SEQUENCE LISTING

[0001] This application is accompanied by a sequence listing entitled

GSK1007PROV_ST25.txt, created July 23, 2021, which is approximately 1 kilobyte in size. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted herewith via EFS-Web, and is in compliance with 37 C.F.R. §1.824(a)(2)-(6) and (b).

BACKGROUND OF THE INVENTION

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Assays for routine quantitation of retrovirus particles are essential tools in research areas addressing the biology of retroviruses and the development and use of viral vectors for gene transfer. Direct measurement of retroviral proteins, such as the use of enzyme linked immunoassays (ELISA) of retroviral Gag proteins, are often expensive and require extensive dilution of samples because of the limited range of antigen concentration that provides assay linearity. Reverse transcriptase polymerase chain reaction (RT-PCR) based assays can also be used for retroviral quantification, but require sequence specific primer design and RNA isolation procedures which can be expensive and labor intensive. Thus, biochemical assays have been developed which estimate the amount of virion-associated reverse transcriptase (RT-assays) that allow for universal quantification of retroviral particles. These assays involve measuring the enzymatic activity of RT, using an RNA template which is reverse transcribed in vitro by the retroviral enzyme in the presence of a measurable label. The resulting fluorescence is then quantified and when compared to a standard curve, provides a measure of the amount of virions present (see, Arnold et al., Biotechniques, 25:98-106, 1998).

[0004] This basic assay has been adapted in multiple ways, termed generally “product enhanced reverse transcriptase” (PERT) assays, to provide high sensitivity and broad quantitive capacity. Such adaptions have included fluorogenic 5’-nuclease chemistry (F-PERT). PERT and F-PERT assays can be used to detect low amounts of retroviruses in biological products, HIV in serum samples, replication competent retrovirus in cultures producing vectors for gene transfer, and endogenous retroviruses. SYBR Green I-based systems offer a popular alternative to fluorescent probe-based real time PCR because it is a general double-stranded DNA binding molecule that is not sequence specific. This directly measurable dye has been adapted into a one-step PERT assay (see, Pizzato et al., J. of Virol. Meth., 156 (2009), 1-7, discussing PERT assays in general and SYBR Green 1 -based assays in particular). Further, this general SYBR Green 1 -based PERT assay has been specifically adapted to provide a method of quantifying HIV, lenti-, and retroviral vectors (see, Vermeire et al., PLOS One, 7(12):e505859, 2012) through the development of a standardized approach using commercially available reagents. Notably, Vermeire et al. does explicitly teach the requirement of lysing the viral supernatant prior to the performance of the assay, see Vermeire et al., Figure 1, pg. 4. It remains that any gains in efficiency of this assay are desirable, given the high throughput nature of its practice and the many possible applications for the assay in numerous commercial vector production and medical diagnostic settings.

[0005] As evident from these teachings, there remains a need in the art for methods of titrating retrovirus concentration and screening cells lines for retroviral production which are faster and more efficient than those presently available. These improved methods, as well as other uses of the method strategies provided here, should be apparent to those skilled in the art from the present teachings.

SUMMARY OF THE INVENTION

[0006] This summary is provided to introduce a selection of concepts that are further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0007] In a first embodiment, the present invention provides a method of quantifying a retrovirus or a retroviral vector comprising the steps of combining unlysed, cell-free viral supernatant directly with a reaction mix to form a test sample; subjecting the sample to quantitative PCR (qPCR) reactions comprising the steps of reverse transcription, denaturation, annealing and amplification; optionally combining the annealing and amplification step; acquiring the fluorescence level of the test sample; and comparing the reverse transcriptase concentration in the test sample to a standardized reverse transcriptase concentration curve to determine the quantity of reverse transcriptase, and therefore the relative quantity of retrovirus or retroviral vector, present in the test sample.

[0008] In a further embodiment, the method comprises the use of a reaction mix that includes a directly measurable dye. In one embodiment of the present invention, the measurable dye is SYBR Green or a dye derived from SYBR Green. Further, the reaction mix can exclude external reverse transcriptase or RNAase and can further comprise template RNA and forward and reverse primers specific for the template RNA. In the method of the present invention, the template RNA can be from a bacteriophage, such as MS2.

[0009] In a further embodiment, the reaction mix can comprises a fluorogenic probe rather than the directly measurable dye.

