Provided are methods of detecting replication competent retrovirus in a sample containing a cell transduced with a viral vector particle encoding a recombinant and/or heterologous molecule, e.g., heterologous gene product. The methods may include assessing transcription of one or more target genes, such as viral genes, that are expressed in a retrovirus but not expressed in the viral vector particle. Replication competent retrovirus may be determined to be present if the levels of RNA of the one or more target genes is higher than a reference value, which can be measured directly or indirectly, including from a positive control sample containing RNA from the respective target gene at a known level and/or at or above the limit of detection of the assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. application Ser. No. 16/320,076 filed Jan. 23, 2019 entitled “Methods for Assessing the Presence or Absence of Replication Competent Virus”, from U.S. provisional application No. 62/369,024 filed Jul. 29, 2016, entitled “Methods for Assessing the Presence or Absence of Replication Competent Virus”, and from U.S. provisional application No. 62/448,954 filed on Jan. 20, 2017, entitled “Methods for Assessing the Presence or Absence of Replication Competent Virus”, the contents of which are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042005610SeqList.TXT, created Sep. 26, 2022, which is 40,511 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

FIELD

[0003] The present disclosure relates to methods of detecting or confirming the absence of replication competent retrovirus. The methods may include assessing RNA levels of one or more target genes, such as viral genes, e.g. structural or packaging genes, from which gene products are expressed in certain cells infected with a replication-competent retrovirus, such as a gammaretrovirus or lentivirus, but not present in a viral vector used to transduce cells with a heterologous nucleic acid and not, or not expected to be, present and/or expressed in cells not containing replication-competent retrovirus. Replication competent retrovirus may be determined to be present if RNA levels of the one or more target genes is higher than a reference value, which can be measured directly or indirectly, e.g. from a positive control sample containing the target gene.

BACKGROUND

[0004] Retroviral vector particles, such as gammaretroviral and lentiviral vector particles, are used in various clinical applications, including for the introduction of therapeutic genes into cells and/or subjects. Such viral vector particles are engineered to be replication defective, however, in many instances it may be desirable or even necessary to verify the absence of replication competent virus (e.g., as replication competent retrovirus (RCR) or replication competent lentivirus (RCL)) in a sample or composition, such as a therapeutic or pharmaceutical composition formulated for administration. For example, in certain applications, methods are used to verify or confirm that no RCR has resulted during generation or processing steps, such as through homologous or non-homologous recombination between the transfer vector, packaging components, and/or endogenous viral elements in the cells used for production of the viral vector particles. Various methods are available for such confirmation and verification, such as to verify the absence of replication competent virus, for example during or after generation and processing, in formulated therapeutic compositions and/or drug products for administration, such as engineered cells and/or in samples from subjects, e.g., those having received therapies containing cells transduced with viral vector particles. However, existing methods can be overly time consuming and/or carry a risk of false positive results. There is a need for improved methods are needed for detecting RCR.

SUMMARY

[0005] Provided herein are methods including: a) determining a level of a parameter in a test sample, wherein the parameter or level indicates or correlates (optionally positively or inversely) with a presence, absence, or amount or concentration of a viral RNA in a biological sample, the biological sample including at least one cell that contains a heterologous nucleic acid and/or a nucleic acid encoding a heterologous protein, wherein: the presence, absence or amount or concentration of the viral RNA in the biological sample indicates a presence or absence of, or risk of, a replication competent virus in the biological sample, or a sample from which the biological sample is derived; and/or the viral RNA is required for, or encodes a gene product or specifically identifiable portion thereof that is required for, replication competency of a replication competent virus.

[0006] In some embodiments, the method further includes determining the presence, absence, concentration, or amount of the viral RNA in the biological sample or risk thereof, based on said level so-determined. In some embodiments, the method further includes the steps of: b) comparing the level of the parameter, determined in (a), to a first reference value for the parameter. In some embodiments, the comparison indicates the presence, absence, concentration or amount of the viral RNA in the biological sample or sample derived therefrom, or risk of any of the foregoing.

[0007] In some embodiments, the method further includes determining the presence, absence, or amount or concentration of the viral RNA, or risk of any of the foregoing, in the biological sample or portion thereof; and/or determining whether replication competent virus is (or is potentially or is likely to be) present or at risk for being present in the biological sample, or a portion thereof.

