Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

Systemic administration of a novel Beclin 1-derived peptide significantly upregulates autophagy in the spinal motor neurons of autophagy reporter mice.

Autophagy, an intracellular degradation system, plays a vital role in protecting cells by clearing damaged organelles, pathogens, and protein aggregates. Autophagy upregulation through pharmacological interventions has gained significant attention as a potential therapeutic avenue for proteinopathies. Here, we report the development of an autophagy-inducing peptide (BCN4) derived from the Beclin 1 protein, the master regulator of autophagy. To deliver the BCN4 into cells and the central nervous system (CNS), it was conjugated to our previously developed cell and blood-brain barrier-penetrating peptide (CPP). CPP-BCN4 significantly upregulated autophagy and reduced protein aggregates in motor neuron (MN)-like cells. Moreover, its systemic administration in a reporter mouse model of autophagy resulted in a significant increase in autophagy activity in the spinal MNs. Therefore, this novel autophagy-inducing peptide with a demonstrated ability to upregulate autophagy in the CNS has significant potential for the treatment of various neurodegenerative diseases with protein aggregates as a characteristic feature.

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation.

Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133-150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133-150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The in vitro analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.

Endosomal escape cell-penetrating peptides significantly enhance pharmacological effectiveness and CNS activity of systemically administered antisense oligonucleotides.

Antisense oligonucleotides (ASOs) are an emerging class of gene-specific therapeutics for diseases associated with the central nervous system (CNS). However, ASO delivery across the blood-brain barrier (BBB) to their CNS target cells remains a major challenge. Since ASOs are mainly taken up into the brain capillary endothelial cells interface through endosomal routes, entrapment in the endosomal compartment is a major obstacle for efficient CNS delivery of ASOs. Therefore, we evaluated the effectiveness of a panel of cell-penetrating peptides (CPPs) bearing several endosomal escape domains for the intracellular delivery, endosomal release and antisense activity of FDA-approved Spinraza (Nusinersen), an ASO used to treat spinal muscular atrophy (SMA). We identified a CPP, HA2-ApoE(131-150), which, when conjugated to Nusinersen, showed efficient endosomal escape capability and significantly increased the level of full-length functional mRNA of the survival motor neuron 2 (SMN2) gene in SMA patient-derived fibroblasts. Treatment of SMN2 transgenic adult mice with this CPP-PMO conjugate resulted in a significant increase in the level of full-length SMN2 in the brain and spinal cord. This work provides proof-of-principle that integration of endosomal escape domains with CPPs enables higher cytosolic delivery of ASOs, and more importantly enhances the efficiency of BBB-permeability and CNS activity of systemically administered ASOs.

Amyotrophic Lateral Sclerosis and Autophagy: Dysfunction and Therapeutic Targeting.

Over the past 20 years, there has been a drastically increased understanding of the genetic basis of Amyotrophic Lateral Sclerosis. Despite the identification of more than 40 different ALS-causing mutations, the accumulation of neurotoxic misfolded proteins, inclusions, and aggregates within motor neurons is the main pathological hallmark in all cases of ALS. These protein aggregates are proposed to disrupt cellular processes and ultimately result in neurodegeneration. One of the main reasons implicated in the accumulation of protein aggregates may be defective autophagy, a highly conserved intracellular "clearance" system delivering misfolded proteins, aggregates, and damaged organelles to lysosomes for degradation. Autophagy is one of the primary stress response mechanisms activated in highly sensitive and specialised neurons following insult to ensure their survival. The upregulation of autophagy through pharmacological autophagy-inducing agents has largely been shown to reduce intracellular protein aggregate levels and disease phenotypes in different in vitro and in vivo models of neurodegenerative diseases. In this review, we explore the intriguing interface between ALS and autophagy, provide a most comprehensive summary of autophagy-targeted drugs that have been examined or are being developed as potential treatments for ALS to date, and discuss potential therapeutic strategies for targeting autophagy in ALS.

Correlation of the Ratio of Upper Third to Lower Third Circumferences of the Chest with Obstructive Pattern in Spirometry.

BACKGROUND: Obstructive Lung Diseases (OLDs), could lead to progressive hyperinflation of the lungs that cause increased work of breathing, impaired gas exchanges and functional limitations in patients. In this study, thoracic circumference of patients in upper and lower third were measured directly and the association of the upper to lower third width of chest with spirometric parameters was evaluated. MATERIALS AND METHODS: In this cross-sectional study, five hundred twenty nine consecutive patients, with obstructive pattern in spirometry (FEV1/FEVC<70% and FEV1<80%), and 143 controls with normal spirometry were entered. Demographic and clinical data including age, sex, smoking, type, duration and severity of disease and spirometric characteristics were recorded. Upper Third circumference of Chest (UTCC) at axillary level, and Lower Third circumference of Chest (LTCC) at lower rib edge, were measured with an ordinary tape meter. Asthma Control Test (ACT) questionnaire for asthmatic and COPD Assessment Test (CAT) questionnaire in COPD patients were completed. RESULTS: We found that in patients with UTCC/LTCC ratio > 0.8, UTCC had significant correlation with FEV1 and FEV1/FVC (R: 0.069, 0.055); Moreover significant correlation was found in UTCC, LTCC and UTCC/LTCC ratio with ACT score in this subgroup (R: -0.123, -0.092, -0.124)On the other hand in patients with UTCC/LTCC ratio > 0.9, UTCC and LTCC had significant correlation with FEV1 (R: 0.07, 0.051). CONCLUSION: UTCC/LTCC ratio > 0.8, may be a predictor of obstructive pattern in patients. This is more important in some occations, for example during preoprative evaluation of a patient in an emergency conditions which there is no enough time for performing appropriate diagnostic tests such as spirometry to reveal the type and severity of obstructive pulmonary diseases.