[0010] In embodiments of the present invention, the retrovirus being titrated can be any retrovirus or retroviral vector. In particular, the retrovirus can be human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); feline immunodeficiency virus (FIV), Moloney murine leukemia virus (MMLV); and murine stem cell virus (MSCV), EIAV (Equine infectious anemia virus), prototype foamy virus (PFV), feline foamy virus, simian foamy virus, HTLV-l(human T cell lymphotropic virus type 1), HTLV-2 (human t cell lymphotropic virus type 2), and Rous sarcoma virus or retroviral vectors derived from such viruses.

[0011] A further aspect of the present method is the use of primers that are constructed such that they are most efficient at high cycling temperatures. High cycling temperatures can mean the amplification steps are performed at no less than about 65° C. In a specific embodiment of the present method, the amplification steps comprises about thirty rounds of subjecting the test sample to i) 5 seconds at 95° C and ii) 15 seconds at 65° C.

[0012] A still further aspect of the present invention is a method of rapidly processing a screen for an appropriate cell line for the production of a retroviral vector comprising the steps of combining unlysed, cell-free supernatant directly with a reaction mix to form a test sample; subjecting the sample to quantitative PCR (qPCR) reactions comprising the steps of reverse transcription, denaturation, annealing and amplification; optionally combining the annealing and amplification step; acquiring the fluorescence level of the test sample; comparing the reverse transcriptase concentration in the test sample to a standardized reverse transcriptase concentration curve to determine the quantity of reverse transcriptase, and therefore the quantity of retrovirus or retroviral vector, present in the test sample, and selecting the cell line producing the desired quantity of retrovirus or retroviral vector for further production uses.

[0013] In a further embodiment, the method of rapidly processing the screen for an appropriate cell line comprises the use of a reaction mix that includes a directly measurable dye. In one embodiment of the present invention, the measurable dye is SYBR Green or a dye derived from SYBR Green. Further, the reaction mix can exclude external reverse transcriptase or RNAase and can further comprise template RNA and forward and reverse primers specific for the template RNA. In the method of the present invention, the template RNA can be from a bacteriophage, such as MS2.

[0014] In a further embodiment, the reaction mix for the method of rapidly processing the screen for an appropriate cell line the reaction mix can comprises a fluorogenic probe rather than the directly measurable dye.

[0015] A further aspect of the present method for screening for an appropriate cell line is the use of primers that are constructed such that they are most efficient at high cycling temperatures. High cycling temperatures can mean the amplification steps are performed at no less than about 65° C. In a specific embodiment of the present method, the amplification steps comprises about thirty rounds of subjecting the test sample to i) 5 seconds at 95° C and ii) 15 seconds at 65° C.

[0016] A still further embodiment of the present invention is a method of quantifying a retrovirus or retroviral vector comprising the steps of combining unlysed, cell-free supernatant directly with a reaction mix to form a test sample wherein the reaction mix comprises MS2 template RNA and forward and reverse primers specific for the template RNA; where the forward and reverse primers are most efficient at no less than 65° C; subjecting the sample to quantitative PCR (qPCR) reactions comprising the steps of reverse transcription, denaturation, annealing, and amplification steps; wherein the amplification step comprises about thirty rounds of subjecting the test sample to i) 5 seconds at 95° C and ii) 15 seconds at 65° C; acquiring the fluorescence level of the test sample; and comparing the reverse transcriptase concentration in the test sample to a standardized reverse transcriptase concentration curve to determine the quantity of retrovirus or retroviral vector present in the test sample.

BRIEF DESCRIPTION OF THE FIGURES [0017] Representative embodiments of Assay Methods For Titration Of Viral Vectors are described with reference to the following figures.

[0018] Figure 1 A provides a typical workflow of the PERT assay of the prior art and

Figure IB provides a typical workflow of the Fast-PERT assay of the present invention. [0019] Figure 2A provides typical thermocycling parameters of the PERT assay of the prior art and Figure 2B provides typical thermocycling parameters of the Fast-PERT assay of the present invention.

[0020] Figure 3 shows example standard curves using 5-fold serial dilutions of recombinant HIV-1 reverse transcriptase with PERT and Fast-PERT assays, providing graphic evidence of similar dynamic range and linearity achieved using either method.

[0021] Figure 4 graphically demonstrates the test assay linearity of Fast-PERT for a variety of in process samples from lentiviral vector manufacture. This graph shows the assay has good linearity for all dilutions of all sample sources within the range of the standard curve.

