Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

Viral Strategies for Targeting the Central and Peripheral Nervous Systems.

Recombinant viruses allow for targeted transgene expression in specific cell populations throughout the nervous system. The adeno-associated virus (AAV) is among the most commonly used viruses for neuroscience research. Recombinant AAVs (rAAVs) are highly versatile and can package most cargo composed of desired genes within the capsid's ∼5-kb carrying capacity. Numerous regulatory elements and intersectional strategies have been validated in rAAVs to enable cell type-specific expression. rAAVs can be delivered to specific neuronal populations or globally throughout the animal. The AAV capsids have natural cell type or tissue tropism and trafficking that can be modified for increased specificity. Here, we describe recently engineered AAV capsids and associated cargo that have extended the utility of AAVs in targeting molecularly defined neurons throughout the nervous system, which will further facilitate neuronal circuit interrogation and discovery.

Systemic delivery of oncolytic herpes virus using CAR-T cells enhances targeting of antitumor immuno-virotherapy.

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors.

Targeted Nanocarriers for Systemic Delivery of IRAK4 Inhibitors to Inflamed Tissues.

Persistent and uncontrolled inflammation is the root cause of various debilitating diseases. Given that interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical modulator of inflammation, inhibition of its activity with selective drug molecules (IRAK4 inhibitors) represents a promising therapeutic strategy for inflammatory disorders. To exploit the full potential of this treatment approach, drug carriers for efficient delivery of IRAK4 inhibitors to inflamed tissues are essential. Herein, the first nanoparticle-based platform for the targeted systemic delivery of a clinically tested IRAK4 inhibitor, PF-06650833, with limited aqueous solubility (57 µg mL-1 ) is presented. The developed nanocarriers increase the intrinsic aqueous dispersibility of this IRAK4 inhibitor by 40 times. A targeting peptide on the surface of nanocarriers significantly enhances their accumulation after intravenous injection in inflamed tissues of mice with induced paw edema and ulcerative colitis when compared to non-targeted counterparts. The delivered IRAK4 inhibitor markedly abates inflammation and dramatically suppresses paw edema, mitigates colitis symptoms, and reduces proinflammatory cytokine levels in the affected tissues. Importantly, repeated injections of IRAK4 inhibitor-loaded nanocarriers have no acute toxic effect on major organs of mice. Therefore, the developed nanocarriers have the potential to significantly improve the therapeutic efficacy of IRAK4 inhibitors for different inflammatory diseases.

Utilizing adeno-associated virus as a vector in treating genetic disorders or human cancers.

Clinical data from over two decades, involving more than 3000 treated patients, demonstrate that adeno-associated virus (AAV) gene therapy is a safe, effective, and well-tolerated therapeutic method. Clinical trials using AAV-mediated gene delivery to accessible tissues have led to successful treatments for numerous monogenic disorders and advancements in tissue engineering. Although the US Food and Drug Administration (FDA) has approved AAV for clinical use, systemic administration remains a significant challenge. In this review, we delve into AAV biology, focusing on current manufacturing technologies and transgene engineering strategies. We examine the use of AAVs in ongoing clinical trials for ocular, neurological, and hematological disorders, as well as cancers. By discussing recent advancements and current challenges in the field, we aim to provide valuable insights for researchers and clinicians navigating the evolving landscape of AAV-based gene therapy.

AAV-aMTD-Parkin, a therapeutic gene delivery cargo, enhances motor and cognitive functions in Parkinson's and Alzheimer's diseases.

Neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD), have a global prevalence and profoundly impact both motor and cognitive functions. Although adeno-associated virus (AAV)-based gene therapy has shown promise, its application for treating central nervous system (CNS) diseases faces several challenges, including effective delivery of AAV vectors across the blood-brain barrier, determining optimal dosages, and achieving targeted distribution. To address these challenges, we have developed a fusion delivery therapeutic cargo called AAV-aMTD-Parkin, which combines a hydrophobic cell-penetrating peptide sequence with the DNA sequences of AAV and Parkin. By employing this fusion delivery platform at lower dosages compared to zolgensma, we have achieved significant enhancements in cell and tissue permeability, while reducing the occurrence of common pathological protein aggregates. Consequently, motor and cognitive functions were restored in animal models of PD and AD. With its dual functionality in addressing PD and AD, AAV-aMTD-Parkin holds immense potential as a novel class of therapeutic biologics for prevalent CNS diseases.

Customizing biomimetic surface attributes of dendritic lipopeptide nanoplatforms for extended circulation.

The pressing demand for innovative approaches to create delivery systems with heightened drug loading and prolonged circulation has spurred numerous efforts, yielding some successes but accompanied by constraints. Our study proposes employing dendritic lipopeptide with precisely balanced opposing charges to extend blood residency for biomimetic nanoplatforms. Neutrally mixed-charged zwitterionic nanoparticles (NNPs) achieved a notable 19 % simvastatin loading content and kept stable even after one-month storage at 4 °C. These nanoplatforms demonstrated low cytotoxicity in NIH-3T3 and L02 cells and negligible hemolysis (<5 %). NNPs inhibited protein adhesion (>95 %) from positively and negatively charged sources through surface hydration. In comparison to positively charged CNPs, NNPs demonstrated an 86 % decrease in phagocytic rate by BMDMs, highlighting their efficacy. Importantly, NNPs showed prolonged circulation compared to CNPs and free simvastatin. These findings highlight the potential of this biomimetic nanoplatform for future therapeutic applications with enhanced drug loading and circulation traits.

Systemic Delivery of a STING Agonist-Loaded Positively Charged Liposome Selectively Targets Tumor Immune Microenvironment and Suppresses Tumor Angiogenesis.

Although stimulator of interferon genes (STING) agonists has shown great promise in preclinical studies, the clinical development of STING agonist therapy is challenged by its limited systemic delivery. Here, positively charged fusogenic liposomes loaded with a STING agonist (PoSTING) are designed for systemic delivery and to preferentially target the tumor microenvironment. When PoSTING is administered intravenously, it selectively targets not only tumor cells but also immune and tumor endothelial cells (ECs). In particular, delivery of STING agonists to tumor ECs normalizes abnormal tumor vasculatures, induces intratumoral STING activation, and elicits robust anti-tumor T cell immunity within the tumor microenvironment. Therefore, PoSTING can be used as a systemic delivery platform to overcome the limitations of using STING agonists in clinical trials.

Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice.

Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.

Cannabidiol for the Treatment of Brain Disorders: Therapeutic Potential and Routes of Administration.

The use of cannabidiol (CBD) for treating brain disorders has gained increasing interest. While the mechanism of action of CBD in these conditions is still under investigation, CBD has been shown to affect numerous different drug targets in the brain that are involved in brain disorders. Here we review the preclinical and clinical evidence on the potential therapeutic use of CBD in treating various brain disorders. Moreover, we also examine various drug delivery approaches that have been applied to CBD. Due to the slow absorption and low bioavailability with the current oral CBD therapy, more efficient routes of administration to bypass hepatic metabolism, particularly pulmonary delivery, should be considered. Comparison of pharmacokinetic studies of different delivery routes highlight the advantages of intranasal and inhalation drug delivery over other routes of administration (oral, injection, sublingual, buccal, and transdermal) for treating brain disorders. These two routes of delivery, being non-invasive and able to achieve fast absorption and increase bioavailability, are attracting increasing interest for CBD applications, with more research and development expected in the near future.

Poly(aspartic acid)-Based Polymeric Nanoparticle for Local and Systemic mRNA Delivery.

