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Real-Time Quantitative PCR for the Design of Lentiviral Vector Analytical Assays

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Real-Time Quantitative PCR for the Design of Lentiviral Vector Analytical Assays Gene Therapy (2005) 12, S36–S50 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt CONFERENCE PAPER Real-time quantitative PCR for the design of lentiviral vector analytical assays C Delenda1 and C Gaillard2 1Genethon, CNRS UMR 8115, 1bis rue de l’Internationale, Evry Cedex, France; and 2GenoSafe, 1 rue Pierre Fontaine, Evry Cedex, France From the recent and emerging concerns for approving context, have been included in the effort to dress an lentiviral vector-mediated gene transfer in human clinical exhaustive list. Also, great variations have been observed applications, several analytical methods have been applied from interlaboratory results, we have tempted to compare in preclinical models to address the lentiviral vector load between them the different analytical methods that have in batches, cells or tissues. This review points out the oldest been used to consider (i) the titration of lentiviral vector generation methods (blots, RT activity, standard PCR) as batches, (ii) the absence of the susceptible emerging well as a full description of the newest real-time quantitative replicative lentiviruses or (iii) the lentiviral vector biodistribu- PCR (qPCR) applications. Combinations of primer and probe tion in the organism. sequences, which have worked in the lentiviral amplification Gene Therapy (2005) 12, S36–S50. doi:10.1038/sj.gt.3302614 Keywords: real-time PCR; lentiviral vector; standardization Introduction This review points out the major progress undertaken for the standardization of some of these technical Lentiviral-derived transfer vectors have gained increased expertises. Associated with conventional detection attention because their karyophilic properties allow their methods, techniques derived from qPCR have been use for the transduction of quiescent cells.1,2 The applied in the lentiviral vector context for the evaluation acceptance of their use in clinical settings will require of titration, RCL and biodistribution in animal models. profound and vigorous assays in order to evaluate the quality as well as the quantity of lentiviral particles in vector batches. Description of pre-existing lentiviral vector Validation and safety tests that will be required for the use of lentiviral vectors in human gene transfer should titration methods include (i) the search for susceptible emerging replicating The main options for titering lentiviral vectors include forms (assays for replication-competent lentiviruses assessment of the reverse transcriptase (RT) activity and (RCL)), (ii) several sterility tests in order to detect the viral RNA content in supernatants, as well as the remnants from mycoplasma, fungi or bacteria as well number of proviral DNA and the efficacy of transgene as contaminants originating from adventitious agents, expression in transduced target cells. Specifically dedi- (iii) the determination of the viral vector titers as well cated to lentiviral vectors derived from the human as (iv) in vivo preclinical studies for demonstrating immunodeficiency virus (HIV), an enzyme-linked the proof of complementation concept. In addition, the immunosorbent assay (ELISA) directed against the p24 organ biodistribution of lentiviral vectors should also capsid protein has also been routinely employed for be addressed. vector titer determination. In this concern, GenoSafes www.GenoSafe.com, a service company, has been recently founded by Gene- Lentiviral vector titration from transgene protein thon to provide gene transfer safety and quality tests for expression preclinical and clinical studies. Overall, this company is dedicated to vector characterization, containing titration For lentiviral vectors containing a reporter transgene and replication-competent virus experiments as part expression cassette, ‘infectious’ particles (ip) are usually of quality control for preclinical products, and safety, determined by evaluating the percentage of target cells preclinical in vivo studies, as well as gene therapy- that express the transgene protein. specific patient follow-up. GenoSafes operates under Depending on the type of reporter used, lentiviral validated GLP conditions. ‘infectious’ units are defined in several ways. Colony- forming units are generally used for drug selection genes.3 Relative luciferase units are defined for renilla as well as firefly luciferase gene activities.4–6 The term of Correspondence: Dr C Delenda, Genethon, CNRS UMR 8115, 1bis rue de transducing unit used in the context of lentiviral vector l’Internationale, 91002 Evry Cedex, France gene transfer has been routinely employed, especially for qPCR applied to lentiviral analytical assays C Delenda and C Gaillard S37 