DESCRIPTION OF THE INVENTION

[0022] In the summary and this detailed description, each numerical value should be read once as modified by the term "about" (unless already expressly so modified), and then read again as not so modified unless otherwise indicated in context. Also, in the summary and this detailed description, it should be understood that a physical range listed or described as being useful, suitable, or the like, is intended that any and every value within the range, including the end points, is to be considered as having been stated. For example, "a range of from 1 to 10" is to be read as indicating each and every possible number along the continuum between about 1 and about 10. Thus, even if specific data points within the range, or even no data points within the range, are explicitly identified or refer to only a few specific data points, it is to be understood that inventors appreciate and understand that any and all data points within the range are to be considered to have been specified, and that inventors possessed knowledge of the entire range and all points within the range.

[0023] Prior to setting forth the invention in detail, it may be helpful to the understanding of one of ordinary skill to define the following terms:

[0024] The term "cell-free" is used to denote supernatant or another form of a test sample that is substantially free of whole cells. The methods of achieving a cell-free sample are well known to one of ordinary skill and can include, but are not limited to, centrifugation, filtration, or leaving the sample on the bench for a suitable time period, for example, but not limited to five minutes, followed by removal of the liquid remaining above the settled materials.

[0025] The term “directly measurable dye” is used to denote a chemical with color changing properties that binds to other molecules where the amount of the dye present can be measured through the color change of the molecule and therefore be a measurement of the amount of the molecule that the dye binds to. Particularly useful in this context are those directly measurable dyes that bind dsDNA, such as SYBR Green I.

[0026] The term “Fast-PERT” is used to denote the improved method of the present invention which provides significant time saving and other efficiencies as compared to the standard PERT assay.

[0027] The term “high cycling temperature” is used to denote a range of thermocycling settings that are generally above the standard temperatures used in qPCR protocols.

[0028] The term “quantitative PCR (qPCR)” is used to denote a polymerase chain reaction wherein the goal of the process is to quantify the amount of target DNA present in the sample. This is done through the real time monitoring of the fluorescent signal detected in the test sample. The point at which the fluorescence intensity increases above the detectable level corresponds proportionally to the initial number of template DNA molecules in the sample. In general, qPCR comprises the steps of reverse transcription, denaturation, annealing, and amplification, where the annealing and amplification step can be optionally combined.

[0029] The term “PERT” stands for product enhanced reverse transcriptase assay. It is a reverse transcription assay that has increased sensitivity due to the addition of PCR amplification of the produced cDNA. This assay has additional enhancement of both accuracy and linear range through the use of qPCR for fast cDNA quantification. F-PERT, using cDNA- specific fluorogenic labeled probes for quantification, and SG-PERT, using SYBR Green I dye incorporation for quantification, are two nonlimiting types of PERT assays.

[0030] The term “retrovirus” is used to denote any of a group of RNA viruses which insert a DNA copy of their genome into the host cell to replicate. The viruses of the genus Lentivirus are representative examples.

[0031] The term “template RNA” is used to denote exogenous RNA that is added to a reaction mixture to serve as the starting template for the reverse transcriptase reaction in reverse transcription qPCR, e.g. it acts as the template for the production of the cDNA.

[0032] The term “unlysed” is used to denote a supernatant or another form of a test sample that has not undergone a lysis reaction through the deliberate use of a lysis buffer (e.g. including but not limited to use of a dedicated lysis buffer that utilizes detergent, such as TritonX-100 or any other anionic or ionic detergent or other physical lysis means) within the method.

[0033] The term “vector” is used to denote to a nucleic acid molecule which is able to artificially carry foreign (i.e. exogenous) genetic material into another cell, where it can be replicated and/or expressed. A “retroviral vector” is such a molecule that has been adapted from or incorporates within it nucleic acid sequences which are derived from retroviral genomes. One non-limiting example are lentiviral vectors, such as those based upon Human Immunodeficiency Virus Type 1 (HIV-1), which widely used as they are able to integrate into non-proliferating cells. Reviews of lentiviral vectors can be found in Sakuma et al., Biochem. J. 443(3): 603-18 (2012) and Picanqo-Castro et al., Exp. Opin. Therap. Patents 18(5): 525-539 (2008).

[0034] All references cited herein are incorporated by reference in their entirety.

[0035] The present invention is based in part upon the discoveries that the PERT assay methods of the prior art can be made more efficient through the elimination of the lysis step for the test sample, the elimination of RNAase inhibitor in the reaction mix, and the use of high temperature annealing primers to shorten the thermocycling time involved.

