Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2

Abstract

We demonstrate the rapid and reliable quantification of the recombinant, but not in the wild-type AAV-2 prepphysical AAV-2 (adeno-associated virus type 2) particles arations. Moreover, additional expression of VP proteins via a novel ELISA based on a monoclonal antibody which during rAAV production was found to result in an excessive selectively recognizes assembled AAV-2 capsids. Titration capsid formation, whilst yielding only minor increases in of a variety of recombinant AAV-2 (rAAV) preparations DNA-containing or transducing rAAV particles. We conrevealed that at least 80% of all particles were empty, comclude that encapsidation of viral genomes rather than cappared with a maximum of 50% in wild-type AAV-2 stocks, sid assembly can be limiting for rAAV production, provided indicating that the recombinant genomes were less that a critical level of VP expression is maintained. The efficiently encapsidated. This finding was confirmed upon feasibility of quantifying AAV-2 capsid numbers via the titration of CsCl gradient fractions from recombinant and ELISA allows determination of physical to DNA-containing wild-type AAV-2 stocks. ELISA-based measurement of or infectious particle ratios. These are important paracapsid numbers revealed a large number of physical parmeters which should help to optimize and standardize the ticles with low densities corresponding to empty capsids in production and application of recombinant AAV-2.Keywords: AAV-2; recombinant AAV-2; AAV-2 titration; AAV-2 ELISA Gene therapy vectors derived from the human parvovirus AAV-2 (adeno-associated virus type 2) have gained attention owing to a unique combination of attractive features. Wild-type AAV-2 is nonpathogenic in humans and naturally defective, requiring coinfection with a helpervirus (eg adenovirus) for a productive infection. 1 Recombinant AAV-2 can infect both dividing and nondividing cells in vitro and in vivo [2][3][4][5][6] and have the potential for sitespecific integration into chromosome 19.7-9 Long-term expression of heterologous genes transduced by rAAV has been observed in a variety of human cells and tissues, such as muscle, 10,11 lung, 12 central nervous system, 13-15 retina 16 and liver. 17 An important prerequisite for the testing of AAV-2 vectors in preclinical and clinical studies is the accurate and reliable titration of the recombinant virus particles. Precise information on rAAV titers is not only crucial for the careful planning and execution of such studies, but also for comparing results amongst laboratories. In brief, presently available methods for rAAV titration can be divided into biological and physical assays. Biological assays rely on infection of cultured cells followed by events that depend on the biological functionality of the 18-20 These two types of assay yield titers of infectious or transducing particles, respectively. In contrast, physical methods are independent of biological functions of the recombinant viruses. Typically, viral DNA is extracted from the rAAV particles via enzymatic digestion ...

Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid: Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection

“…Binding of MAbs to AAV serotypes and AAV-2 insertion mutants was quantified in an ELISA. Titers of determined CsCl-purified particles of AAV serotypes and AAV-2 insertion mutants were determined by counting of negatively stained particles in electron micrographs (14). Particles were diluted in PBS, and equal amounts (8 ϫ 10 7 particles/well) were used to coat the wells of microtiter plates (Polysorb; Nunc Nalgene International, Roskilde, Denmark) overnight at 4°C.…”

“…After blocking, hybridoma supernatants diluted in PBS containing 0.05% Tween 20 were incubated for 1 h at 37°C, followed by another hour of incubation with a biotinylated anti-mouse secondary antibody (Amersham Pharmacia). Detection and quantification were performed as described previously (14).…”

“…Hybridoma supernatant (final dilution, 1:10) was preincubated with 10 9 AAV-2 particles for 30 min on ice in HEPESbuffered saline containing 1% bovine serum albumin before a 90-min incubation with HeLa cells at 4°C was performed. Cells were washed with PBS and fixed in methanol for 15 min at Ϫ20°C, and bound AAV-2 particles were detected by an A20 enzyme-linked immunosorbent assay (ELISA) (14).…”

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

“…Yield (vg/cell) Scale-up Reference 293, triple transfection 10-10 3 Small scale Salvetti et al (1998);Xiao et al (1998), Matsushita et al (1998), Grimm et al (1999), Collaco et al (1999). HeLa based producer cell, rAAV production induced by infection with wt Ad5 10 4 -10 6…”

Construcción de un vector de expresión derivado de virus adenoasociados para corregir in vitro el defecto genético de la enfermedad de Morquio A

“…El número de partículas virales se determinó comparando la absorbancia obtenida por las muestras contra una curva de calibración realizada con una suspensión viral suministrada en el estuche. Este método permite la captura de partículas virales completas, pero no de las proteínas aisladas de la cápside (28).…”