[0008] In some embodiments, the biological sample is deemed to have, to potentially have, or to be at risk for, presence of the replication competent virus, and/or to have the presence or at least a threshold amount of the viral RNA, if, and optionally only if, the level of the parameter determined in (a) is at or is above the first reference value, optionally wherein the level of the parameter positively correlates with the amount of the RNA in the biological sample; and/or the biological sample is deemed to have, or to be at risk for, the presence of the replication competent virus, and/or to have the presence or at least a threshold amount of the viral RNA, if, and optionally only if, the level of the parameter determined in (a) is at or below the first reference value, optionally wherein the level of the parameter inversely correlates with the amount of viral RNA in the biological sample; the biological sample, and/or one or more of the at least one cell, is deemed RCR negative, or is deemed to not have, to not be at risk for, or to not potentially contain the presence of replication competent virus, if, and optionally only if, the level of the parameter determined in (a) is below the first reference value, optionally wherein the level of the parameter positively correlates with the amount of the RNA in the biological sample; and/or the biological sample and/or one or more of the at least one cell is deemed not to have or not to be at risk for the presence of the replication competent virus, and/or not to have the presence or at least a threshold amount of the viral RNA, if, and optionally only if, the level of the parameter determined in (a) is above the first reference value, optionally wherein the level of the parameter inversely correlates with the amount of viral RNA in the biological sample.

[0009] In some embodiments, the biological sample is so-deemed negative, not to have, or not to be at risk, even if another replication competent-required viral RNA is determined to be present in the biological sample; the biological sample is so-deemed negative, not to have, or not to be at risk, even if a level, at or above a second reference value, of a second parameter that is positively correlated with an amount of another replication competent-required viral RNA in the biological sample, is or has been detected in the test sample or a second test sample derived from or containing nucleic acid from the biological sample; and/or the biological sample is so-deemed negative, not to have, or not to be at risk, even if a level, at or below a second reference value, of a second parameter negatively correlated with an amount of another replication competent-required viral RNA, is or has been detected in the test sample or another test sample derived from or containing nucleic acid from the biological sample.

[0010] In some embodiments, the biological sample is deemed to have, to potentially have, or to be at risk for the presence of the replication competent virus, if (and optionally only if) the presence of the viral RNA in the biological sample is determined, and/or if (and optionally only if) an amount, which optionally is at or above a threshold amount, of the viral RNA is determined to be in the biological sample; the biological sample is deemed to potentially have or to be at risk for the presence of the replication competent virus if (and optionally only if) the presence of the viral RNA in the biological sample is determined, and/or if (and optionally only if) an amount, which optionally is at or above a threshold amount, of the viral RNA is determined to be in the biological sample, but is not deemed to contain the presence of the replication competent virus without further indication of risk and/or without assessing another parameter indicative of another viral RNA; and/or the biological sample is deemed not to have, not to potentially have, and/or not to be at risk for the presence of the replication competent virus, if the absence of the viral RNA in the biological sample is determined, and/or if an amount (or no more than the amount), which amount optionally is at or below a threshold amount, of the viral RNA is determined to be in the biological sample, optionally even if the presence of another RNA required for replication competency of the virus is determined to be present in the biological sample; and/or the biological sample is deemed replication competent virus negative if the parameter and/or the viral RNA is undetectable or is not detected in the test sample and/or is not determined to be present in the biological sample.

[0011] In some embodiments, the first reference value is a value at or approximately at or just above a threshold level or a minimum detectable level or readout corresponding thereto; the first reference value is a value of the parameter detected in, and/or a value of a parameter indicative of an amount of RNA in, a positive control sample; and/or the level of the parameter indicates the presence or the absence of the viral RNA in the biological sample; and/or the viral RNA includes a nucleic acid encoding a first viral gene; and/or the heterologous nucleic acid encodes a heterologous gene product.

[0012] In some embodiments, the parameter assessed in the test sample is or includes an amount or relative amount of the viral RNA, or a product expressed therefrom or from a viral gene corresponding to the RNA, which optionally is a relative copy number, or a relative weight, or is or includes a concentration, or relative concentration, of the viral RNA, or of a product expressed therefrom or from a viral gene corresponding to the RNA. In some embodiments, the amount is an absolute or relative amount. In some embodiments, the parameter and/or level is or includes a surrogate or relative value, which optionally is a cycle threshold (Ct) value.

[0013] In some embodiments, the viral RNA or the expression thereof is determined to be present or at risk of being present in the biological sample or in the test sample, if the CT value for the test sample is below a first reference value, which is a reference Ct score. In some embodiments, wherein the test sample is or is derived from the biological sample or a portion thereof.