A. Description of PERT

[0036] The goal of reverse transcriptase (RT) assays is the measurement of the amount of enzyme present by measuring the amount of cDNA that is reverse transcribed during a reaction with the test sample. In all reverse transcriptase assays, an exogenous RNA template is added to the viral supernatant to estimate RT activity by determining the amount of RNA that is converted to cDNA by the retroviral RT. In the first generation RT assays, cDNA production was monitored through measuring labeled nucleotide incorporation. Sensitivity was greatly increased when a PCR amplification step of the synthesized cDNA was introduced prior to product detection. These types of assays are known as product-enhanced RT (PERT) assays. The newest PERT generation uses integrated qPCR techniques for fast cDNA quantification, further increasing the accuracy and linear range of the assays. The qPCR based PERT assays can use cDNA-specific fluorogenic labeled probes for signal generation (F-PERT) (see, Brorson et al., Biotechnol. Prog. 17: 188-196 (2001); Brorson et al. Biologicals, 30:15-26 (2002); Lovatt et al, J. Virolo. Meth.82: 185-200 (1999)). Alternatively, a one-step PERT assay using the more accessible and cost-efficient SYBR Green I chemistry (SG-PERT) can also be used (see, Pizzato et al., J. of Virol. Meth., 156:1-7 (2009)).

[0037] The first step of the prior art PERT assay, after thawing of samples and standards and optional dilution or clarification of the test sample through centrifugation and/or filtration, is a lysis step, see Figure 1 A. This can be accomplished by mixing a small amount, such as 5pL of the viral supernatant sample, with 5 pL of 2X concentrated lysis buffer, which includes RNAse inhibitor. One example of such a lysis buffer in the prior art includes .25% Triton X-100, 50 mM KC1, 100 mM Tris-HCL (pH 7.4), and 40% glycerol (see, Vermeire and Verhasselt, Bio-Protocol 3(l l):e780, June 5, 2013). RNAase inhibitor is then added to 0.4 U/pL. After the mixture of the lysis buffer and the sample, the mix is can be incubated at room temperature for a period of time, such as 10 minutes. This step can be further followed by a dilution with water of the lysate. Next, PCR master mix is prepared and the diluted lysate sample is added to the mix volume.

[0038] The PCR master mix utilized in the PERT assay, such as SG-PERT, can include the SYBR Green I dye containing PCR master mix, forward primer, reverse primer, MS2 template RNA and RNase inhibitor. This assay can be set up in 384 well plates, for example, if a LightCycler 480 PCR machine (Roche, Basel, Switzerland) is being utilized or in 96 well plates if an ABI 7300 real-time PCR system (Applied Biosystems, Bedford, MA) is being utilized. Practically, the particular plate set up is unimportant as long as there is a match between the plate utilized and the machine doing the measuring.

[0039] The next step in the standard PERT assay is qPCR, which is performed as well known in the art, for example, utilizing the PERT thermocycling process disclosed in Figure 2A. In general, the process steps involved are reverse transcription, denaturation, annealing and amplification. Optionally, the annealing and amplification step can be combined. The goal of these steps are to provide the concentration of cDNA present in the reaction mixture through the measurement of fluorescence. The point at which the fluorescence intensity increases above the detectable level corresponds proportionally to the initial number of template DNA molecules in the sample. The process of qPCR is well established in the art and is disclosed in at least U.S. Patent Nos. 5219727; 5538848; 5863736; and 6180349 (see also, Arya et al., Exp. Rev. Mol. Diag. 5(2): 209-19 (2005)). For embodiments of the methods of the present invention, this process can involve thermocycling steps which are particularly preferred for RT-qPCR measurements, for example, a 20 minutes at 42° C reverse transcription step, a 2 minutes at 95° C heat inactivation and denaturation step, followed by 40 cycles of annealing and elongation steps including 5 seconds at 95° C, 15 seconds at 55° C, and 30 seconds at 72° C (see, Vermeire et al., PLOS One, 7(12):e505859, 2012 and Vermeire and Verhasselt, Bio-Protocol 3(l l):e780, June 5, 2013). As discussed more fully below and well known to one of ordinary skill, the time and temperature of the thermocycling steps necessary for effective concentration analysis is very dependent on the specific primer sequences that are selected, as well as the choices of the template RNA. This thermocycling phase is followed by data analysis.

[0040] The data analysis step involves comparing a known reverse transcriptase concentration-fluorescence curve to the fluorescence curve provided by the qPCR process to provide the concentration of RT found in the test sample. Preferably, the standardization curve is produced at the same time as the experimental samples are tested and can be done using a serial dilution of any known RT, such as HIV-1 RT. For example, HIV-1 RT can be diluted to between 6.4 to 20,000 U per mL of PCR reaction (see Figure 3) and a curve produced from this dilution series. Comparison to the sample curve provides a likely RT concentration within the sample, given the observed fluorescence. Because the number of RT molecules per virion is expected to be similar between virions of the same retrovirus, RT activity provides a relative quantity of viral particles in the test sample (see, for example, Yi et al, Virologica Sinica, 29(5):314-1317 (2014) discussing quantification for prototype foamy virus (PFV) using PERT). Absolute quantities of infectious units or particle number may be estimated using correlations between RT activity and functional or ELISA based titration methods.

