3 research outputs found
Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome
Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome. Keywords: AAV vector, Crigler-Najjar syndrome, UGT1A1, liver gene transfer, long-term safet
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 vectorsâAAVMYO2 and AAVMYO3âprolonged 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
Rational Design of Bisubstrate-Type Analogues as Inhibitors of DNA Methyltransferases in Cancer Cells
Aberrant DNA hypermethylation
of promoter of tumor suppressor genes
is commonly observed in cancer, and its inhibition by small molecules
is promising for their reactivation. Here we designed bisubstrate
analogues-based inhibitors, by mimicking each substrate, the <i>S</i>-adenosyl-l-methionine and the deoxycytidine,
and linking them together. This approach resulted in quinazolineâquinoline
derivatives as potent inhibitors of DNMT3A and DNMT1, some showing
certain isoform selectivity. We highlighted the importance of (i)
the nature and rigidity of the linker between the two moieties for
inhibition, as (ii) the presence of the nitrogen on the quinoline
group, and (iii) of a hydrophobic group on the quinazoline. The most
potent inhibitors induced demethylation of <i>CDKN2A</i> promoter in colon carcinoma HCT116 cells and its reactivation after
7 days of treatment. Furthermore, in a leukemia cell model system,
we found a correlation between demethylation of the promoter induced
by the treatment, chromatin opening at the promoter, and the reactivation
of a reporter gene