[0014] Also provided herein are methods including a) determining or assessing a first level of a first parameter in a test sample, wherein said first level or first parameter correlates (optionally positively or inversely) with a presence, absence, amount or concentration, in a biological sample, of a first viral RNA and/or a first nucleic acid encoding a first viral gene that encodes the first viral RNA; b) determining or assessing a second level of a second parameter in a test sample, which optionally is the same or a different test sample, wherein said second level or second parameter correlates (optionally positively or inversely) with a presence, absence, amount or concentration, in the biological sample, of a second viral RNA distinct from the first viral RNA and/or a second nucleic acid encoding a second and distinct viral gene encoding said second viral RNA; wherein at least one cell that contains a heterologous nucleic acid, a heterologous gene product, and/or a nucleic acid encoding a heterologous protein and/or has been transduced with a viral vector; wherein: the presence, absence or amount or concentration of the first viral RNA or gene or the second viral RNA or gene, and/or the presence, absence, or amount or concentration of both the first and the second viral RNA or gene, in the biological sample indicates a presence or absence of, or risk of, a replication competent virus in the biological sample, or a sample from which the biological sample is derived; and/or the first viral RNA, the second viral RNA, and/or both the first and the second viral RNA, is required for, or encodes a gene product or specifically identifiable portion thereof that is required for, replication competency of a replication competent virus.

[0015] In some embodiments, the method further includes determining the presence, absence, concentration, or amount of the first and/or the second viral RNA or gene product in the biological sample, or risk thereof, based on said level(s) so-determined. In some embodiments, the method further includes c) comparing the level of the first and/or the second parameter determined in (a) and/or (b) to a first and/or a second reference value, wherein the comparison optionally indicates the presence, absence, concentration or amount of the viral RNA in the biological sample or sample derived therefrom, or risk of any of the foregoing.

[0016] In some embodiments, the method further includes c) determining the presence, absence, or amount or concentration of the first viral RNA or expression, or risk of any of the foregoing, in the biological sample or portion thereof, and/or determining the presence, absence, or amount or concentration of the second viral RNA or expression, or risk of any of the foregoing, in the biological sample or portion thereof; and/or d) determining whether a replication competent virus is (or is potentially or is likely to be) present or at risk for being present in the biological sample or a portion thereof, optionally based on the determination in c).

[0017] In some embodiments, the viral RNA is from and/or the first viral RNA is from and/or the first viral gene is env, gag, pol, or rev; and/or the viral RNA is from and/or the second viral RNA is from, and/or the second viral gene is, env, gag, pol, or rev.

[0018] Also provided herein are methods including a) assessing a level of a first parameter in a test sample, which is indicative of or correlates (optionally positively or inversely) with of an amount or presence or absence of a first viral RNA in a biological sample, wherein the first viral RNA is from a first viral gene that is an env gene, and assessing a level of a second parameter in a test sample, which optionally is the same or a different test sample, wherein the second viral parameter is indicative of or correlates (optionally positively or inversely) with a presence, absence or amount of a second viral RNA in the biological sample, wherein the second viral RNA is from a gene selected from a gag, pol, and rev gene, wherein the biological sample includes a cell transduced with a viral vector particle, optionally including a heterologous gene product and/or includes a cell with a heterologous gene product; wherein at least one cell that contains a heterologous nucleic acid, a heterologous gene product, and/or a nucleic acid encoding a heterologous protein and/or has been transduced with a viral vector; wherein: the presence, absence or amount or concentration of the first viral RNA or gene or the second viral RNA or gene, and/or the presence, absence, or amount or concentration of both the first and the second viral RNA or gene, in the biological sample indicates a presence or absence of, or risk of, a replication competent virus in the biological sample, or a sample from which the biological sample is derived; and/or the first viral RNA, the second viral RNA, and/or both the first and the second viral RNA, is required for, or encodes a gene product or specifically identifiable portion thereof that is required for, replication competency of a replication competent virus.

[0019] In some embodiments, parameter is a presence or absence of viral RNA. In some embodiments, the reference value, the first reference value and/or the second reference value is or corresponds to a threshold level or a minimum detectable level. In some embodiments, the reference value, the first reference value and/or the second reference value is a value corresponding to or for the parameter in a positive control test sample, which optionally contains a known amount or concentration of the viral RNA, the second viral RNA and/or the first viral RNA.