[0041] The data analysis step can be enhanced by particular considerations that are well known in the art. For example, various corrections concerning the data can be utilized to increase the reliability of the results such as arithmetic background correction and automatic noise band adjustment. It should be noted that the maximum number of fit points fitting the linear portion of the curve can be added for calculation of crossing points. The standard curve can be obtained using known concentrations of recombinant RT or through the use of defined virus suspension to allow an estimate of absolute quantification or data comparison between experiments. It is recommended that a minimum of four standard samples are included in the assay. The threshold line for crossing point calculation can be automatically defined by software in order to minimize errors of the standard curve. These and other well-known aspects to the handling of RT-PCR, qPCR, and PERT data, many inherent in the software utilized for these analyses, are standard knowledge for one of ordinary skill in this area.

B. Improvements provided by Fast-PERT

[0042] The following describes differences between Fast-PERT and standard PERT assays. These differences are also illustrated through comparison of Figure 1 A with Figure IB and Figure 2A with Figure 2B. [0043] Elimination of the lysis step. One of the time saving discoveries provided by the method of the present invention is the ability to eliminate the lysis step, and several related test sample manipulation steps, from the prior art PERT assay method. The present method begins with unlysed, cell-free test samples, such as viral supernatant or cell line supernatant to which the reaction mixture is directly added. The need for a lysis step is consistent in the prior art and would be expected, given the expected need to lyse the viral coat in order to access the RT that is being measured by the assay. However, the present disclosure and the data provided here indicates that a dedicated lysis step is unexpectedly not needed as sufficient access to the RT is provided merely through exposure to the reaction mix. This unexpected result, contrary to all teachings in the prior art, provides for significant time savings in practicing the Fast- PERT method as well as savings in reduced use of reagents and plasticware. Eliminating this step means there is less manipulation of the sample, thereby reducing the likelihood of mistakes and reducing the amount of variability due to cumulative pipetting errors. Overall, manually preparing 24 thawed samples for thermocycling takes approximately 10 minutes using Fast- PERT compared with 45 minutes using standard PERT.

[0044] Elimination of the use of RNAse inhibitor in the reaction mix. The present method also allows for the possible elimination of the use of RNAse inhibitor in the reaction mix. The need for RNAase inhibitor in the reaction mix is consistent in the prior art and would be expected, given the expected need to avoid degradation of the RNA present in the reaction. However, the present disclosure and the data provided here indicates that RNAase inhibitor is unexpectedly not needed for an effective assay process. The elimination of this component provides further savings on reagents and reduces manipulation steps as well as the possible pipetting errors accompanying the additional steps when using the Fast PERT method.

[0045] Reduced thermocycling time through use of high temperature primers and combined cycling steps. The present method saves significant amount of time by utilizing primers for the qPCR step which have been specifically selected for efficient function for high temperature thermocycling steps. Broadly speaking, primer annealing occurs typically at around 55° C but can be as high as about 70° C or as low as about 45° C. Duplex dissociation occurs typically around 94° C but can be lower depending on factors such as the length and percentage of guanine-cytosine base pairings (GC content) in the amplicon. In particular, the high temperature primers contemplated by the present method are efficient at no less than about 65° Celsius for the annealing step. Methods to adapt or select primer sequences that function more effectively at higher temperatures are known in the art and generally involve primers with higher percentages of guanine (G) and cytosine (C) content than those primers that function more effectively at lower temperatures. Thus, the description of the primers to be utilized within the present invention can be through GC content, if that is the characteristic that is being manipulated to arrive at higher Tm values. For example, primers can have GC content that range from about 60% to about 80%, with values of about 70% exemplified in the primers of Table 1 below.

[0046] The primers of Table 1 provide two exemplary sets of primers for the MS2 genome where the set comprising SEQ ID NO: 3 and SEQ ID NO: 4 have higher melting temperatures than those of the prior art.. For these particular primers, higher melting temperatures was achieved using choices in GC content, but a common other approach to achieve this is to increase the length of the primer. If primer length is the primary consideration for increasing Tm, then GC content can be a larger range, for example, can go as low as about 30%. Generally, primers are recommended to not go much below 18 nucleotides, thus increasing the length of primers to increase Tm will involve nucleotide counts above 18, for example about 25 to about 40 nucleotides.