the expression of living color and lacZ genes.7,8 Protocols Whereas the sensitivity of first generation RT assays for the determination of functional viral titers by drug- ranges from 104 to 106 viral particles,27 it decreases to resistance colony assay, expression of green fluorescent 1–10 virions when PERT assays are used.18,20,21 Such protein (GFP) and b-galactosidase staining can be found assays, which are based on the endogenous RT activity of in Tonini et al.9 retrovirions, have been commonly employed for the Additionally, infectious titers of vectors containing verification of the absence of retroviral-based adventi- nonreporter gene cassettes have been determined by tious viruses in different biological materials,28,29 but FACS analysis using primary antibodies directed against they have also served for isolating lentiviral packaging the transgene protein. However, this application is cell clones able to secrete functional empty capsids, limiting as it requires antibody for all type of transgene where the term ‘functional’ argued for RT encapsida- and because it is restricted to some specific cellular tion.30 compartments, predominantly at the plasma membrane. By comparing different analytical methods for the Nevertheless, mild permeabilization of the plasma titration of lentiviral vectors derived from the equine membrane has already been described to allow cellular infectious anemia virus (EIAV), Martin-Rendon et al31 antibody uptake and vector recognition within the have shown that there was no correlation between PERT cytoplasmic compartment.10,11 assays and eGFP titers, by contrast to the results obtained Several optimizations undertaken in some of these with a RT-PCR titration method (cf Quantification of reporter genes have been addressed in the context of the viral RNA genomes). lentiviral gene transfer. To improve the LacZ gene expression in mammalian cells, Anson and Limberis12 have codon optimized the Escherichia coli LacZ gene. HIV-1-based vector titration from capsid content From this mutagenesis, they have observed a 15-fold measurement increase in b-galactosidase expression as well as a five- The assay commonly used for titering HIV-1-derived fold enhancement in HIV-1 vector titers, apparently due vectors that do not contain a reporter gene cassette is to the new opportunity to detect transduced cells that based on an ELISA test directed toward the capsid did not highly express the transgene. Concerning the protein (p24). This approach is generally used to GFP reporter protein, a lower cytotoxic mutant (from determine the number of ‘physical’ particles (pp), but the sea pansy Renilla reniformis) has been compared to the is limited by the variable amount of free, nonparticle- original enhanced GFP (eGFP) from the jellyfish Aequorae associated p24 proteins that are produced by standard victoria. However, in vivo lentiviral gene transfer studies plasmid cotransfection methods of lentiviral production. did not show a clear advantage in term of cytotoxicity of Based on the estimation that a HIV core particle is this newly used GFP.13,14 Whereas one report has shown composed of around two thousands capsid proteins,32 evidence of rejection in the retinal epithelium of rats 1 fg of p24 therefore represents around 12 pp. From this when using the eGFP reporter transgene,15 Duisit et al13 calculation, it is therefore able to depict the ip/pp ratio did not observe neither sign of rejection nor difference in for any HIV-1-based lentiviral vector production, as from the long-term expression. In addition, although cytotoxi- the moment when ip titer measurements are possible. city has been observed after intracorneal injection of cats, The ip/pp ratio estimation is routinely employed for it was only noticed in conditions where the eGFP protein quality control validation of the different batches being has been overexpressed, and was solved by lentiviral produced. In fact, it allows the rejection of vector batches vector dose reduction.14 having a too great quantity of non-ip, which may result from gag-pol polyprotein overexpression and/or matura- Lentiviral vector titration from RT activity tion problems. Although concrete specifications are not The first generation of RT assays measures the incorpora- formally described in the litterature, it is generally tion of radiolabelled deoxyribonucleoside triphosphates accepted to consider that a relevant lentiviral vector into DNA using viral genomic RNA as template.16 The batch is defined when the ip/pp ratio is contained in a quantity of incorporated radiolabelled nucleotides is window of 1/100–1/1000. then measured by a scintillation counter or by auto- radiography. This RT protocol dedicated for retroviral and lentiviral vector titration can be found in Tonini Lentiviral vector titration from viral nucleic acid et al.17 An additional radiolabelled technique
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