In gene therapy, viruses are used as vectors to deliver genes into cells. In the case of an inherited disease, where a mutated gene causes production of an abnormal protein or even completely disables the production of a protein, the goal of gene therapy is to correct the underlying defect by introducing a functional copy of the gene into the patient’s own cells. While multiple viruses can be used as vectors, adeno-associated virus (AAV) is especially well suited for treating diseases of the retina that lead to severe vision loss or blindness. AAV is a small, non enveloped and non-replicating virus with only two native genes, which makes it easy to work with from a vector-engineering standpoint. AAV elicits only a low immune response and has never been linked to disease in humans, and to date, AAV vectors have been safe for use in gene therapy. Engineered, or recombinant, AAV vectors have no viral genes remaining, virtually eliminating the possibility that any viral genes will cause an adverse event in a patient.
Until recently, there was a lack of manufacturing infrastructure to support the reliable and reproducible commercial scale production of AAV vectors for use in human gene therapy clinical trials and future marketed products. Applied Genetic Technologies Corporation (AGTC), a clinical-stage biotechnology company, has developed a scalable manufacturing platform to produce AAV-based gene therapies for a wide range of planned indications. These include programs at multiple stages of development, from preclinical proof-of-concept studies to Phase 1 and Phase 2 clinical trials, and many are focused on product candidates that are designed to transform the lives of patients with severe ophthalmology diseases.
PRODUCING COMMERCIAL SCALE QUANTITIES OF AAV VECTOR
A commercial-scale gene therapy platform needs to couple high production capacity with downstream purification methods that efficiently clear the raw materials used during manufacture. AGTC’s Herpes-Assisted Vector Expansion (HAVE) method achieves this balance in four key steps.
STEP 1 – Use Helper Viruses to Make Each Component Of The AAV Vector
The first step involves preparation of the genetic materials that will be combined to form the recombinant AAV vector, the therapeutic gene and the native AAV genes that are needed to create the actual virus particle (or capsid). Each of these genetic materials is inserted into separate Herpes Simplex Virus (HSV) lines, called helpers. HSV helpers are non-replicating on their own, and can only be propagated when introduced into specialized, or complementing, cell lines (Figure 1). After being propagated in separate complementing cell cultures, each HSV helper batch (one carrying the native AAV genes and the other carrying the therapeutic gene) is harvested and prepared for the next step.
STEP 2 – Introduce Each HSV Helper; Into The Same Cell, Which Will Then Produce the AAV Vector
The two HSV helpers are co-introduced into cells that will produce AAV vector. In the HAVE method, the cells are derived from a baby hamster kidney (BHK) line and expanded in suspension culture before addition of the HSV helpers (Figure 2). Once the cells are expanded to the production volume, the HSV helpers are introduced into the culture. As the culture continues to incubate, the BHK cells package therapeutic genes from one HSV helper into AAV capsids that are built from proteins produced by the other HSV helper. The therapeutic gene is flanked by packaging signals that have been specifically separated from the native AAV genes. This ensures that only the therapeutic gene will be packaged into the recombinant AAV vector. As many as 2.4 x 1014 vector copies per liter have been produced with this method.1 Because BHK cells are not complementing, HSV helpers will not replicate in this culture.