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.
Abstract Context For more precise, personalized care in prostate cancer (PC), a new classification based on molecular features relevant for prognostication and treatment stratification is needed. ...Genomic aberrations in the DNA damage repair pathway are common in PC, particularly in late-stage disease, and may be relevant for treatment stratification. Objective To review current knowledge on the prevalence and clinical significance of aberrations in DNA repair genes in PC, particularly in metastatic disease. Evidence acquisition A literature search up to July 2016 was conducted, including clinical trials and preclinical basic research studies. Keywords included DNA repair, BRCA, ATM, CRPC, prostate cancer, PARP, platinum, predictive biomarkers , and hereditary cancer. Evidence synthesis We review how the DNA repair pathway is relevant to prostate carcinogenesis and progression. Data on how this may be relevant to hereditary cancer and genetic counseling are included, as well as data from clinical trials of PARP inhibitors and platinum therapeutics in PC. Conclusions Relevant studies have identified genomic defects in DNA repair in PCs in 20–30% of advanced castration-resistant PC cases, a proportion of which are germline aberrations and heritable. Phase 1/2 clinical trial data, and other supporting clinical data, support the development of PARP inhibitors and DNA-damaging agents in this molecularly defined subgroup of PC following success in other cancer types. These studies may be an opportunity to improve patient care with personalized therapeutic strategies. Patient summary Key literature on how genomic defects in the DNA damage repair pathway are relevant for prostate cancer biology and clinical management is reviewed. Potential implications for future changes in patient care are discussed.
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 (dSaCas9
) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9
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 dSaCas9
and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9
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 dSaCas9
. 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.
A family of pH-responsive diblock polymers composed of poly(ethylene glycol)-b-(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate), PEG-(DMAEMA-co-BMA), was reversible ...addition–fragmentation chain transfer (RAFT) synthesized with 0–75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and in vivo bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer “50B”) in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. In vitro delivery of siRNA with polymer 50B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0B) NPs (p < 0.05), due to improved stability and a reduced rate of renal clearance. The 50B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0B (p < 0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery in vivo.
Engineering Delivery Vehicles for Genome Editing Nelson, Christopher E; Gersbach, Charles A
Annual review of chemical and biomolecular engineering,
06/2016, Volume:
7, Issue:
1
Journal Article
Peer reviewed
Open access
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.
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.
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Family caregivers of patients with chronic critical illness experience significant psychological distress.
To determine whether family informational and emotional support meetings led by palliative ...care clinicians improve family anxiety and depression.
A multicenter randomized clinical trial conducted from October 2010 through November 2014 in 4 medical intensive care units (ICUs). Adult patients (aged ≥21 years) requiring 7 days of mechanical ventilation were randomized and their family surrogate decision makers were enrolled in the study. Observers were blinded to group allocation for the measurement of the primary outcomes.
At least 2 structured family meetings led by palliative care specialists and provision of an informational brochure (intervention) compared with provision of an informational brochure and routine family meetings conducted by ICU teams (control). There were 130 patients with 184 family surrogate decision makers in the intervention group and 126 patients with 181 family surrogate decision makers in the control group.
The primary outcome was Hospital Anxiety and Depression Scale symptom score (HADS; score range, 0 best to 42 worst; minimal clinically important difference, 1.5) obtained during 3-month follow-up interviews with the surrogate decision makers. Secondary outcomes included posttraumatic stress disorder experienced by the family and measured by the Impact of Events Scale-Revised (IES-R; total score range, 0 best to 88 worst), discussion of patient preferences, hospital length of stay, and 90-day survival.
Among 365 family surrogate decision makers (mean age, 51 years; 71% female), 312 completed the study. At 3 months, there was no significant difference in anxiety and depression symptoms between surrogate decision makers in the intervention group and the control group (adjusted mean HADS score, 12.2 vs 11.4, respectively; between-group difference, 0.8 95% CI, -0.9 to 2.6; P = .34). Posttraumatic stress disorder symptoms were higher in the intervention group (adjusted mean IES-R score, 25.9) compared with the control group (adjusted mean IES-R score, 21.3) (between-group difference, 4.60 95% CI, 0.01 to 9.10; P = .0495). There was no difference between groups regarding the discussion of patient preferences (intervention, 75%; control, 83%; odds ratio, 0.63 95% CI, 0.34 to 1.16; P = .14). The median number of hospital days for patients in the intervention vs the control group (19 days vs 23 days, respectively; between-group difference, -4 days 95% CI, -6 to 3 days; P = .51) and 90-day survival (hazard ratio, 0.95 95% CI, 0.65 to 1.38, P = .96) were not significantly different.
Among families of patients with chronic critical illness, the use of palliative care-led informational and emotional support meetings compared with usual care did not reduce anxiety or depression symptoms and may have increased posttraumatic stress disorder symptoms. These findings do not support routine or mandatory palliative care-led discussion of goals of care for all families of patients with chronic critical illness.
clinicaltrials.gov Identifier: NCT01230099.
Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers ...have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.
Comprehensive genomic characterization of prostate cancer has identified recurrent alterations in genes involved in androgen signaling, DNA repair, and PI3K signaling, among others. However, larger ...and uniform genomic analysis may identify additional recurrently mutated genes at lower frequencies. Here we aggregate and uniformly analyze exome sequencing data from 1,013 prostate cancers. We identify and validate a new class of E26 transformation-specific (ETS)-fusion-negative tumors defined by mutations in epigenetic regulators, as well as alterations in pathways not previously implicated in prostate cancer, such as the spliceosome pathway. We find that the incidence of significantly mutated genes (SMGs) follows a long-tail distribution, with many genes mutated in less than 3% of cases. We identify a total of 97 SMGs, including 70 not previously implicated in prostate cancer, such as the ubiquitin ligase CUL3 and the transcription factor SPEN. Finally, comparing primary and metastatic prostate cancer identifies a set of genomic markers that may inform risk stratification.
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