AAV Capsid Engineering
Gene therapy (GT) is bound to be the future of biomedicine. Recombinant AAVs (rAAVs) are the most promising vectors because they transduce dividing and quiescent cells, have long-term gene expression in vivo, and low toxicity and inﬂammation in target tissues. Currently AAV is a popularly used vector in gene therapy clinical trials.
GT AAV vectors contain two parts, the capsid and the gene of interest (GOI). The capsids of AAV vectors are derived either from natural AAV viral isolates or by engineering capsids through rational design, directed evolution, or computer-guided technologies. Capsid quality and target cell tropism are important features of rAAV products to achieve safety and efficacy in gene therapy. This is particularly important for the development of rAAV to deliver a GOI into retinal cells for the treatment of ocular diseases. Our AAV capsid engineering technologies provide an effective solution for a simple administration of rAAV vector via an intravitreal (IVT) route with high efficiency of transduction of retinal cells. Use of minimal capsid formation protein (MCFP) helps us greatly to achieve high efficiency and accuracy of candidate screening for AAV and its receptor binding (Fig. 1).
VLP formed by MCFP
AAV VP1 complexed with AAV receptor
AAVR interaction loop
Predicted AAVR binding region
FIG. 1. Engineered AAV2 Capsid Structures
Note: the upper left image shows the predicted structure of the engineered AAV VP1; The upper right image is the predicted structure of a short sequence capable of forming a viral-like particle (VLP)
Engineered AAV capsids are selected using rodent and large animal model systems to determine the locality of AAV vectors and cell types transduced in retinal layers. Avirmax’s proprietary capsid delivers GOI targeting retinal cells efficiently via IVT administration. The target gene (green fluorescent protein, GFP) carried by AAV vectors were detected in the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS), outer segment (OS), and retina pigment epithelium (RPE) layer (Fig. 2).
FIG. 2. Confocal image showing retina locality and cell types transduced 25 days prior by AAV2-GFP vector delivered intravitreally
Enhanced Gene Expression Technology
Avirmax’s proprietary transgene optimization technology has demonstrated a significant enhancement of gene expression for its lead GOIs. Below is an example of target gene expression before and after optimization using the Enhanced Gene Expression Technology
FIG. 3. Comparison of Target Gene Expression before and after Optimization Using the Enhanced Gene Expression Technology
Equal amount of plasmid DNA (A) or AAV vectors (B) carrying a target gene was used to transfect/transduce HEK293 cells for 48 hours and media were collected and analyzed with a specific antibody against the target protein. Arrow indicates the molecular weight of the target protein.
Avirmax Generic rAAV Manufacturing Technologies
Avirmax is building an internal cGMP facility for manufacturing clinical material to support its preclinical and clinical programs. Our production system is based on the Bac-to-AAV technology which was initially invented by our co-founder, Dr. Haifeng Chen. It utilizes the engineered artificial intron consisting of insect promoter in both the AAV rep and cap sequences, coupled with versatile baculovirus expression system and the suspension insect culture to dramatically enhance the stability of recombinant baculoviruses, the infectivity of AAV vectors, and the AAV production yield that reaches as high as 1e+15 vg/L.
FIG. 4. The outline of Avirmax’s Generic Sf9-AAV Production Platform for Large Scale Manufacturing
The Bac-to-AAV technology has been utilized by numerous gene therapy companies to produce clinical material for clinical development at early and late phases in the industry.
Avirmax has developed a robust, simple and cost-effective manufacturing technologies for fast production of high quality and titer rAAV stocks using Sf9 cell system. We can achieve a productivity of 1e+15 to 1e+17 vg/batch within about two months from a plasmid DNA of GOI and defined serotype of vector capsid. This process is easy to be scaled up for large scale production for late phase development and commercial launch.
FIG. 5. Some Process Development Batches of rAAV Yield of Final Purified Product (AAV2* indicated an engineered capsid)
The Sf9 cell culture provides higher pathogen safety assurance for gene therapy products than those manufactured using human or other mammalian cell culture systems (i.e. HEK293, HeLa-3 cells). Sf9 cell culture hardly supports mammalian virus prorogation and it is operated under BSL-1 biological safety environment during production of the recombinant protein (i.e. monoclonal antibodies) using CHO, BHK cells.