Viral Vectors For Gene Delivery Research Articles

Genome length determination in adeno-associated virus vectors with mass photometry

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the AAV capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (%CV <2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV <2.9%) considering a calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with a precision of %CV <0.7% and an accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their used calibration materials and efforts.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

Nano-carrier for gene delivery and bioimaging based on pentaetheylenehexamine modified carbon dots

Carbon dots (CDs) have attracted much attention due to their excellent properties and applications, especially the use for gene delivery. Considering the risks and concerns involved in the use of viral vectors for gene delivery in vivo, non-viral vectors such as CDs have gradually become an ideal alternative due to their biocompatibility and low toxicity. Therefore, in this study, the potential to apply CDs as a non-viral vector for gene delivery was investigated. The CDs were prepared using citric acid and pentaethylenehexamine (PEHA) as precursors via a one-step microwave-mediated approach. The optical, structural, and morphological properties of PEHA-derived CDs (PCDs) were characterized by ultra-violet spectroscopy (UV–vis), photoluminescence (PL), Fourier Transform Infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), zeta potential, circular dichroism spectrometry, atomic force (AFM) and transmission electron microscopies (TEM). The analysis demonstrated that the as-prepared PCDs were rich in amine groups and were positively charged. Subsequently, gel retardation assay showed that PCDs could non-covalently bind with DNA at a mass ratio of 2:1 (PCDs: DNA). Additionally, PCDs possessed a tremendously lower cytotoxicity compared with polyethylenimine (PEI), a popular precursor/dopant for many CDs preparations, and their plasmid composite showed a high transfection efficiency. Meanwhile, PCDs were also observed to cross the blood–brain barrier (BBB) by using a zebrafish model. In conclusion, these results significantly indicate that PCDs are a potential non-viral nucleic acid/gene vector to gene therapy. Also, PCDs can be utilized in drug delivery for treating brain diseases, such as Alzheimer’s disease and brain tumors.

Viral and non-viral gene therapy using 3D (bio)printing.

The overall success in launching discovered drugs is tightly restricted to the high rate of late-stage failures, which ultimately inhibits the distribution of medicines in markets. As a result, it is imperative that methods reliably predict the effectiveness and, more critically, the toxicity of medicine early in the drug development process before clinical trials be continuously innovated. We must stay up to date with the fast appearance of new infections and diseases by rapidly developing the requisite vaccinations and medicines. Modern in vitro models of disease may be used as an alternative to traditional disease models, and advanced technology can be used for the creation of pharmaceuticals as well as cells, drugs, and gene delivery systems to expedite the drug discovery procedure. Furthermore, in vitro models that mimic the spatial and chemical characteristics of native tissues, such as a 3D bioprinting system or other technologies, have proven to be more effective for drug screening than traditional 2D models. Viral and non-viral gene delivery vectors are a hopeful tool for combinatorial gene therapy, suggesting a quick way of simultaneously deliver multiple genes. A 3D bioprinting system embraces an excellent potential for gene delivery into the different cells or tissues for different diseases, in tissue engineering and regeneration medicine, in which the precise nucleic acid is located in the 3D printed tissues and scaffolds. Non-viral nanocarriers, in combination with 3D printed scaffolds, are applied to their delivery of genes and controlled release properties. There remains, however, a big obstacle in reaching the full potential of 3D models because of a lack of in vitro manufacturing of live tissues. Bioprinting advancements have made it possible to create biomimetic constructions that may be used in various drug discovery research applications. 3D bioprinting also benefits vaccinations, medicines, and relevant delivery methods because of its flexibility and adaptability. This review discusses the potential of 3D bioprinting technologies for pharmaceutical studies.

