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.
STEP 3 – Collect And Purify the AAV Vector
The vectors are released with a detergent step that lyses the cells, then purified with column chromatography.
STEP 4 – Prepare the Final Product
The purified vector is concentrated, formulated and filtered, then dispensed to containers that are appropriate for storage and shipping.
PURIFYING THE HARVEST FOR SAFE USE IN HUMAN GENE THERAPY
The downstream purification processes that comprise Step 3 are designed to clear raw materials that were used during manufacturing. This especially includes clearance of the HSV helper input. To terminate the culture, a nonionic detergent is added that lyses the BHK cells and releases the AAV vectors. The detergent also inactivates any HSV that failed to enter cells but instead remained free in the culture medium.
Once released, the AAV vectors are purified with serial column chromatography (Figure 3). The details of the column purification process often vary across gene therapy product candidates, especially if each AAV vector contains a distinct set of capsid proteins. The choice of AAV capsid proteins is a critical part of the vector design process because they determine which cell types will accept the vector and, by extension, the therapeutic gene. Ion exchange chromatography purifies vectors based on overall charge, which depends on the AAV capsid.
Cation exchange columns bind targets with a net positive charge and anion exchange columns bind targets with a net negative charge.These columns can be made with Convective Interaction Media (CIM) to enhance the efficiency and speed of purification. Columns made of CIM are cast as single units of homogenous methacrylate matrix that houses an interconnected network of pores to facilitate the continuous flow of large biomolecules, such as viruses. The other form of column purification is based on AVB Sepharose affinity chromatography. With high affinity for AAV capsid proteins, AVB Sepharose holds vectors in the column while everything else is washed away.
When put to the test in validation studies, these purification methods eliminated all detectable HSV used in the production process. In a program outside of ophthalmology, AGTC has developed an AAV-based gene therapy product candidate for the treatment of patients with inherited α1-antitrypsin (AAT) deficiency. AAT protein normally functions to prevent lung tissue damage and inherited AAT deficiencies are associated with an increased risk of developing emphysema and liver disease. After expansion in a BHK suspension culture, AGTC’s gene therapy product candidate rAAV1-CB-hAAT was (1) released from cells with the addition of a nonionic detergent, (2) concentrated, and then purified with passage through (3) a CIM anion exchange column and (4) an AVB Sepharose affinity column. The validation study was conducted to quantify the efficiency of HSV clearance at the end of each step. The pre-purification input was spiked with normal enveloped replication-competent HSV to simulate the worst-case scenario that large amounts of replication competent HSV are present in the culture medium. The study showed that the detergent treatment cleared at least6.84 log10 HSV from the spiked harvest, the CIM anion exchange column removed an additional 4.34 log10 HSV, and the AVB Sepharose affinity column removed a final 2.86 log10 HSV. All combined, the purification process cleared a total of at least14.04 log10 HSV from the spiked harvest.2
WHAT THIS MEANS
But what does this mean? What do these clearance values say about the safety of rAAV1-CB-hAAT gene therapy for patients with AAT deficiency?
To answer these questions, start with a 100-liter production batch where 1.2 x 1012 HSV are added to a BHK cell culture. The purification process clears more than14.04 log10 HSV, leaving less than 1.09 x 10-2 HSV in the bulk product. The bulk product is formulated and filtered into a 450 mL volume, which would then contain less than 2.42x10-5 HSV per mL.2 In a clinical trial of AAT deficiency patients, the volume of the largest single rAAV1-CB-hAAT dose was 100 x 1.35 mL.3 According to the above assessment this dose would have contained less than 3.27x10-3 HSV.2
In a second validation study, AGTC measured HSV clearance during purification of an AAV-based gene therapy product candidate that has been developed for the treatment of patients with X-linked retinoschisis (XLRS), an inherited retinal disease that can lead to significant reductions in vision and other serious ocular complications. XLRS is caused by mutations in the RS1 gene that encodes the retinoschisin protein. Retinoschisin is expressed and secreted from specific cells in the retina, including photoreceptors, but binds to surface of many cells across the retinal layers. Mutated forms of retinoschisin are unable to bind properly, and this leads to splitting of the layers of the retina.
