicity and development of resistance mechanisms in the tumor. In colorectal cancer (CRC), MET is a promising novel strategy to treat locally advanced malignancies when used as neoadjuvant chemotherapy (NAC). However, so far there are no preclinical studies to assess the impact of MET NAC in CRC to assess the benefits and challenges of this approach. Here, we used a primary model of CRC (via azoxymethane) to analyze longitudinal changes in angiogenesis in primary tumors under MET and MTD NAC using a combination of diffuse reflectance spectroscopy and mRNA expression (via qPCR). Our results show that MET and MTD NAC lead to increased mean tissue oxygen saturation (8% and 5%, respectively) and oxyhemoglobin (15% and 10%) between weeks 2 and 5 of NAC, and that such increases are caused by distinct molecular signatures in the angiogenic program. Specifically, we find that in the MET group there is a sustained increase in Hif-1a, Aldoa, and Pgk1 expression, suggesting upregulated glycolysis, whereas MTD NAC causes a significant reduction in the expression of the aforementioned genes and of Vegf, leading to vascular remodeling in MTD-treated tumors. Taken together, this study demonstrates the ability of combined optical and molecular methodologies to provide a holistic picture of tumor response to therapy in CRC in a minimally invasive manner.

Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models.

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.

Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells.

Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.

Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1-6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.

AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.

RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.

CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.

Genome engineering: a new approach to gene therapy for neuromuscular disorders.

For many neuromuscular disorders, including Duchenne muscular dystrophy, spinal muscular atrophy and myotonic dystrophy, the genetic causes are well known. Gene therapy holds promise for the treatment of these monogenic neuromuscular diseases, and many such therapies have made substantial strides toward clinical translation. Recently, genome engineering tools, including targeted gene editing and gene regulation, have become available to correct the underlying genetic mutations that cause these diseases. In particular, meganucleases, zinc finger nucleases, TALENs, and the CRISPR-Cas9 system have been harnessed to make targeted and specific modifications to the genome. However, for most gene therapy applications, including genome engineering, gene delivery remains the primary hurdle to clinical translation. In preclinical models, genome engineering tools have been delivered via gene-modified cells or by non-viral or viral vectors to correct a diverse array of genetic diseases. In light of the positive results of these studies, genome engineering therapies are being enthusiastically explored for several genetic neuromuscular disorders. This Review summarizes the genome engineering strategies that are currently under preclinical evaluation for the treatment of degenerative neuromuscular disorders, with a focus on the molecular tools that show the greatest potential for clinical translation of these therapies.

Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma.

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.

Local Delivery of PHD2 siRNA from ROS-Degradable Scaffolds to Promote Diabetic Wound Healing.

Small interfering RNA (siRNA) delivered from reactive oxygen species-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2, thereby increasing the expression of progrowth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

Engineering Delivery Vehicles for Genome Editing.

The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.

Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.

Technologies for controlled, local delivery of siRNA.

The discovery of RNAi in the late 1990s unlocked a new realm of therapeutic possibilities by enabling potent and specific silencing of theoretically any desired genetic target. Better elucidation of the mechanism of action, the impact of chemical modifications that stabilize and reduce nonspecific effects of siRNA molecules, and the key design considerations for effective delivery systems has spurred progress toward developing clinically-successful siRNA therapies. A logical aim for initial siRNA translation is local therapies, as delivering siRNA directly to its site of action helps to ensure that a sufficient dose reaches the target tissue, lessens the potential for off-target side effects, and circumvents the substantial systemic delivery barriers. While locally injected or topically applied siRNA has progressed into numerous clinical trials, an enormous opportunity exists to develop sustained-release, local delivery systems that enable both spatial and temporal control of gene silencing. This review focuses on material platforms that establish both localized and controlled gene silencing, with emphasis on the systems that show most promise for clinical translation.

MiRNA inhibition in tissue engineering and regenerative medicine.

MicroRNAs (miRNAs) are noncoding RNAs that provide an endogenous negative feedback mechanism for translation of messenger RNA (mRNA) into protein. Single miRNAs can regulate hundreds of mRNAs, enabling miRNAs to orchestrate robust biological responses by simultaneously impacting multiple gene networks. MiRNAs can act as master regulators of normal and pathological tissue development, homeostasis, and repair, which has motivated expanding efforts toward the development of technologies for therapeutically modulating miRNA activity for regenerative medicine and tissue engineering applications. This review highlights the tools currently available for miRNA inhibition and their recent therapeutic applications for improving tissue repair.

Dual MMP7-proximity-activated and folate receptor-targeted nanoparticles for siRNA delivery.

A dual-targeted siRNA nanocarrier has been synthesized and validated that is selectively activated in environments where there is colocalization of two breast cancer hallmarks, elevated matrix metalloproteinase (MMP) activity and folate receptor overexpression. This siRNA nanocarrier is self-assembled from two polymers containing the same pH-responsive, endosomolytic core-forming block but varying hydrophilic, corona-forming blocks. The corona block of one polymer consists of a 2 kDa PEG attached to a terminal folic acid (FA); the second polymer contains a larger (Y-shaped, 20 kDa) PEG attached to the core block by a proximity-activated targeting (PAT), MMP7-cleavable peptide. In mixed micelle smart polymer nanoparticles (SPNs) formed from the FA- and PAT-based polymers, the proteolytically removable PEG on the PAT polymers shields nonspecific SPN interactions with cells or proteins. When the PAT element is cleaved within an MMP-rich environment, the PEG shielding is removed, exposing the underlying FA and making it accessible for folate receptor-mediated SPN uptake. Characterization of mixed micelles prepared from these two polymers revealed that uptake and siRNA knockdown bioactivity of a 50% FA/50% PAT formulation was dependent on both proteolytic activation and FA receptor engagement. MMP activation and delivery of this formulation to breast cancer cells expressing the FA receptor achieved greater than 50% protein-level knockdown of a model gene with undetectable cytotoxicity. This modular nanoparticle design represents a new paradigm in cell-selective siRNA delivery and allows for stoichiometric tuning of dual-targeting components to achieve superior targeting specificity.

ROS-responsive microspheres for on demand antioxidant therapy in a model of diabetic peripheral arterial disease.

A new microparticle-based delivery system was synthesized from reactive oxygen species (ROS)-responsive poly(propylene sulfide) (PPS) and tested for "on demand" antioxidant therapy. PPS is hydrophobic but undergoes a phase change to become hydrophilic upon oxidation and thus provides a useful platform for ROS-demanded drug release. This platform was tested for delivery of the promising anti-inflammatory and antioxidant therapeutic molecule curcumin, which is currently limited in use in its free form due to poor pharmacokinetic properties. PPS microspheres efficiently encapsulated curcumin through oil-in-water emulsion and provided sustained, on demand release that was modulated in vitro by hydrogen peroxide concentration. The cytocompatible, curcumin-loaded microspheres preferentially targeted and scavenged intracellular ROS in activated macrophages, reduced in vitro cell death in the presence of cytotoxic levels of ROS, and decreased tissue-level ROS in vivo in the diabetic mouse hind limb ischemia model of peripheral arterial disease. Interestingly, due to the ROS scavenging behavior of PPS, the blank microparticles also showed inherent therapeutic properties that were synergistic with the effects of curcumin in these assays. Functionally, local delivery of curcumin-PPS microspheres accelerated recovery from hind limb ischemia in diabetic mice, as demonstrated using non-invasive imaging techniques. This work demonstrates the potential for PPS microspheres as a generalizable vehicle for ROS-demanded drug release and establishes the utility of this platform for improving local curcumin bioavailability for treatment of chronic inflammatory diseases.