[Gene-based therapies of spinal muscular atrophy: a piece of history of medicine]. / Thérapies géniques de l'amyotrophie spinale infantile - Un morceau d'histoire de la médecine.

It is worth stating that a generation is needed to bring about a new family of drugs. After the deciphering of the genetic cause in 1995, two innovative classes of therapeutics are now available for spinal muscular atrophy (SMA): the repeated administration of antisens oligonucleotides and the one-shot administration of a scAAV9-SMN as a gene therapy. By addressing the genetic mechanisms of the disease, these drugs fundamentally change its course. These major advances in an extremely severe disease, often fatal before the age of 18 months in the type 1 form (50% of patients), pave the way for the treatment of other serious pathologies of the nervous or neuromuscular system, and provide unambiguous evidence of the effectiveness of these new classes of drugs called to address a number of genetic or acquired diseases. These breakthroughs raise also new scientific and technological questions (limited production yields of gene therapy drugs) but also ethical issues (access of patients to these innovative therapies) that resonate beyond this disease alone.

TITLE: Thérapies géniques de l'amyotrophie spinale infantile - Un morceau d'histoire de la médecine. ABSTRACT: On convient de dire qu'une génération est nécessaire pour faire émerger une nouvelle famille de médicaments. L'amyotrophie spinale infantile (SMA), après l'élucidation du gène causal en 1995, dispose depuis peu de deux classes innovantes de thérapeutiques : l'administration répétée d'oligonucléotides antisens et l'administration unique d'une thérapie génique par scAAV9-SMN. En s'adressant aux mécanismes génétiques de la maladie, elles en modifient fondamentalement le cours. Ces avancées majeures dans une maladie extrêmement sévère, mortelle souvent avant l'âge de 18 mois dans les formes de type 1 (50 % des malades), ouvrent la voie pour d'autres pathologies graves du système nerveux ou neuromusculaire, et apportent une preuve déterminante de l'efficacité de ces classes nouvelles de produits appelés à s'adresser à de nombreuses maladies génétiques ou acquises. Elles génèrent aussi de nouvelles questions d'ordre scientifique et technologique (capacités limitées de production des quantités nécessaires en thérapie génique) mais également d'ordre éthique (conditions d'accès des malades à ces thérapies innovantes), qui résonnent au-delà de cette seule maladie.

Gene Therapy for Retinal Degeneration.

Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.

Vector production in an academic environment: a tool to assess production costs.

Generating gene and cell therapy products under good manufacturing practices is a complex process. When determining the cost of these products, researchers must consider the large number of supplies used for manufacturing and the personnel and facility costs to generate vector and maintain a cleanroom facility. To facilitate cost estimates, the Indiana University Vector Production Facility teamed with the Indiana University Kelley School of Business to develop a costing tool that, in turn, provides pricing. The tool is designed in Microsoft Excel and is customizable to meet the needs of other core facilities. It is available from the National Gene Vector Biorepository. The tool allows cost determinations using three different costing methods and was developed in an effort to meet the A21 circular requirements for U.S. core facilities performing work for federally funded projects. The costing tool analysis reveals that the cost of vector production does not have a linear relationship with batch size. For example, increasing the production from 9 to18 liters of a retroviral vector product increases total costs a modest 1.2-fold rather than doubling in total cost. The analysis discussed in this article will help core facilities and investigators plan a cost-effective strategy for gene and cell therapy production.

A novel adenovirus vector for easy cloning in the E3 region downstream of the CMV promoter.

The construction of expression vectors derived from the human adenovirus type 5 (Ad5), usually based on homologous recombination, is time consuming as a shuttle plasmid has to be selected before recombination with the viral genome. Here, we describe a method allowing direct cloning of a transgene in the E3 region of the Ad5 genome already containing the immediate early CMV promoter upstream of three unique restriction sites. This allowed the construction of recombinant adenoviral genomes in just one step, reducing considerably the time of selection and, of course, production of the corresponding vectors. Using this vector, we produced recombinant adenoviruses, each giving high-level expression of the transgene in the transduced cells.

Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system.

BACKGROUND: The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined. METHODS: The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale. RESULTS: An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter). CONCLUSION: These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization.