NZ712211B2 - Thymidine kinase diagnostic assay for gene therapy applications - Google Patents

Abstract

Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases (HSV-TK) are provided, including their use in diagnostic and therapeutic applications. The HSV-TK polynucleotide encodes a mutated form of HSV-TK comprising mutations at amino acid residues 32, 33, and 168, wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisting of glycine, serine, glutamic acid, an acidic amino acid and cysteine, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK. Also provided are gene therapeutic systems, including viral and retroviral particles. mino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisting of glycine, serine, glutamic acid, an acidic amino acid and cysteine, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK. Also provided are gene therapeutic systems, including viral and retroviral particles.

Description

(12) Granted patent specificaon (19) NZ (11) 712211 (13) B2 (47) Publicaon date: 2021.12.24 (54) THYMIDINE KINASE DIAGNOSTIC ASSAY FOR GENE THERAPY APPLICATIONS (51) Internaonal Patent Classificaon(s): A01N 63/00 A61K 48/00 A61K 49/00 C12N 15/00 (22) Filing date: (73) Owner(s): 2014.03.14 GENVIVO, INC. (23) Complete specificaon filing date: (74) t: 2014.03.14 PHILLIPS ORMONDE FITZPATRICK (30) Internaonal Priority Data: (72) Inventor(s): US 61/784,901 3.14 LEVY, John, P.

REED, Rebecca, A. (86) Internaonal Applicaon No.: MCNULTY, Joseph JOHNSON, Robert, G., Jr. (87) Internaonal Publicaon number: WO/2014/153205 (57) ct: Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases (HSV-TK) are ed, including their use in diagnosc and therapeuc applicaons. The HSV-TK polynucleode encodes a d form of HSV-TK comprising mutaons at amino acid residues 32, 33, and 168, wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisng of glycine, serine, glutamic acid, an acidic amino acid and cysteine, and wherein the mutated form of HSV-TK increases cell kill acvity relave to a wild-type HSV-TK. Also provided are gene euc systems, including viral and retroviral parcles. 712211 B2 INE KINASE DIAGNOSTIC ASSAY FOR GENE THERAPY APPLICATIONS CROSS-REFERENCE This application claims the benefit of US. Provisional Application No. 61/784,901, filed on March 14, 2013, which is incorporated herein by reference in its entirety.

This application is related to the following co-pending patent applications: application Serial No. [not yet assigned], Attorney Docket No. 30863-722201, filed the same day herewith, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Proliferative diseases, such as cancer, pose a serious challenge to society. Cancerous growths, including malignant cancerous growths, possess unique characteristics such as uncontrollable cell proliferation resulting in, for example, unregulated growth of malignant tissue, an ability to invade local and even remote s, lack of differentiation, lack of able symptoms and most significantly, the lack of effective y and tion.

Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not clearly defined but mechanisms such as genetic susceptibility, chromosome breakage disorders, viruses, environmental factors and immunologic ers have all been linked to a malignant cell growth and transformation. Cancer encompasses a large category of medical conditions, affecting millions of individuals worldwide. Cancer cells can arise in almost any organ and/or tissue of the body. Worldwide, more than 10 million people are diagnosed with cancer every year and it is estimated that this number will grow to 15 n new cases every year by 2020. Cancer causes six million deaths every year or 12% of the deaths worldwide.

SUMMARY OF THE INVENTION Provided herein are methods and compositions for identifying subjects or patients that are capable of benefitting from gene therapy treatment. More specifically, ed herein are methods and compositions for identifying ts or patients that express in ent quantities a therapeutic protein included in a gene therapy agent. Preferably the therapeutic protein is an enzyme, more specifically viral thymidine kinase or mutant viral thymidine .

Accordingly, provided herein are methods for identifying a patient capable of benefitting from gene therapy treatment comprising administering a gene therapy retroviral particle comprising an HSV-TK polynucleotide to the t; administering to the patient a substrate of HSV-TK attached to a radioactive tracer; ing the relative amount and location of the ctive signal present in the patient; and determining the location of lesions in the patient, wherein patients with: radioactive signals above a n threshold, and location of the radioactive signal correlating with lesions measured in step (d) of the patient, are identified as capable of benefitting from gene therapy treatment.