[0020] In some embodiments, the biological sample is deemed to contain or to be at risk for containing or to potentially contain a replication competent virus if the method determines the presence or an amount above a threshold amount of the first and the second viral RNA in the biological sample, and optionally not if the method determines the presence or an amount above a threshold amount for one but not both the first and second RNAs; and/or the biological sample is deemed negative for or not to contain or to not be at risk for containing or not potentially containing a replication competent virus, if the method determines the absence of, the absence of a detectable amount of, or an amount below a threshold amount of, the first viral RNA, or the second viral RNA, and/or both the first and the second viral RNA, in the biological sample.

[0021] In some embodiments, the biological sample is deemed replication competent virus negative if the level of viral RNA encoding the first and second viral genes is undetectable in the test sample and/or is not determined to be present or present above a threshold level in the biological sample.

[0022] In some embodiments, the method further includes a step of isolating RNA from the biological sample or a portion therefrom or a sample or portion thereof derived from the biological sample, prior to step a), wherein the RNA contains RNA from one or more of the at least one cell.

[0023] In some embodiments, the replication competent virus is or includes a retrovirus, and/or wherein the viral RNA or the first and/or the second viral RNA is expressed by a retrovirus. In some embodiments, the retrovirus is a gammaretrovirus. In some embodiments, the retrovirus is a lentivirus.

[0024] In some embodiments, the biological sample is from a human or mammal and/or the one or more cell is a primary cell, which is optionally a human or mammalian cell. In some embodiments, the one or more cell and/or biological sample is from a master cell bank (MCB), a working cell bank (WCB), or a cell line, or a sample thereof. In some embodiments, the at least one cell or biological sample is or is from a cryopreserved material (CMAT), a cryopreserved drug product (CDP), or a formulated drug product (FDP) for autologous cell therapy, or a sample thereof. In some embodiments, the at least one cell or biological sample is or is from a cell that is undergoing expansion, such as ex-vivo expansion, after transduction.

[0025] In some embodiments, the heterologous nucleic acid or heterologous gene product is or encodes a recombinant receptor, a chimeric receptor, optionally a chimeric antigen receptor, or a transgenic T cell receptor.

[0026] In some embodiments, the determining or assessing is carried out using one or more oligonucleotide primers specific for a sequence of the viral RNA, the first viral RNA and/or the second viral RNA. In some embodiments, the level of viral RNA encoding the second viral gene is assessed using one or more oligonucleotide primers specific for a sequence of the second viral gene. In some embodiments, the level of viral RNA is assessed by real-time polymerase chain reaction (PCR). In some embodiments, the determining or assessing includes carrying out reverse transcriptase quantitative PCR (RT-qPCR).

[0027] In some embodiments, the viral RNA and/or the first and/or second viral RNA and/or viral RNA and/or gene is from a retrovirus. In some embodiments, the viral RNA, the first viral RNA and/or second viral RNA (or gene encoding any of the foregoing) is or is encoded by a gene involved in virion replication and/or packaging. In some embodiments, the viral RNA and/or the first viral RNA and/or the second viral RNA (or gene encoding one or more of the foregoing) is not a gene encoded by a transfer vector that has been used to transduce the transduced cell. In some embodiments, the viral RNA, the first viral RNA and/or the second viral RNA encodes a viral surface protein, an envelope protein, a group-specific antigen, a virally-derived polymerase, a virally-derived reverse transcriptase, a virally-derived regulatory element, a trans activator of transcription, or a response element.

[0028] In some embodiments, the gag gene is selected from the group consisting of murine leukemia virus (MMLV) gag and Human Immunodeficiency Virus (HIV) gag. In some embodiments, the env gene is selected from GaLV env and VSVG. In some embodiments, the one or more oligonucleotide primers specific for a sequence of the first viral gene include one or more sequences set forth in SEQ ID NOs: 4-5. In some embodiments, the one or more oligonucleotide primers specific for a sequence of the first viral gene include one or more sequences set forth in SEQ ID NOs: 16-24. In some embodiments, the one or more oligonucleotide primers specific for a sequence of the second viral gene include one or more sequences set forth in SEQ ID NOs: 4-5. In some embodiments, the one or more oligonucleotide primers specific for a sequence of the second viral gene include one or more sequences set forth in SEQ ID NOs: 16-24.

