Copackaging of Multiple Adeno-Associated Viral Vectors in a Single Production Step

Abstract

Limiting factors in large preclinical and clinical studies utilizing adeno-associated virus (AAV) for gene therapy are focused on the restrictive packaging capacity, the overall yields, and the versatility of the production methods for single AAV vector production. Furthermore, applications where multiple vectors are needed to provide long expression cassettes, whether because of long cDNA sequences or the need of different regulatory elements, require that each vector be packaged and characterized separately, directly affecting labor and cost associated with such manufacturing strategies. To overcome these limitations, we propose a novel method of vector production that allows for the packaging of multiple expression cassettes in a single transfection step.Here we combined two expression cassettes in predetermined ratios before transfection and empirically demonstrate that the output vector recapitulates the predicted ratios. Titration by quantitative polymerase chain reaction of AAV vector genome copies using shared or unique genetic elements allowed for delineation of the individual vector contribution to the total preparation that showed the predicted differential packaging outcomes. By copackaging green fluorescent protein (GFP) and mCherry constructs, we demonstrate that both vector genome and infectious titers reiterated the ratios utilized to produce the constructs by transfection. Copackaged therapeutic constructs that only differ in transcriptional elements produced a heterogeneous vector population of both constructs in the predefined ratios. This study shows feasibility and reproducibility of a method that allows for two constructs, differing in either transgene or transcription elements, to be efficiently copackaged and characterized simultaneously, reducing cost of manufacturing and release testing.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease

“…51,88 The present study included, consistent reproducibility of the predicted packaging ratios in AAV2, AAV8, and AAV9 shows that the process is serotype-independent; production levels at surface areas ranging from 148 to 6,320 cm 2 using roller bottles or flasks highlight the scalability and flexibility in manufacturing; purification of copackaged vectors are not affected whether performing cesium-chloride or iodixanol centrifugation; and careful titration of the expression plasmids results in consistent ratios across preparations. 51,88,92 Rearrangement of the plasmid genome brought on by homologous recombination during packaging is a concern but seems to occur at a negligible rate as the output ratios are highly consistent of the predicted ratios. 51 Once the manufacturing process is defined, the copackaged candidate vector can therefore be treated as a single product for toxicology, stability, sterility, and release testing as performed for any current AAV clinical trial rather than as two independent products.…”

“…51,88,92 Rearrangement of the plasmid genome brought on by homologous recombination during packaging is a concern but seems to occur at a negligible rate as the output ratios are highly consistent of the predicted ratios. 51 Once the manufacturing process is defined, the copackaged candidate vector can therefore be treated as a single product for toxicology, stability, sterility, and release testing as performed for any current AAV clinical trial rather than as two independent products. Dose assessment and identity studies will be necessary for clinical protocol validation but inclusion of next-generation sequencing would confirm if the ratio has been maintained and indicate if homologous recombination occurred.…”

“…3C), corroborating previous observations that AAV can be faithfully copackaged at predetermined ratios and directly compared with admixed preparations within margins of pipetting or titration error. 51 Eight weeks post-gene transfer (5 · 10 13 vg/kg; n = 4-6/group), GAA activity was significantly increased in all treated mice in the heart, diaphragm, liver, quadriceps, and sciatic nerve compared with vehicle-treated mice (Fig. 4A).…”

“…51 Copackaging of AAV vectors was performed by combining the expression plasmids pTR-DES-coGAA and pTR-LSPcoGAA at a molar ratio of 9 DES:1 LSP before transfection with the capsid plasmids rep2/cap9 (courtesy of Dr. James Wilson, University of Pennsylvania) and the adenovirus helper plasmid pXX6-80 (courtesy of Dr. Xiao Xiao, University of North Carolina) in two 6,320 cm 2 cell factories of HEK293 cells. AAV was purified via iodixanol step gradients, buffer exchanged in lactated Ringer's solution, and titrated as described.…”

“…Additionally, we have previously described a method of AAV production that allows for the manufacturing of multiple vectors in a single preparation. 51 With the hypothesis that the DES:LSP ratio would confer adequate cardiac and neuromuscular expression based on previous publications, we copackaged these constructs at that ratio to produce a single gene therapy product (copackaged DES:LSP). 11,46,47 We hypothesized that this would have the same benefits as producing these vectors separately and combining them but would provide a quicker and more costeffective method of production.…”