Abstract 15042: Cardiac-Enhanced Adeno-Associated Viral Vector Achieves Long-Term Transgene Expression in Transplanted Porcine Hearts When Administered During Ex Vivo Perfusion

Introduction: Use of gene therapy for cardiac transplantation is limited by the lack of an effective recombinant Adeno-associated viral vector (rAAV) gene delivery strategy. Ex vivo normothermic perfusion has been increasingly used in clinical practice and is an effective platform for achieving robust global gene delivery to a donor heart. Here we utilized a rationally designed cardiac-enhanced AAV3b variant, SASTG, to deliver the gene firefly luciferase to porcine hearts and evaluated transgene expression 30-days post-transplantation. Methods: Heterotopic heart transplantation was performed using blood-type and MHC matched Yucatan pigs (n=4). Donor hearts underwent ex vivo perfusion for 2-hours with perfusate containing the rAAV SASTG-Luciferase. Three different doses of rAAV were used: 2x10 13 , 8x10 13 , 1x10 14 viral genome copies (VGC). Recipients were survived for 30 days at which time they were euthanized and donor and recipient hearts were examined for overall levels of luciferase activity, luciferase DNA copy number, and histology for luciferase expression on immunostaining and evidence of inflammation. Results: There was a dose-dependent response in luciferase activity observed in the donor heart ventricles with the lowest activity detected at the 2x10 13 VGC dose (1.5-9.9x10 3 RLU/mg) mid-level activity at the 8x10 13 VGC dose (32.7-71.2x10 3 RLU/mg) and highest activity at the 1x10 14 VGC dose (27.6-278.1x10 3 RLU/mg). Luciferase DNA was detectable in a dose-dependent manner in the donor heart ventricles (0.02-131.1x10 3 VGC/pg of DNA), but undetectable in all native hearts. Finally, immunostaining demonstrated diffuse luciferase expression in the donor heart but none in the recipient hearts. There was also no evidence of inflammation in any of the donor hearts. Conclusions: The cardiac-enhanced AAV3b variant, SASTG, is associated with robust, global transgene expression in porcine cardiac allografts when administered during ex vivo perfusion.

Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders.

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors—AAVMYO2 and AAVMYO3—prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work...

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

The Genomics of Auditory Function and Disease.

Current estimates suggest that nearly half a billion people worldwide are affected by hearing loss. Because of the major psychological, social, economic, and health ramifications, considerable efforts have been invested in identifying the genes and molecular pathways involved in hearing loss, whether genetic or environmental, to promote prevention, improve rehabilitation, and develop therapeutics. Genomic sequencing technologies have led to the discovery of genes associated with hearing loss. Studies of the transcriptome and epigenome of the inner ear have characterized key regulators and pathways involved in the development of the inner ear and have paved the way for their use in regenerative medicine. In parallel, the immense preclinical success of using viral vectors for gene delivery in animal models of hearing loss has motivated the industry to work on translating such approaches into the clinic. Here, we review the recent advances in the genomics of auditory function and dysfunction, from patient diagnostics to epigenetics and gene therapy.

Solutions against emerging infectious and noninfectious human diseases through the application of baculovirus technologies.

Baculoviruses are insect pathogens widely used as biotechnological tools in different fields of life sciences and technologies. The particular biology of these entities (biosafety viruses 1; large circular double-stranded DNA genomes, infective per se; generally of narrow host range on insect larvae; many of the latter being pests in agriculture) and the availability of molecular-biology procedures (e.g., genetic engineering to edit their genomes) and cellular resources (availability of cell lines that grow under in vitro culture conditions) have enabled the application of baculoviruses as active ingredients in pest control, as systems for the expression of recombinant proteins (Baculovirus Expression Vector Systems—BEVS) and as viral vectors for gene delivery in mammals or to display antigenic proteins (Baculoviruses applied on mammals—BacMam). Accordingly, BEVS and BacMam technologies have been introduced in academia because of their availability as commercial systems and ease of use and have also reached the human pharmaceutical industry, as incomparable tools in the development of biological products such as diagnostic kits, vaccines, protein therapies, and—though still in the conceptual stage involving animal models—gene therapies. Among all the baculovirus species, the Autographa californica multiple nucleopolyhedrovirus has been the most highly exploited in the above utilities for the human-biotechnology field. This review highlights the main achievements (in their different stages of development) of the use of BEVS and BacMam technologies for the generation of products for infectious and noninfectious human diseases.Key points• Baculoviruses can assist as biotechnological tools in human health problems.• Vaccines and diagnosis reagents produced in the baculovirus platform are described.• The use of recombinant baculovirus for gene therapy–based treatment is reviewed.

