Two questions, what do you consider typical titer for AAV production and what can you do to increase it? (2024)

FAQs

What is a typical AAV titer? ›

Typically, AAV production titers are around 10e11 to 10e12 in VG/ml and 10e8 to 10e9 TU/ml. To put these numbers into perspective, it's important to keep in mind that two AAV serotypes will most likely not give the same yield, even when using the same production process.

In contrast to the AAV2 capsid, the AAV9 capsid interacts with galactose and AAV1,5,4, and 6 capsids interact with sialic acid proteoglycans as their primary attachments for cellular entry. This differential preference for cellular receptors and co-receptors dictates the tropism of a given AAV serotype. Figure 2.

Physical titer: the concentration of viral particles containing viral genomes. Physical titers are measured by quantifying the concentration of viral genomes (by qPCR or other DNA quantification methods - see below), since each viral particle typically contains one viral genome.

We guarantee a total titer of 1x10E11, although average yield is usually ~1-4x10E12 vg/ml in 1-1.3ml. Yield is dependent on customer construct.

Traditionally, AAV genome titer is determined by qPCR using a plasmid DNA standard.

Because AAV vectors lack any coding viral sequence, the main sources of foreign antigens brought in during gene transfer, aside from contaminant carryovers from the production and purification process, are derived from initial input of viral capsid and from the transgene product.

Eleven serotypes of AAV have thus far been identified, with the best characterized and most commonly used being AAV2. These serotypes differ in their tropism, or the types of cells they infect, making AAV a very useful system for preferentially transducing specific cell types.

In order to generate infectious AAV particles, a helper plasmid (encoding adenovirus E4, E2A and VA) and a RepCap plasmid (encoding AAV Rep and respective capsid protein which determines the AAV serotype) are used in conjunction with the plasmid that encodes the viral genome (typically called cis-plasmid or transfer ...

AAV Tropisms

AAV2 is probably the most widely used AAV serotype for in vitro and in vivo gene delivery. However, many other serotypes of AAV have been isolated, and every year there are new serotypes being discovered. Among them, AAV1, AAV2, AAV5, as well as AAV7, 8 and 9 etc, have been used for gene delivery studies.

To calculate the viral titer, it is first necessary to determine the number of Transduction Units (TU) used to infect the cells. When the percentage of infected cells is at or below 20%, the number of integrations is approximately equal to the number of transduced cells.

What is the difference between physical titer and infectious titer? ›

Physical titer is strictly a measurement of how much virus is present, and is usually expressed as the number of viral particles per mL (VP/mL). Functional titer, also known as infectious titer, is the measurement of how much virus actually infects a target cell.

Viral infectivity is defined as the number of virus particles capable to invade a host cell. This is determined by using susceptible cells to the specific virus by measuring the viral infectivity.

Currently, AAV yield is increasing by using various cell lines, assembly systems, transfection types, and downstream stage optimization. However, several other approaches based on the modification of the rAAV genome or structure can be used to obtain a higher titer of pure and functional AAV viral particles.

This AAV production system is referred to as 'helper-free' and consists of three different plasmids encoding essential viral and helper genes: pHelper, AAV trans-plasmid comprising AAV replication (Rep) and capsid (Cap) genes, and AAV cis-plasmid encoding the gene of interest, promoter, and inverse terminal repeats ( ...

Two to five days after transfection, the AAV-containing cells and media are harvested and purified. In total, this process takes 4-7 days, not counting the time needed to expand the HEK293 cells. Check out the Addgene AAV Production protocol for more details.

The assay could detect viral titres between 0.75 and 8.5 log₁₀/TCID₅₀/midge.

The titer range for lentivirus can vary depending on factors such as the viral vector design, production method, and purification process. A typical titer range for lentivirus preparations used in research or gene therapy applications may range from 10^6 to 10^9 TU/mL or vg/mL.

Formula for virus titer calculation: titer = {(F × Cn) /V} × DF F: The frequency of GFP-positive cells determined by flow cytometry; Cn: The total number of target cells infected. V: The volume of the inoculum.