Factors Influencing the Titer and Infectivity of Lentiviral Vectors

Abstract

Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34 ؉ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34 ؉ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G glycoprotein (VSV-G). OVERVIEW SUMMARYWe have analyzed several variables involved in the design and production of lentiviral vectors for the effect they have on vector titer and infectivity. Wide variability in the amounts of input transfer vector, packaging, and envelope plasmids, as well as changes to the transfer vector construct, are tolerated with no significant change in vector infectivity. We found that inclusion of the central polypurine tract, concentration by ultracentrifugation, and collection in serum-free medium increase the infectivity of vectors on HEK293 cells and human umbilical cord blood CD34 ؉ HPCs. In addition, use of the RD114 envelope instead of VSV-G dramatically increases the infectivity of lentiviral vectors when they are used to transduce CD34 ؉ HPCs, but the titers and infectivities of RD114-pseudotyped vectors as determined on HEK293 cells is misleading, because of poor performance of the RD114 envelope on these cells.

Altering the Tropism of Lentiviral Vectors through Pseudotyping

“…Virus particle numbers can be determined using real-time PCR based on strong-stop cDNA present in virions [Scherr, 2001]. Alternatively, the amount of virus proteins present in virus cores such as p24 Gag are determined by ELISA to arrive at relative particle titers [Logan, 2004]. Functional titration assays are based on vector-encoded reporter gene expression.…”

Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics

“…85,86 Alternatively, the amount of virus proteins present in virus cores such as p24 gag are determined by enzyme-linked immunosorbent assay to arrive at relative particle titers. 87 Titration assays based on vector-encoded reporter gene expression using fluorescence-activated cell sorting analysis of cells transduced with varying amounts of the virus have been widely used. 88 An important factor is the cell line used for titration, as receptors for a given glycoprotein may vary from cell line to cell line.…”

Stable gene expression occurs from a minority of integrated HIV-1-based vectors: transcriptional silencing is present in the majority

“…Firstly, the study shows that vector utility may be critically affected by poor efficiency of gene expression. Thus, to enhance the utility of a vector, manipulating the promoter and enhancer, in addition to efforts to improve viral titre 40 and transduction efficiency 41 may be a fruitful avenue of investigation. Secondly, the estimation of the risk of gene therapy and, in particular, the number of integrations required before a cell expresses the transgene to a sufficiently high level has to be determined and this impacts on the risk of insertional oncogenesis caused by retroviral gene vectors.…”