Molecular Therapy-Methods & Clinical Development最新文献

Mesial temporal lobe epilepsy (mTLE) is the most prevalent type of epilepsy in adults. First and subsequent generations of anti-epileptic therapy regimens fail to decrease seizures in a large number of patients suffering from mTLE, leaving surgical ablation of part of the hippocampus as the only therapeutic option to potentially reach seizure freedom. GluK2 has recently been identified as a promising target for the treatment of mTLE using gene therapy. Here, we engineered an adeno-associated virus serotype 9 vector expressing a cluster of two synthetic microRNAs (miRNAs), expressed from the human synapsin promoter, that target GRIK2 mRNA. Intra-hippocampal delivery of this vector in a mouse model of mTLE significantly reduced GRIK2 expression and daily seizure frequency. This treatment also improved the animals' health, reduced their anxiety, and restored working memory. Focal administration of the vector to the hippocampus of cynomolgus monkeys in GLP toxicology studies led to the selective transduction of hippocampal neurons with little exposure elsewhere in the brain and no transduction outside the central nervous system. Expression of miRNAs in hippocampal neurons resulted in substantially decreased GRIK2 mRNA expression. These data suggest that the intra-hippocampal delivery of a GMP-grade AAV9 coding for a synthetic miRNAs targeting GRIK2 is a promising treatment strategy for mTLE.

To achieve cell type-specific gene expression, using target cell type-tropic different adeno-associated virus (AAV) capsids is advantageous. However, their tropism across brain cell types in nonhuman primates has not been fully elucidated. We assessed the tropism of nine AAV serotype capsids (AAV1, 2, 5, 6, 7, 8, 9, rh10, and DJ) expressing enhanced green fluorescent protein (EGFP) by chicken β-actin hybrid (CBh) promoter in marmoset cerebral cortical cells. All nine AAV capsid vectors, especially AAV9 and AAVrh10, caused highly neuron-selective EGFP expression. Some AAV capsids, including AAV5, induced EGFP expression to a lesser extent in oligodendrocytes. Different ubiquitous cytomegalovirus (CMV) and CMV early enhancer/chicken β actin (CAG) promoters exhibited similar neuron-predominant transgene expression. Conversely, all nine AAV capsid vectors with the astrocyte-specific hGFA(ABC1D) promoter selectively expressed EGFP in astrocytes, except AAV5, which modestly expressed EGFP in oligodendrocytes. Oligodendrocyte-specific mouse myelin basic protein (mMBP) promoter in AAV5 vectors expressed EGFP in oligodendrocytes specifically and efficiently. The following are optimal combinations of capsids and promoters for cell type-specific expression: AAV9 or AAVrh10 and ubiquitous CBh or CMV promoter for neuron-specific transgene expression; AAV2 or AAV7 and hGFA(ABC1D) promoters for astrocyte-specific transgene expression; and AAV5 and mMBP promoters for oligodendrocyte-specific transgene expression.

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPC) but have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage Single-Stranded Virus (SSV)-seq, an NGS-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34⁺ hematopoietic stem and progenitor cells (HSPC) with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential.

Chronic kidney disease (CKD) poses a significant global health challenge, projected to become one of the leading causes of death by 2040. Current treatments primarily manage complications and slow progression, highlighting the urgent need for personalized therapies targeting the disease-causing genes. Our increased understanding on the underlying genomic changes that leads to kidney diseases coupled with recent successful gene therapies targeting specific kidney cells have turned gene therapy and genome editing into a promising therapeutic approach for treating kidney disease. This review paper will reflect on different delivery routes and system that can be exploited to target specific kidney cells, and the ways that gene therapy can be used to improve kidney health.

Therapeutic antibodies (Ab) have revolutionized the management of multiple illnesses including respiratory tract infections (RTIs). However, anti-infectious Ab displayed several limitations including antigen restrictiveness, narrowed therapeutic windows, and limited dose in the vicinity of the target when delivered by parenteral routes. Strategies enhancing further Ab-dependent containment of infection are currently needed. Here we showed that a combination of inhaled anti-infectious Ab and probiotics is an efficient formulation to protect against lung infection. Using a mouse model of Pseudomonas aeruginosa-induced pneumonia, we demonstrated a synergistic effect reducing both bacterial burden and pro-inflammatory response affording protection against primary and secondary infections. This is the first study showing that the local combination in the airways of anti-infective Ab and probiotics subverts suboptimal potency of Ab monotherapy and provides protection against respiratory pathogen.