[0047] In particular, in one embodiment of the present invention, at least one or both primers involved in the qPCR process have melting temperatures above about 70° Celsius which allows the entire thermocycling process to be efficiently performed at or above 65° Celsius. Embodiments of the present invention are also contemplated where the annealing step can be performed at any range of temperature from about 65° to about 75°, including performance of the annealing step (or combination of the annealing and elongation step, see discussion below) at 65°, 66°, 67°, 68°, 69°, 70°, 71°, 72°, 73°, 74°, or 75° or any fraction thereof between those temperatures. It is within the purview of one of ordinary skill to select similar primers for other template RNA sequences or even to provide an artificially constructed template RNA that comprises the sequences needed to have primers that function well at high thermocycling temperatures. However, as taught herein by the present inventors, the commonly used MS2 viral genome includes sites that can bind primers with the desired high temperature specificity without the need of building from scratch a suitable template RNA.

Table 1 - Exemplary primer sequences for standard PERT and Fast-PERT.

¹ Determined using OligoEvaluator™ (Sigma Aldrich, St. Louis, MO)

² MS2 genome, NCBI accession number MK213795.1

[0048] Considerations for developing the template RNA and appropriate primer sequences take into account the time saving resulting from the use of higher melting temperatures for the selected primers. This can be accomplished through judicious selection of particular RNA templates that comprise sequences that accommodate the higher melting temperature goal. Template RNA for RT-PCR is traditionally derived from positive-sense single stranded bacteriophage RNA genomes, as such sources are relatively short commercially available RNA with known sequences. This selection also has the advantage of simplifying setting up the PERT assay in high throughput settings, as specialized RNA template preparations are not needed. As described above, MS2 genome is a very common choice, but other sources of RNA are within the scope of the present invention. For example, the RNA template could be specially synthesized for use in the present invention. Alternatively, other RNA-based bacteriophages could be utilized, such as those within the family Leviviridae including the genuses Levivirus and Allolevivirus, particularly if sequences with the desired high melting temperatures are within such genomes. See, Tars K. (2020) ssRNA Phages: Life Cycle, Structure and Applications. In: Witzany G. (eds) Biocommunication of Phages. Springer, Cham., for more information about possible bacteriophage RNA templates. Further, mRNA for the production of particular virus proteins can be utilized, for example, mRNA enriched for the production of glycoprotein D (gD) produced by the US6 gene of herpes simplex virus 1, has been isolated from gD producing transfectants I143tk cells, and utilized as an RNA template in a PERT assay (see, Macchi et ak, Pathogens, 9(12): 1047 (2020)). Practically, any RNA sequence with the desired length and sequence characteristics to allow for high temperature primers to be designed and utilized can be used as template RNA in the present methods.

[0049] Selection of templates and design of possible primers with desired Tm characteristics utilize skills sets well known in the art and step-by-step guides are widely available from manufacturers of PCR devices. See, for example, Guidelines for real-time RT-PCR assay optimization by Qiagen (http s : // www .qiagen.com/- /media/project/qiagen/qiagen-home/content-worlds/pcr/12-real -time-qpcr/prom-l 1405- 001-an-guidelinesfor-rtpcr-0917-ww.pdf); Basic Principles of RT-qPCR by Thermo-Fisher (https://www.thermofisher.com/us/en/home/brands/thermo-scien tific/molecular- biology/molecular-biology-learning-center/molecular-biology- resource-library/spotlight- articles/basic-principles-rt-qpcr.html), and A Step-by-Step Guide to Designing qPCR primers by New England Biolabs (https://bitesizebio.com/10041/designing-qpcr-primers/).

Briefly, prime considerations for primer design in general include template length, with about 18 nucleotides commonly considered a minimum, as described previously. Melting temperature (Tm), also as discussed previously, is a second important consideration. In particular, one exemplary method of figuring the melting temperature of primer sequences longer than 13 nucleotides is calculated by

[0050] Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC); where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

[0051] However, it should be noted that there are many approaches utilized (see,

Panjkovich and Melo, Bioinformatics, 21(6):711-722 (2005) for comparisons of various approaches) and thus, one of ordinary skill can select the method that best suits the goals of the Fast-PERT assay. GC content, directly related to Tm as more GC increases the Tm as can be seen by the above equation, is a still further consideration. A further consideration is that runs of identical nucleotides, such as multiple guanosines, should be avoided. Primers that can form dimers and primers that include palindromes are not ideal because of the production of secondary structures, either between primers or internally, reducing efficiency. Each of these design considerations are within the purview of one of ordinary skill, particularly when combined with the specific characteristics desired to perform the Fast-PERT method, as presently disclosed.

[0052] A further time saving measure utilized by the methods of the present invention is the combination of the annealing and elongation steps into one step during qPCR thermocycling. This combination reduces temperature changes during the qPCR process and the time involved in heating or cooling the blocks holding the samples, while still maintaining efficient elongation by the thermostable polymerase, of which a nonlimiting example is the Taq polymerase. This effect is discussed in Montgomery et ah, J. Mol. Diagnos. 16(3) (2014). A thermocycling process with an exemplary combination of these steps is illustrated in Figure 2B and described in Example 1. C. Uses of the Fast-PERT Assay

[0053] Fast-PERT was specifically developed to aid in the quick titration of viral concentrations during commercial vector production, in particular lentiviral vector production. The ability of the present method to function effectively in the vector manufacturing setting is experimentally shown in Example 3 and illustrated in Figure 4. In particular, samples taken from a range of time points during the manufacturing process including clarification step, nuclease treatment step, chromatography 1 step, chromatography 2 step, and at the concentration step were shown to have good linearity for all dilutions within range of the standard curve. Even the presence of the nuclease treatment, a sugar non-specific nuclease step, did not inhibit the qPCR reaction at appropriate sample dilutions despite the absence of RNAse inhibitor. Thus, this assay has proven useful in making viral titration more efficient during all stages of viral vector commercial production. A particular embodiment is the use of this assay to understand the volume needed to aliquot the produced vectors into single dose amounts at the end of the process or to normalize load prior to chromatography.

[0054] This assay is also extremely effective in providing a means to do high throughput screens of various candidate cell lines that are producing viral vectors, to easily understand the viral production level of each potential cell line so that decisions can be made as to which will continue on in the selection process. Notably, the simplification of the process is an important impact because it allows for more efficient automation of the high throughput method. There are cost and environmental savings based on using less plasticware or glassware and further, less reagants. Also, by reducing the amount of time each titration process takes, more cell lines can be screened in the same time, thereby increasing the number of candidate cell lines that can be screened in the allotted time or reducing the amount of time needed to screen a given number of cell lines. Therefore, this is a second possible application of the Fast- PERT method to viral vector commercial production. However, even though this assay has significant utility in the viral vector commercial manufacturing setting, this is far from the only uses of the Fast-PERT assay even within pharmaceutical production.

[0055] The assay of the present invention can also be utilized for the screening of small molecule drug candidates on viral production. Essentially, the drug candidates are administered to retroviral producing model systems, and the assay is used to quickly measure the amount of virus that is present after exposure to the drug. The most significant impact is the simplification of the process, allowing for easy automation of its execution. Cost and environmental savings plus less reagent use contribute to the economic applicability of this method to this use. Also, in high throughput screening settings, the time savings in quantification of the virus again allows for either the ability to screen more drug candidates in the same time period or, alternatively, the ability to screen more candidates in a set time. This application of the present Fast-PERT method is a further contemplated embodiment of its uses in pharmaceutical commercial settings.

[0056] The Fast-PERT assay also has applicability in the patient diagnostic setting. For example PERT has been utilized in the detection and quantification of HIV-1 particles in patient plasma samples (see, Boni et al., J. Med. Virol., 49(l):23-8 (1996)), and Fast-PERT can be similarly utilized for these and other related diagnostic measures involving retroviruses. It has distinct advantages of being likely more sensitive than RNA PCR and not impacted by sequence subtypes due to the use of SYBR Green I rather than a sequence specific quantification method. Fast-PERT brings these advantages as well as time savings. Since the description by Boni et al., the PERT assay has continued to be used in this setting, and more recently adapted for use on blood samples (see, Macchi et al., Pathogens, 9(12): 1047 (2020)). Results demonstrated that plasma HIV-1 RT levels, expressed as cycle threshold values obtained with the real time RT quantitative PCR assay described by Macchi et al., were inversely and highly significantly correlated with the plasma HIV-l-RNA levels of the patients. Such an assay can be adapted to a Fast-PERT assay as described in the present specification, thereby reducing the time needed for performance.

[0057] PERT assays have also been utilized in biological product safety testing.

Specifically, the testing has been used in three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load; and (iii) monitoring levels of infectious retroviral generation in cell lines that contain endogenous retroviruses (see, Chang and Dusing, Dev. Biol. (Basel); 123:91-7 (2006)). A particular area of concern is the present of adventitious retroviruses in human vaccine production (see, Andre et al., Biologicals, 28(2):67-80 (2000)). Assays for the detection of RT activity are used as general methods for detection of both known and unknown retroviruses in viral production in mammalian cell based production systems, such as chicken cells. As such, in each of these areas of product testing, a conversion of a traditional PERT assay to Fast-PERT could provide savings without altering the ability of the assay to function in the product safety testing area. Therefore, the use in product safety testing, both biological or otherwise, involving the detection of retroviruses is a still further contemplated use of the methods of the present invention. [0058] The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1 - Fast-PERT Assay

[0059] Cell-free viral supernatant was generally used as input for the assay with HIV-

1 recombinant RT (Abeam, Cambridge, UK) as standard. The HIV-1 RT was stored in aliquots at -80°C in 2 mU/mL in RT storage buffer (0.25% Triton X-100, 100 mM Tris-HCl, 50 mM KC1 and 40% glycerol, all from Sigma-Aldrich, UK). To generate the standard curve, an aliquot of 2 mU/mL HIV-1 RT was diluted 1 in 100 with nuclease-free water (Ambion, UK) followed by a 5-fold serial dilution in nuclease-free water to achieve a 6-point standard curve with concentrations between 20 000 pU/mL and 6.4 pU/mL. Unconcentrated test samples were typically diluted 1 in 100 with nuclease-free water while concentrated samples were typically diluted 1 in 2500 with nuclease-free water. For 96-well plate Fast-PERT assays using either the Quantstudio 12k flex or StepOnePlus instruments (Thermofisher, UK), 5 pL of diluted sample or standard was transferred to a MicroAmp Optical Fast 96-Well Reaction Plate (Thermofisher) that already contained 15 pL of a PCR master reaction mix consisting of 10 pL 2x PowerUp SYBR Green master mix and 5 pL of Fast-PERT mix (containing 2 pM forward and reverse primers and 0.1 pL MS2 RNA. The reaction was carried out according to the following program: 42° C for five minutes; 95° C for five minutes; then 32 cycles of 95° C for five seconds and 65° C for fifteen seconds. Fluorescence acquisition was done at the end of the 65° C hold.

[0060] All reagents were kept on ice or on a cooling block during preparation of the assay. For each sample Fast-PERT was performed in triplicate reactions. Cycles of quantification (Cq) values were generated by the software of the qPCR instruments after manually setting the threshold to 0.132.

[0061] The general workflow for this assay is visually presented in Figure IB. The thermocycling conditions are visually presented in Figure 2B.

Example 2 - Standard Curve Production and Assay Comparison

[0062] To perform absolute quantification of RT activity values, a standard curve of 5-fold serial dilutions of recombinant HIV-1 RT was run in parallel with each assay and values were extrapolated from the obtained Cq values. Dilutions were aliquoted for use in different assays, to avoid loss of RT activity by repeated freeze-thaw cycles.

[0063] Following the sample preparation and thermal cycling described in Example 1,

Figures IB and Figures 2B (Fast-PERT) and those described by Vermeire et al., PLOS One, 7(12):e505859, 2012, also described in Figures 1 A and 2A (PERT), the results for the produced standard curves using the recombinant HIV-1 RT are in Figure 3. As shown, using manual pipetting by experienced operators, similar dynamic range and linearity was achieved using either approach. Quantification of RT activity was linear between at least 6.4 and 20 000 pU RT activity per mL (concentration of samples added to PCR reaction). All R ² values (goodness of fit) were greater than 0.99. PCR efficiencies were similar (97 and 100% for Fast-PERT, 96, 97 and 99% using standard PERT). In Figure 3, the mean Ct value from triplicate reactions is shown from independent assay replicates (PERT n=3, Fast-PERT n=2)

Example 3 - Fast-PERT during Commercial Viral Vector Production

[0064] Various in-process samples from a 2-stage chromatography downstream process of clinically relevant lentiviral vector were serial diluted to test assay linearity of Fast- PERT using typical samples generated during lentiviral vector manufacture. The process was performed as described in Example 1, Figures 1A and 2A. Results are shown in Figure 4. One of the samples was obtained following a sugar non-specific nuclease treatment step, but assay inhibition at low dilutions was not apparent. There was good linearity for all dilutions within range of the standard curve. The R ² goodness of fit for the line of best fit was greater than 0.99 for all samples and PCR efficiency ranged from 93 to 103%. In Figure 4, mean averages from triplicate PCR reactions are shown for samples with Ct values within the range of the reference standards (6.4 to 20 000 pU RT/mL).

[0065] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.