Viral vector gene delivery: balancing safety, efficacy and dose

Thijs Gerritzen is a curious biotechnologist currently working as Principal Scientist Process Development at Amarna Therapeutics in Leiden, The Netherlands. His work focuses on the development of a high-yielding and scalable manufacturing process for replication deficient SV40-based gene therapies. Amarna Therapeutics is a Dutch biotech company, which develops next-generation gene therapies, based on a new proprietary, non-immunogenic viral vector and production platform. Dr Gerritzen holds a PhD in BioProcess Engineering from Wageningen University in a joint project with the BioProcess Engineering group and Intravacc. During his industrial PhD project, he studied the production of bioengineered bacterial outer membrane vesicles as vaccine platform. Besides working as a scientist, Thijs is a rookie dad and likes to spend his time with his family and to explore the world with his 6-month old.

Cell Fate Reprogramming in the Era of Cancer Immunotherapy.

Advances in understanding how cancer cells interact with the immune system allowed the development of immunotherapeutic strategies, harnessing patients’ immune system to fight cancer. Dendritic cell-based vaccines are being explored to reactivate anti-tumor adaptive immunity. Immune checkpoint inhibitors and chimeric antigen receptor T-cells (CAR T) were however the main approaches that catapulted the therapeutic success of immunotherapy. Despite their success across a broad range of human cancers, many challenges remain for basic understanding and clinical progress as only a minority of patients benefit from immunotherapy. In addition, cellular immunotherapies face important limitations imposed by the availability and quality of immune cells isolated from donors. Cell fate reprogramming is offering interesting alternatives to meet these challenges. Induced pluripotent stem cell (iPSC) technology not only enables studying immune cell specification but also serves as a platform for the differentiation of a myriad of clinically useful immune cells including T-cells, NK cells, or monocytes at scale. Moreover, the utilization of iPSCs allows introduction of genetic modifications and generation of T/NK cells with enhanced anti-tumor properties. Immune cells, such as macrophages and dendritic cells, can also be generated by direct cellular reprogramming employing lineage-specific master regulators bypassing the pluripotent stage. Thus, the cellular reprogramming toolbox is now providing the means to address the potential of patient-tailored immune cell types for cancer immunotherapy. In parallel, development of viral vectors for gene delivery has opened the door for in vivo reprogramming in regenerative medicine, an elegant strategy circumventing the current limitations of in vitro cell manipulation. An analogous paradigm has been recently developed in cancer immunotherapy by the generation of CAR T-cells in vivo. These new ideas on endogenous reprogramming, cross-fertilized from the fields of regenerative medicine and gene therapy, are opening exciting avenues for direct modulation of immune or tumor cells in situ, widening our strategies to remove cancer immunotherapy roadblocks. Here, we review current strategies for cancer immunotherapy, summarize technologies for generation of immune cells by cell fate reprogramming as well as highlight the future potential of inducing these unique cell identities in vivo, providing new and exciting tools for the fast-paced field of cancer immunotherapy.

Viral vector gene delivery of the novel chaperone protein SRCP1 to modify insoluble protein in in vitro and in vivo models of ALS

Protein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington’s disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1-G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

Membrane-bound MMP-14 protease-activatable adeno-associated viral vectors for gene delivery to pancreatic tumors.

Adeno-associated virus’ (AAV) relatively simple structure makes it accommodating for engineering into controllable delivery platforms. Cancer, such as pancreatic ductal adenocarcinoma (PDAC), are often characterized by upregulation of membrane-bound proteins, such as MMP-14, that propagate survival integrin signaling. In order to target tumors, we have engineered an MMP-14 protease-activatable AAV vector that responds to both membrane-bound and extracellularly active MMPs. This ‘provector’ was generated by inserting a tetra-aspartic acid inactivating motif flanked by the MMP-14 cleavage sequence IPESLRAG into the capsid subunits. The MMP-14 provector shows lower background transduction than previously developed provectors, leading to a 9.5-fold increase in transduction ability. In a murine model of PDAC, the MMP-14 provector shows increased delivery to an allograft tumor. This proof-of-concept study illustrates the possibilities of membrane-bound protease-activatable gene therapies to target tumors.

