Replicationcompetent or Wildtype AAV

Wild-type AAV is not a human pathogen but generation of wild-type or rcAAV during vector production needs to be avoided for several reasons. The presence of wild-type AAV in vector preparations may increase the likelihood of vector mobilization following a helper virus infection in the patient, which could increase the likelihood of cellular immune responses to AAV proteins and can cause significant alterations in the biology of the vector because of the pleiotropic effects of the rep proteins. The earliest AAV vectors (4,5) were produced by cotransfection with helper plasmids that had overlapping homology with the vector, and this generated vector particles contaminated with wild-type AAV due to homologous recombination. Reduction of the overlapping AAV sequence homology between the vector and helper plasmids reduced, but did not eliminate, generation of wild-type AAV (98,107,108).

A combination of vector plasmid and packaging plasmid in which the AAV region containing the P5 promoter was not present in either plasmid prevented generation of wt AAV but some pseudo wild-type AAV (rcAAV) was generated at very low frequency by nonhomologous recombination (98,108). This nonhomologous recombination was decreased to unde-tectable levels in a packaging system (split-gene packaging) carrying rep and cap genes in separate cassettes (98) so that 3 or 4 recombination events would be required to generate rcAAV. An alternate approach to decreasing pseudo wild-type or recombinant AAV is to insert a large intron within the rep gene in the helper plasmid so that any recombinants would tend to be too large to package in AAV particles (122).

It is likely that all vector production systems may have a propensity to generate pseudo wild-type AAV or other recombinants of AAV at some low frequency because it is not possible to eliminate nonhomologous recombination in DNA. This may be more likely in DNA transfection systems, especially in view of the very large genome numbers that are normally introduced into transfected cells. Thus, packaging systems in which transfection is avoided may help to reduce the frequency of such recombination. Standardized assays to analyze and evaluate such rcAAV or recombinant species in AAV vector preparations have not yet been developed. However, to detect replication competent species, an assay that employs 2 cycles of amplification in non-rep/cap expressing cells, and then a sensitive readout such as hybridization or PCR (rather than rep or cap immunoassays), is likely to be required.

E. Purification

Historically, AAV was purified by proteolytic digestion of cell lysates in the presence of detergents followed by banding in CsCl gradients to concentrate and purify the particles and separate adenovirus particles. Significant progress has been made in the downstream processing of AAV vectors, and this has led to much higher quality and purity. This is critically important for preclinical studies and clinical trials. The reliance upon the original CsCl centrifugation techniques is being abandoned because it is a cumbersome procedure that does not provide high purity, it may inactivate some AAV vector, and it is difficult to envision its use for commercial production. Several groups have employed nonionic iodixonal gradients as an initial bulk-recovery method (123,124). A variety of chromatographic methods, including ion exchange resins and antibody, heparin, or sialic acid affinity resins, in both conventional and high-performance liquid chromatography formats, have been employed (106,125-134). Chromatographic procedures will be generally more acceptable for the biopharmaceu-tical manufacturing as therapeutic applications are developed for AAV vectors.

F. Assay of AAV Vectors

Characterization and testing of AAV vectors are critical elements for clinical development. Although there is variability in assay methods and properties measured, the minimal testing should include measure of vector strength by determining the concentration of capsids containing vector genomes, a measure of total capsids, a measure of ''infectivity'', and an assessment of vector purity. The efficiency of transgene expression or potency of the vector should also be measured. In some cases, it is difficult to measure potency using in vitro assays because transgene expression may be mediated by weak or tissue-specific promoters.

Early in the development of AAV vectors, the concentration was measured using dot-blot hybridization of vector genomes and comparing the signal intensity with a standard curve derived from a plasmid containing the vector genome (135). Recently, more precise and quantitative assays have largely supplanted the dot blot assay. Measurement of vector concentration by real-time PCR amplification (TaqMan) of the vector genome has resulted in increased precision and accuracy (126). These assays measure the number of vector genomes that are encapsidated and thus protected from digestion by DNAse. Consequently, vector concentration is expressed in units of DNAse-resistant particles (DRPs). This type of assay can be used to facilitate reliable quantitation of vectors during process development for production of multiple AAV serotypes (118).

Infectivity measurements of vector preparations are important mainly to establish a consistency of vector manufacturing rather than being an indication of vector potency in vivo. By their nature, AAV vectors are replication defective, so in vitro measurements of infectivity are artificial and generally require complementation with rep and cap as well as helper virus functions. Initially, vectors were measured by an infectious center assay in which wtAAV as a source of rep and cap and adenovirus were added, along with dilutions of vectors to infect cells (136). Cells that were replicating the vector genome were then scored by probe hybridization to individual cells collected on a filter, hence the term ''infectious center''. The development of cell lines containing rep and cap (137) has allowed infectivity measurements of vectors to be performed in a 96-well format in an assay that yields data with higher precision than the infectious center assay, and is amenable to a high-throughput format that can be used for purification development (138). In conjunction with the real-time PCR assay, this type of infectivity assay yields data on particle to infectivity (P:I) ratios of vectors with high precision, thus aiding process development. It is important to recognize that the apparent particle to infectivity ratio of vectors based on different serotypes of AAV may be quite different due to the natural variation that exists in receptor usage and potentially in intracellular trafficking among the serotypes. A cell line containing rep and cap may be more efficiently transduced by one serotype as compared with a second serotype, yielding differences in P:I ratios that may not be reflective of differences in vector potency in vivo but rather a difference in the ability of a given serotype to efficiently infect the rep/cap-expressing cell line used in the assay.

Other aspects of vector preparations are also worth measuring. These include a measure of the ratio of empty to full capsids such as a capsid enzyme-linked immunosorbent assay (139), negative-stain electron microscopy analysis (131,132), or a measure of total protein compared with concentration of genome-containing vectors and an assessment of vector purity, usually by acrylamide gel electrophoresis analysis. For clinical development it is important to measure the residual contaminants, including host cell protein, DNA, and serum components. In cases where the vector is manufactured from a cell line of tumor origin (e.g., HeLa cells), determination of the residual level of host cell DNA is an important part of the release testing. If the vector is of high purity, its aggregation state can be assessed by using dynamic laser light scattering. This method enables a measurement of the hydrodynamic radius and provides a size distribution of particles in solution and the relative amount of particles in each size class. This measure is useful for vectors at high concentration and can aid in development of formulations that maintain vectors in a monomeric, nonaggregated state.

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