Recently, therapeutics based on mRNA (mRNA) have attracted significant interest for vaccines, cancer immunotherapy, and gene editing. However, the lack of biocompatible vehicles capable of delivering mRNA to the target tissue and efficiently expressing the encoded proteins impedes the development of mRNA-based therapies for a variety of diseases. Herein, we report mRNA-loaded polymeric nanoparticles based on diethylenetriamine-substituted poly(aspartic acid) that induce protein expression in the lungs and muscles following intravenous and intramuscular injections, respectively. Animal studies revealed that the amount of polyethylene glycol (PEG) on the nanoparticle surface affects the translation of the delivered mRNA into the encoded protein in the target tissue. After systemic administration, only mRNA-loaded nanoparticles modified with PEG at a molar ratio of 1:1 (PEG/polymer) induce protein expression in the lungs. In contrast, protein expression was detected only following intramuscular injection of mRNA-loaded nanoparticles with a PEG/polymer ratio of 10:1. These findings suggest that the PEG density on the surface of poly(aspartic acid)-based nanoparticles should be optimized for different delivery routes depending on the purpose of the mRNA treatment.

Local oncolytic adenovirotherapy produces an abscopal effect via tumor-derived extracellular vesicles.

Extracellular vesicles (EVs) play important roles in various intercellular communication processes. The abscopal effect is an interesting phenomenon in cancer treatment, in which immune activation is generally considered a main factor. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), and occasionally observed therapeutic effects on distal tumors after local treatment in immunodeficient mice. In this study, we hypothesized that EVs may be involved in the abscopal effect of OBP-301. EVs isolated from the supernatant of HCT116 human colon carcinoma cells treated with OBP-301 were confirmed to contain OBP-301, and they showed cytotoxic activity (apoptosis and autophagy) similar to OBP-301. In bilateral subcutaneous HCT116 and CT26 tumor models, intratumoral administration of OBP-301 produced potent antitumor effects on tumors that were not directly treated with OBP-301, involving direct mediation by tumor-derived EVs containing OBP-301. This indicates that immune activation is not the main factor in this abscopal effect. Moreover, tumor-derived EVs exhibited high tumor tropism in orthotopic HCT116 rectal tumors, in which adenovirus E1A and adenovirus type 5 proteins were observed in metastatic liver tumors after localized rectal tumor treatment. In conclusion, local treatment with OBP-301 has the potential to produce abscopal effects via tumor-derived EVs.

Mesenchymal stromal cell delivery of oncolytic immunotherapy improves CAR-T cell antitumor activity.

The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids in vitro and suppresses tumor growth in two orthotopic lung cancer models in vivo. Treatment with CAd MSCs increases the overall numbers of human T cells in vivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.

Single Micelle Vectors based on Lipid/Block Copolymer Compositions as mRNA Formulations for Efficient Cancer Immunogene Therapy.

Immunogene therapy provides a new strategy for the treatment of colorectal cancer. Compared to plasmid DNA, mRNA possesses several advantages as a therapeutic nucleic acid material and shows high potential in cancer therapy. Although efforts have been made to conquer the limited efficiency of mRNA delivery, most of the current mRNA vectors possess complex structures or compositions, which introduces additional toxicity and hinders their further clinical application. Hence, it is highly necessary to develop potent mRNA delivery systems with simple structures. Here, we report efficient mRNA delivery using the biodegradable micelle delivery system of DMP (DOTAP-mPEG-PCL). Biodegradable DMP micelles were simply prepared by the self-assembly of cationic lipid DOTAP and the diblock polymer monomethoxy poly(ethylene glycol)-poly(ε-caprolactone). With an average size of only 30 nm, we proved that these single-structured cationic micelles are highly potent in condensing and protecting mRNA molecules, with a delivery efficiency of 60.59% on C26 mouse colon cancer cells. The micelles triggered specific internalization pathways and were fully degraded in vivo. After binding with IL-22BP (interleukin-22 binding protein)-encoding mRNA, a strongly elevated IL-22BP mRNA level was detected in C26 cells. After intraperitoneal and intratumoral injection of the DMP/mIL-22BP complex, strong inhibition effects on C26 colon cancer models were observed, with high therapeutic efficiency and safety when systemically administrated. These data suggest that the DMP micelle is an advanced single-structured mRNA delivery system with high safety.

