The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.

Perfluorooctanesulfonic acid (PFOS) is a neurotoxic widespread organic contaminant which affects several brain functions including memory, motor coordination and social activity. PFOS has the ability to traverse the placenta and the blood brain barrier (BBB) and cause weight gain in female mice. It's also known that obesity and consumption of a high fat diet have negative effects on the brain, impairs cognition and increases the risk for the development of dementia. The combination effect of developmental exposure to PFOS and the intake of a high-fat diet (HFD) has not been explored. This study investigates the effect of PFOS and /or HFD on weight gain, behavior and transcriptomic and proteomic analysis of adult brain mice. We found that female mice exposed to PFOS alone showed an increase in weight, while HFD expectedly increased body weight. The combination of HFD and PFOS exacerbated generalized behavior such as time spent in the center and rearing, while PFOS alone impacted the distance travelled. These results suggest that PFOS exposure may promote hyperactivity. The combination of PFOS and HFD alter social behavior such as rearing and withdrawal. Although HFD interfered with memory retrieval, biomarkers of dementia did not change except for total Tau and phosphorylated Tau. Tau was impacted by either or both PFOS exposure and HFD. Consistent with behavioral observations, global cerebral transcriptomic analysis showed that PFOS exposure affects calcium signaling, MAPK pathways, ion transmembrane transport, and developmental processes. The combination of HFD with PFOS enhances the effect of PFOS in the brain and affects pathways related to ER stress, axon guidance and extension, and neural migration. Proteomic analysis showed that HFD enhances the impact of PFOS on inflammatory pathways, regulation of cell migration and proliferation, and MAPK signaling pathways. Overall, these data show that PFOS combined with HFD may reprogram the genome and modulate neuromotor development and may promote symptoms linked to attention deficit-hyperactivity disorders (ADHD) and autism spectrum disorders (ASD). Future work will be needed to confirm these connections.

The aging population contributes to an increase in the prevalence of neurodegenerative diseases, such as Parkinson's disease (PD). Due to the progressive nature of these diseases and an incomplete understanding of their pathophysiology, current drugs are inefficient, with a limited efficacy and major side effects. In this study, CRISPR-Cas9 RNA-proteins (RNP) composed of a Cas9 nuclease and single-guide RNA were delivered with a non-viral targeted delivery system to rescue the PD-associated phenotype in neuronal cells. Here, we fused the cell-penetrating amphipathic peptide, PepFect14 (PF14), with a short fragment of the rabies virus glycoprotein (C2) previously shown to have an affinity towards nicotinic acetylcholine receptors expressed on neuronal cells and on the blood–brain barrier. The resultant peptide, C2-PF14, was used to complex with and deliver RNPs to neuronal cells. We observed that RNP/C2-PF14 complexes formed nanosized, monodispersed, and nontoxic nanoparticles that led to a specific delivery into neuronal cells. α-Synuclein (α-syn) plays a major role in the pathology of PD and is considered to be a target for therapy. We demonstrated that CRISPR/Cas9 RNP delivered by C2-PF14 achieved α-syn gene (SNCA) editing in neuronal cells as determined by T7EI assay and western blotting. Furthermore, RNP/C2-PF14 relieved PD-associated toxicity in neuronal cells in vitro. This is a proof-of-concept towards simple and safe targeted genome-editing for treating PD and other neurological disorders.

Parkinson’s disease (PD) is a complex multifactorial disorder that is not yet fully surmised, and it is only when such a disease is tackled on multiple levels simultaneously that we should expect to see fruitful results. Gene therapy is a modern medical practice that theoretically and, so far, practically, has demonstrated its capability in joining the battle against PD and other complex disorders on most if not all fronts. This review discusses how gene therapy can efficiently replace current forms of therapy such as drugs, personalized medicine or invasive surgery. Furthermore, we discuss the importance of enhancing delivery techniques to increase the level of transduction and control of gene expression or tissue specificity. Importantly, the results of current trials establish the safety, efficacy and applicability of gene therapy for PD. Gene therapy’s variety of potential in interfering with PD’s pathology by improving basal ganglial circuitry, enhancing dopamine synthesis, delivering neuroprotection or preventing neurodegeneration may one day achieve symptomatic benefit, disease modification and eradication.

The field of Alzheimer’s disease (AD) has witnessed recent breakthroughs in the development of disease-modifying biologics and diagnostic markers. While immunotherapeutic interventions have provided much-awaited solutions, nucleic acid-based tools represent other avenues of intervention; however, these approaches are costly and invasive, and they have serious side effects. Previously, we have shown in AD animal models that tolfenamic acid (TA) can lower the expression of AD-related genes and their products and subsequently reduce pathological burden and improve cognition. Using TA as a scaffold and the zinc finger domain of SP1 as a pharmacophore, we developed safer and more potent brain-penetrating analogs that interfere with sequence-specific DNA binding at transcription start sites and predominantly modulate the expression of SP1 target genes. More importantly, the proteome of treated cells displayed ~75% of the downregulated products as SP1 targets. Specific levels of SP1-driven genes and AD biomarkers such as amyloid precursor protein (APP) and Tau proteins were also decreased as part of this targeted systemic response. These small molecules, therefore, offer a viable alternative to achieving desired therapeutic outcomes by interfering with both amyloid and Tau pathways with limited off-target systemic changes.

Background: Alzheimer’s disease (AD) is the most common form of dementia, and its prevalence continues to increase with absence of available interventions. Drug repurposing accelerates the drug discovery process as therapeutics with known safety and toxicology profiles are considered. Although the blood‐brain barrier (BBB) prevents uptake of most pharmaceuticals, our laboratory previously showed that tolfenamic acid (TA), a fenamate non‐steroidal anti‐inflammatory drug (NSAID) with known anti‐cancer effects, can cross the BBB and slow the progression of AD (Zawia et al, 2018).Method: In the present study, we test 2‐(2‐Cyanophenylthio) benzoic acid (CBA) and Niflumic acid (NA), small molecule analogs of TA that showed better safety and efficacy at targeting neurodegenerative pathways in vitro, and a similar mechanism of action to TA. We aimed to examine the effects of the analogs on in mice using proteomic and transcriptomic analyses.Single dose study of TA, CBA, and NA was carried out in C57BL/6 mice following oral administration at 100 mg/kg. Differential expression of genes and proteomic biomarkers in mice cortices were analyzed between each treatment group and a corresponding control group.Results: Analysis of the number of perturbed genes revealed that the drugs demonstrate limited off‐target effects. Bioinformatic analyses revealed that the therapeutics targeted cancer and neurodegenerative pathways including autophagy, inflammation, and neuronal trafficking. CBA and NA shared common downstream targets and pathways suggesting that they may have a similar mode of action.Conclusion: We found a set of differentially expressed genes that have been linked to processes involved in AD pathogenesis such as the production and clearance of neurotoxic Aβ peptides, tau phosphorylation, regulation of oxidative stress, and inflammatory processes. These results suggest that the drugs may influence pathways related to the pathogenesis of AD and support the potential of drug repurposing of fenamate NSAIDs such as TA, and its analogs CBA and NA, as a strategy for cancer and neurodegenerative disease therapy. Further research is needed to confirm the effect of the analogs on AD biomarkers and their efficacy as therapeutics for AD.