Gene Therapy最新文献

The success of mRNA vaccines in controlling the COVID 19 pandemic has confirmed the efficacy of synthetically synthesized mRNA in humans and has also provided a blueprint on how to design them in terms of molecular structure and cost. We describe a mRNA vector that, unlike linear mRNAs used in current vaccines/therapeutics, does not require a 5' cap to function. The described mRNA vector initiates translation from an internal ribosomal entry site (IRES) and contains specially designed self-folding secondary structures (hairpins) to protect the 5' end against degradation, dramatically improving its stability. The produced mRNA did not require any additional modifications for functionality. The 5' hairpins completely inhibited cap-dependent translation, and all vectors containing them required an IRES to express protein. When this capless mRNA vector was constructed to express the full-length Influenza A membrane protein hemagglutinin (HA), complexed with pre-formed lipid-based nanoparticles, and then injected into mice as a vaccine, it generated high titers of anti-HA antibodies and protected mice against a lethal dose of Influenza A.

Multiple sclerosis (MS) is an immune-mediated chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than 2.5 million patients worldwide. Chronic demyelination in the CNS has an important role in perpetuating axonal loss and increases difficulty in promoting remyelination. Therefore, regenerative, and neuroprotective strategies are essential to overcome this impediment to rescue axonal integrity and function. Neurotrophin 3 (NT-3) has immunomodulatory and anti-inflammatory properties, in addition to its well-recognized function in nervous system development, myelination, neuroprotection, and regeneration. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of experimental autoimmune encephalomyelitis (EAE) mice, the chronic relapsing mouse model of MS, at 3 weeks post EAE induction. Measurable NT-3 levels were found in serum at 7-weeks post gene delivery. The treated cohort showed improved clinical scores and performed significantly better in rotarod, and grip strength tests compared to their untreated counterparts. Histopathologic studies showed improved remyelination and axon protection. These data correlated with reduced expression of the pro-inflammatory cytokines in brain and spinal cord, and increased percentage of regulatory T cells in the spleens and lymph nodes. Collectively, these findings demonstrate the translational potential of AAV-delivered NT-3 for chronic progressive MS.

Genetic switches that allow for precise control over transgene expression timing or levels may improve the safety and expand the use of adeno-associated viral (AAV) vector-based gene therapy technologies. We previously engineered an efficient RNA switch system that comprises a novel self-cleaving ribozyme (T3H38) and an octaguanidine dendrimer-conjugated morpholino oligonucleotide (v-M8) complementary to the ribozyme. This switch system can be used to efficiently regulate AAV-delivered transgenes with an up to 200-fold regulatory range in mice. However, this switch system has a relatively short induction half-life and only works well when v-M8 was locally but not systemically administered, representing two key limitations of the system. To address these issues, here, we tested replacing the octa-guanidine dendrimer in the v-M8 morpholino oligo with a cell-penetrating peptide (CPP). Two CPP-conjugated morpholino oligos (B-M8 and B-MSP-M8) were synthesized and compared with v-M8 for the induction of T3H38-regulated AAV-luciferase in mice. One of the CPP-conjugated oligos (B-MSP-M8) not only showed significantly improved induction half-life over that of v-M8, but also enabled efficient induction of AAV transgene expression when the oligo was systemically administered. This study improves in vivo performance and broadens the utility of the T3H38 ribozyme-based RNA switch system in gene therapy applications.

Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.

The liver is a unique organ where immunity can be biased toward ineffective response notably in the context of viral infections. Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. In the case of recombinant Adeno-Associated-Virus, used for therapeutic gene transfer, conflicting reports describe tolerance induction to different transgene products while other studies have shown conventional cytotoxic CD8⁺ T cell responses with a rapid loss of transgene expression. We performed a 1 year follow up of 6 non-human primates after all animals received an rAAV8 vector carrying the GFP transgene at doses of 7×10¹² vg/kg. We report that despite anti-GFP peripheral cellular response and loss of hepatic transgene expression, we were still able to detect persisting viral genomes in the liver until 1-year post-injection. These viral genomes were associated with liver inflammation, fibrosis and signs of CD8 T cell exhaustion, including high expression of PD-1. Our study shows that AAV8-mediated gene transfer can results to loss of transgene expression in liver and chronic inflammation several months after gene transfer.