Advances in the Design of (Nano)Formulations for Delivery of Antisense Oligonucleotides and Small Interfering RNA: Focus on the Central Nervous System.

RNA-based therapeutics have emerged as one of the most powerful therapeutic options used for the modulation of gene/protein expression and gene editing with the potential to treat neurodegenerative diseases. However, the delivery of nucleic acids to the central nervous system (CNS), in particular by the systemic route, remains a major hurdle. This review will focus on the strategies for systemic delivery of therapeutic nucleic acids designed to overcome these barriers. Pathways and mechanisms of transport across the blood-brain barrier which could be exploited for delivery are described, focusing in particular on smaller nucleic acids including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). Approaches used to enhance delivery including chemical modifications, nanocarrier systems, and target selection (cell-specific delivery) are critically analyzed. Learnings achieved from a comparison of the successes and failures reported for CNS delivery of ASOs versus siRNA will help identify opportunities for a wider range of nucleic acids and accelerate the clinical translation of these innovative therapies.

Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy.

Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood-brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.

Delivering the right message: Challenges and opportunities in lipid nanoparticles-mediated modified mRNA therapeutics-An innate immune system standpoint.

mRNA molecules hold tremendous potential as a tool for gene therapy of a wide range of diseases. However, the main hurdle in implementation of mRNA for therapeutics, the systemic delivery of mRNA molecules to target cells, remains a challenge. A feasible solution for this challenge relies in the rapidly evolving field of nucleic acid-loaded nanocarriers and specifically in the established family of lipid-based nanoparticles (LNPs). Herein, we will discuss the main factors, which determine the fate of modified mRNA (mmRNA)-loaded LNPs in-vivo, and will focus on their interactions with the innate immune system as a main consideration in the design of lipid-based mmRNA delivery platforms.

Nanoparticles Containing a Combination of a Drug and an Antibody for the Treatment of Breast Cancer Brain Metastases.

In women with human epidermal growth factor 2 (HER2)-positive breast cancer, the improved control of systemic disease with new therapies has unmasked brain metastases that historically would have remained clinically silent. The efficacy of therapeutic agents against brain metastases is limited by their inability to permeate the blood-brain and blood-tumor barriers (BBB and BTB) in therapeutic amounts. Here, we investigate the potential of mucic acid-based, targeted nanoparticles designed to transcytose the BBB/BTB to deliver a small molecule drug, camptothecin (CPT), and therapeutic antibody, Herceptin, to brain metastases in mice. Treatment with BBB-targeted combination CPT/Herceptin nanoparticles significantly inhibits tumor growth compared to free CPT/Herceptin and BBB-targeted nanoparticles carrying CPT alone. Though not as efficacious, BBB-targeted nanoparticles carrying only Herceptin also elicit considerable antitumor activity. These results demonstrate the potential of the targeted nanoparticle system for the delivery of an antibody alone or in combination with other drugs across the BBB/BTB to improve the therapeutic outcome.

siRNA based drug design, quality, delivery and clinical translation.

Gene silencing by RNA interference represents a promising therapeutic approach. The development of carriers, e.g., polymers, lipids, peptides, antibodies, aptamers, small molecules, exosome and red blood cells, is crucial for the systemic delivery of siRNA. Cell-specific targeting ligands in the nano-carriers can improve the pharmacokinetics, biodistribution, and selectivity of siRNA therapeutics. The safety, effectiveness, quality and prosperity of production and manufacturing are important considerations for selecting the appropriate siRNA carriers. Efficacy of systemic delivery of siRNA requires considerations of trafficking through the blood, off-target effects, innate immune response and endosomal escape avoiding lysosomal degradation for entering into RNAi process. Multifunctional nanocarriers with stimuli-responsive properties such as pH, magnetic and photo-sensitive segments can enhance the efficacy of siRNA delivery. The improved preclinical characterization of suitable siRNA drugs, good laboratory practice, that reduce the differences between in vitro and in vivo results may increase the success of siRNA drugs in clinical settings.

Systemic delivery of factor IX messenger RNA for protein replacement therapy.

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.