Inner Ear Gene Therapy in Mouse Models of Genetic Hearing Loss

Hearing loss is the most frequent sensory disorder affecting newborns and children. About 1 newborn in every 500 suffer from congenital hearing loss, with approximately half of these having a genetic cause. In the last few decades, the study of genetic hearing loss and related mouse models has unveiled molecular, cellular, and physiological mechanisms of the disease. In addition, effective and safe viral vectors for gene delivery to the inner ear have been generated. A growing number of approaches, including gene replacement, gene silencing, and gene editing, have proved effective in mouse models. This article briefly introduces basic strategies of gene therapy, viral vectors used and surgical methods for gene delivery, and reviews the current works on mouse modes of genetic hearing loss.

Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development.

Rare monogenic disorders such as lysosomal diseases have been at the forefront in the development of novel treatments where therapeutic options are either limited or unavailable. The increasing number of successful pre-clinical and clinical studies in the last decade demonstrates that gene therapy represents a feasible option to address the unmet medical need of these patients. This article provides a comprehensive overview of the current state of the field, reviewing the most used viral gene delivery vectors in the context of lysosomal storage disorders, a selection of relevant pre-clinical studies and ongoing clinical trials within recent years.

Reovirus directly engages integrin to recruit clathrin for entry into host cells

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Breaking viral vector bioanalysis barriers with centrifugal force

Advances in viral vector gene delivery systems, particularly adeno-associated virus (AAV) and lentivirus (LV), have accelerated the development of new cell and gene therapies. Regulatory programs for accelerated review have added to the demand for the manufacture of viral vectors, exceeding capacity and creating backlogs. Improvements in analysis speed, accuracy, precision, and dynamic range are potential targets for accelerating production timelines. Plate-based enzyme-linked immunosorbent assays (ELISAs), commonly used in analysis of viral vector titer, purity, and potency, have laborious and time-consuming manual processing drawbacks as well as long processing times and poor precision. A novel automated microfluidic, compact disc (CD)-based immunoassay format that uses centrifugal force to precisely control the flow of sample and reagents has been notably effective in accelerating bioanalysis of antibody-based therapeutics with high-precision results. One-hour assay run times and wide dynamic ranges accelerate workflows, and 10 &#181;L sample requirements minimizes consumption of limited production material. These dramatic immunoassay improvements are expected to alleviate the analytical delays in viral vector manufacturing.

Advancements in molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using the insect-cell baculovirus expression platform.

Despite rapid progress in the field, scalable high-yield production of adeno-associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC-BEVS for rAAV production. Since the first report of baculovirus-induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac-vector expression and a reduced number of baculovirus-coinfections. The latter streamlining strategy led to the eventual development of the Two-Bac, One-Bac, and Mono-Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large-scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell-specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges.

Expression of Neprilysin in Skeletal Muscle by Ultrasound-Mediated Gene Transfer (Sonoporation) Reduces Amyloid Burden for AD

Amyloid β (Aβ) accumulation in the brain is considered to be one of the major pathological changes in the progression of Alzheimer’s disease (AD). Neprilysin (NEP) is a zinc metallopeptidase that efficiently degrades Aβ. However, conventional approaches for increasing NEP levels or inducing its activation via viral-vector gene delivery have been shown to be problematic due to complications involving secondary toxicity, immune responses, and/or low gene transfer efficiency. Thus, in the present study, a physical and tractable NEP gene-delivery system via ultrasound (US) combined with microbubbles was developed for AD therapy. We introduced the plasmid, human NEP (hNEP), into skeletal muscle of 6-month-old amyloid precursor protein/presenilin-1 (APP/PS1) AD mice. Interestingly, we found a significantly reduced Aβ burden in the brain at 1 month after the delivery of overexpressed hNEP into skeletal muscle. Moreover, hNEP-treated AD mice exhibited improved performance in the Morris water maze compared to that of untreated AD mice. In addition, there were no apparent injuries in the injected muscle or in the lungs or kidneys at 1 month after the delivery of hNEP into skeletal muscle. These findings suggest that the introduction of hNEP into skeletal muscle via US represents an effective and safe therapeutic strategy for ameliorating AD-like symptoms in APP/PS1 mice, which may have the potential for clinical applications in the future.