[0029] In some embodiments, the assessing or determining includes use of a hydrolysis probe specific for a sequence of the viral RNA, the viral gene, the first viral RNA or the first viral gene, or the second viral RNA or second viral gene. In some embodiments, the hydrolysis probe specific for a sequence of the first viral gene includes a sequence set forth in SEQ ID NO: 6. In some embodiments, the hydrolysis probe specific for a sequence of the first viral gene includes a sequence set forth in SEQ ID NO: 18, 21, or 24.

[0030] In some embodiments, the assessing, determining or detecting includes using a hydrolysis probe specific for a sequence of one or more of the viral RNA, second viral RNA, first viral RNA, and/or second viral gene. In some embodiments, the hydrolysis probe includes a sequence set forth in SEQ ID NO: 6. In some embodiments, the hydrolysis probe specific for a sequence of the first viral gene includes a sequence set forth in SEQ ID NO: 18, 21, or 24.

[0031] In some embodiments, the method further includes assessing in the test sample a level of, or a level of a parameter indicative of or correlative with, an RNA encoding a control gene in the test or biological sample, optionally wherein the control gene is or includes β-actin and/or optionally wherein the level of the parameter or control gene is assessed using one or more oligonucleotide primers specific to a sequence of the control gene, which individually optionally include one or more sequences set forth in SEQ ID NO: 1 or 2 or one of 8-15, optionally wherein the level is assessed using a hydrolysis probe specific for a sequence of the control gene, which optionally includes a sequence set forth in SEQ ID NO: 3, 9, 12, or 15.

[0032] In some embodiments, the assessment or determining includes carrying out a multiplex reaction, wherein optionally the level, the first level, and/or the second level; and optionally the level or parameter indicative or correlative with the control gene, is assessed in the multiplex reaction.

[0033] In some embodiments, the parameter, the first parameter, and/or the second parameter, individually, is or includes an amount or relative amount of the viral RNA (or first or second viral RNA), or a product expressed therefrom or from a viral gene corresponding to the RNA, which optionally is a relative copy number, or a relative weight, or is or includes a concentration, or relative concentration, of the viral RNA (or first or second viral RNA) or of a product expressed therefrom or from a viral gene corresponding to the RNA (or first or viral RNA).

[0034] In some embodiments, the amount is an absolute or relative amount. In some embodiments, the parameter and/or level is or includes a cycle threshold (Ct) value. In some embodiments, the viral RNA or the expression thereof is determined to be present or at risk of being present in the biological sample or in the test sample, if the CT value for the test sample is below a first reference value, which is a reference Ct score.

[0035] In some embodiments, the biological sample and/or the one or more cells is or are from a subject. In some embodiments, said at least one cell includes a plurality of cells, and wherein: said plurality of cells and/or said biological sample includes suspension cells; said plurality of cells and/or said biological sample includes white blood cells; and/or said plurality of cells and/or said biological sample includes T cells or NK cells.

[0036] In some embodiments, one or both of the first test sample and the second test sample, individually is derived from or contains RNA derived from the biological sample or a portion thereof. In some embodiments, the test sample assessed for the first viral RNA and the test sample assessed for the second RNA are the same or are portions of the same sample or composition. In some embodiments, said plurality of cells includes unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells. In some embodiments, said at least one cell is a human cell. In some embodiments, the test sample is or is a portion of the biological sample.

[0037] In some embodiments, acceptance criteria are set to assess validity of the real-time PCR. In some embodiments, the acceptance criteria include a percent efficiency of between or between about 90% and 110%. In some embodiments, the acceptance criteria include an R2 value of about or greater than at or about 0.95, 0.96, 0.97, 0.98, or 0.99. In some embodiments, the methods further include assessing the purity, integrity, and/or concentration of the RNA.

[0038] Also provided are primers including an oligonucleotide including a sequence set forth in any of SEQ ID NOs: 1-24. In some embodiments, the primer includes a fluorescent moiety or label.

[0039] Also provided are kits including one or more primers according. In some embodiments, the kit further includes one or more of nuclease-free water, a reverse transcriptase, a polymerase, deoxynucleotide triphosphates, a buffer, and a DNase.

[0040] In some embodiments, the method is capable of detecting the viral RNA or the first and/or the second viral RNA in a test sample in which at least 5 or at least 10 or at least 20 or at least 50 or at least 100 cells in the sample, or per 10 million cells in the test sample or biological sample; and/or wherein the method is capable of detecting an amount of target RNA that is no more than at or about 1.5 pg, 1 pg, or 0.75 pg or less of the viral target, in the test sample and/or the biological sample.