Osteoarthritis (OA), a prevalent joint disorder, can lead to disability, with no effective treatment available. Interleukin-1 (IL-1) plays a crucial role in the progression of OA, and its receptor antagonist (IL-1Ra), a natural IL-1 inhibitor, represents a promising therapeutic target by obstructing the IL-1 signaling pathway. This study delivered IL-1Ra via adeno-associated virus (AAV), a gene therapy vector enabling long-term protein expression, to treat knee osteoarthritis (KOA) in animal models. scAAV-oIL-1Ra-I1/2 injected directly into the joint in both MMT/ACLT-induced KOA model rat improved abnormal gait (increasing footprint area and pressure), subchondral bone lesions, and significantly reduced cartilage wear and pathological scores. In the MMT-induced KOA rabbit model, weight-bearing asymmetry (indicating pain) improved after 8 weeks of scAAV-oIL-1Ra-I1/2 administration, and X-ray showed decreased K-L scores (severity grade), reduced cartilage loss, and lower pathology scores compared to untreated animals. Additionally, sex-determining region Y-type high mobility group box 9 (SOX9) was co-delivered with IL-1Ra via AAV in ACLT + MMT-induced KOA rats. The combined treatment significantly alleviated subchondral bone lesions, cartilage destruction, synovial inflammation, and pathological scores, demonstrating superior efficacy compared to either treatment administered alone. Co-delivering IL-1Ra and SOX9 inhibited IL-1 mediated inflammatory signaling, maintained cartilage homeostasis, and promoted its repair in KOA models, suggesting potential for clinical use.

The approved intravenous adeno-associated virus (AAV) therapies are limited by the widespread prevalence of pre-existing anti-AAV antibodies in the general population, which are known to restrict patients' ability to receive gene therapy and limit transfection efficacy in vivo. To address this challenge, we have developed a novel recombinant human immunoglobulin G degrading enzyme KJ103, characterized by low immunogenicity and clinical value for the elimination of anti-AAV antibodies in gene transfer. Herein, we conducted two randomized, blinded, placebo-controlled, single ascending dose Phase I studies in China and New Zealand, to evaluate the pharmacokinetics, pharmacodynamics, safety and immunogenicity of KJ103 in healthy volunteers. The results confirmed that KJ103 rapidly reduced IgG and maintained plasma IgG at low levels for one week. Dose of KJ103 ranging from 0.01 to 0.40 mg/kg had a favorable safety and tolerability profile across diverse ethnic and gender groups. KJ103 demonstrated a lower incidence of pre-existing anti-drug antibodies (ADAs) compared to currently approved human IgG degrading enzyme Imlifidase, with most induced ADAs predominantly reverting to baseline six months after administration. These properties are ideal for the management of immune disorders, rejection responses, and immunotherapies where pre-existing antibodies can reduce efficacy. Furthermore, we tested AAV2 neutralizing antibodies to confirm the potential utility of KJ103 in enhancing gene therapy.

Spinal muscular atrophy (SMA) is a progressive disease that affects motor neurons, with symptoms usually starting in infancy or early childhood. Recent breakthroughs in treatments targeting SMA have improved both lifespan and quality of life for infants and children with the disease. Given the impact of these treatments, it is essential to develop methods for managing treatment-induced changes in disease characteristics. Zolgensma® is the first effective and approved gene therapy for SMA caused by biallelic mutation in the SMN1 gene. In three children with SMA treated with Zolgensma®, neuronal, glial, inflammation, and vascular markers in the plasma exhibited a quicker response, emphasizing their potential as valuable biomarkers of treatment efficacy in clinical trials. We chose the novel Nucleic acid Linked Immuno-Sandwich Assay, to investigate a predefined panel of neuroinflammatory markers in plasma samples collected from SMA patients at baseline and six months after Zolgensma® treatment. We identified a set of novel targets whose levels differed between pre and post Zolgensma® treatment group and that were responsive to treatment. Even though our results warrant validation in larger SMA cohorts and longer follow-up time, they may pave the way for a panel of responsive proteins solidifying biomarker endpoints in SMA clinical trials.