After expansion in a BHK suspension culture, AGTC’s XLRS gene therapy product candidate rAAV2tYF-CB-hRS1 was (1) released from cells with the addition of a nonionic detergent and then purified with passage through (2) an AVB Sepharose affinity column and (3) a CIM cation exchange column. As in the above validation study, pre-purification input was spiked with normal enveloped replication-competent HSV to simulate a worst-case scenario. The detergent treatment cleared at least 7.34 log10 HSV from the spiked harvest, the AVB Sepharose column removed an additional 4.54 log10 HSV, and the CIM cation exchange column removed a final 1.41 log10 HSV. The complete purification process cleared a total of at least 13.29 log10 HSV from the original spiked harvest.4
To estimate the residual amount of HSV in a single dose of XLRS gene therapy after purification, start with a 25-liter production batch where 3.0 x 1011 HSV are added to a BHK cell culture. The purification process clears more than 13.29 log10 HSV, leaving less than 1.54 x 10-2 HSV in the bulk product. The bulk product is formulated into a 100 mL volume, which would then contain less than 1.54 x 10-4 HSV per mL. The volume of the highest proposed single dose for a patient is 0.1 mL. According to the above assessment this dose would contain less than 1.54 x 10-5 HSV.4
Findings from preclinical evaluation in mice and large mammals have supported the advancement of rAAV2tYF-CB-hRS1 into clinical studies, and AGTC is currently conducting a Phase 1/2 trial to evaluate the safety and tolerability of rAAV2tYF-CB-hRS1 in patients with XLRS. The trial is a multicenter non-randomized, open-label, dose-escalation trial (NCT02416622) at four study sites in the United States and will enroll a total of 27 patients.
Historical challenges for gene therapy manufacturing have included poor vector quality and a lack of scalable production systems for commercial manufacturing. To address these needs, AGTC has developed the HAVE method to manufacture and purify AAV vectors for use in human gene therapy clinical trials and future marketed products. The downstream purification steps can be customized according to optimized features of the AAV capsid in the product candidate.
Studies showed that the purification process eliminates all infectious HSV helpers, with some steps below the level of detection of the assays used. The results of these studies support the safety of AAV vectors produced by the HAVE method for use in clinical studies, such as the ongoing Phase 1/2 study that is evaluating the safety and tolerability of AGTC’s gene therapy product candidate for XLRS. AGTC has a robust gene therapy development pipeline, including five named ophthalmology development programs across four targets (XLRS, X-linked retinitis pigmentosa, achromatopsia and wet age-related macular degeneration), one non-ophthalmology program (AAT deficiency) and multiple other potential indications for which proof-of-concept data are available. The scalable and customizable design of AGTC’s proprietary HAVE manufacturing platform has the capacity and flexibility to support the development and future commercialization of AAV-based gene therapies across this broad range of programs.
- Thomas DL, Wang L, Niamke J, Liu J, Kang W, Scotti MM, et al. Scalable recombinant adeno-associated virus production using recombinant herpes simplex virus type 1 coinfection of suspension-adapted mammalian cells. Hum Gen Ther. 2009;20(8):861-870.
- Ye GJ, Scotti MM, Thomas DL, Wang L, Knop DR, Chulay JD. Herpes simplex virus clearance during purification of a recombinant adeno-associated virus serotype 1 vector. Hum Gene Ther Clin Dev. 2014;25(4):212-217.
- Flotte TR, Trapnell BC, Humphries M, Carey B, Calcedo R, Rouhani F, et al. Phase 2 clinical trial of recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results. Hum Gene Ther. 22(10):1239-1247.
- Ye GJ, Gaskin C, Butts C, Threadgill R, Knop DR, Chulay JD. Efficient clearance of herpes simplex virus using a GMP-compliant method for production of recombinant adeno-associated virus vectors. Paper presented at: 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13-16, 2015; New Orleans, LA.