In some embodiments, the substrate of HSV-TK is chosen from the group consisting of FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9~([3~fiuoro—l ~hydroxy—2— propoxy]meihyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'- deoxy-2'-fluoro-l-B-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoro-S-methyl-l-beta-D-arabinofuranosyluracil), FHOMP (6- ((l -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4- hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-S -FEP) or 5-(2-)hydroxyethyl)- and 5- (3 -hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains. In yet other embodiments, the ate of HSV- TK is FHBG (9-(4-fluoro(hydroxymethyl)butyl]guanine).

In still other embodiments, the radioactive tracer is 18F, 64Cu, 99mTe, 11C, 14C, 1241, 123I, 131I, 15O, 13N and/or . In other embodiments, the radioactive tracer is 18F.

In one ment, the HSV-TK substrate is [18F]FHBG (9-(4-18F-fluoro (hydroxymethyl)butyl]guanine). In still other embodiments, the radioactive tracer signal is measured using positron emission tomography (PET) ng.

In still other embodiments, the threshold level is at least above 2.0 SUV (standardized uptake value) or at least 20% above ound on a PET scan, or n about 1.0 SUV and about 3.0 SUV, or n about 20% to about 40% above background on a PET scan.

In some embodiments, the methods disclosed herein further comprises treating the patient with the HSV-TK retroviral particle.

In still other embodiments, the viral nuclear localization sequence (NLS) of the encoded HSV-TK polynucleotide is mutated. In yet other embodiments, the thymidine kinase polynucleotide is mutated to include a nuclear export sequence (NES) at or near the amino terminus of the expressed ine kinase protein. In one embodiment, the thymidine kinase polynucleotide is d to increase substrate binding of the expressed thymidine kinase protein. In another embodiment, the on is Al68H.

In still other embodiments, the methods disclosed herein further comprises mutating the thymidine kinase polynucleotide to remove the viral nuclear zation sequence (NLS) and include a nuclear export sequence (NES) at or near the amino terminus of the expressed thymidine kinase protein. In some embodiments, the HSV-thymidine kinase polynucleotide is SEQ ID NO: 18.

In one ment, the methods disclosed herein further comprises a targeting protein expressed on the viral envelope. In some embodiments, the targeting protein binds to collagen, laminin, fibronectin, elastin, aminoglycans, proteoglycans or RGD. In still other embodiments, the targeting protein binds to collagen. In yet other embodiments, the targeting protein is SEQ ID NO: 25.

Also provided herein are methods and compositions for identifying a t or subject in need of treatment for lesions and capable of benefitting from gene therapy treatment: a) administering a gene therapy retroviral particle comprising an HSV-TK cleotide and transducing cells from the patient with the polynucleotide encoding HSV-thymidine ; b) ng the cells with a substrate of HSV-TK attached to a radioactive tracer; c) measuring the ve amount of radioactive signal present in target tissue; d) fying patients wherein the level of radioactively-labelled HSV-TK substrate is above a threshold; e) determining the location of lesions in the t; and f) treating said patient or t with the gene therapy retroviral particle comprising an HSV-TK polynucleotide when the measured radioactive signal in the patient is above a certain old, and the location of the measured radioactive signal correlates with lesions measured in step (e) of the patient.

Provided herein are methods and compositions for measuring the enzymatic activity of a transduced HSV-thymidine, the method comprising: a) administering a gene therapy retroviral particle sing an HSV-TK polynucleotide and transducing cells from the t with the polynucleotide encoding HSV-thymidine kinase; b) treating the cells with a substrate of HSV- TK attached to a radioactive ; and c) measuring the relative amount of ctive signal present in target tissue.

In on, provided herein are methods and compositions for determining the level of a tracer signal in a subject or patient after administration of a gene therapy particle, and selecting the subject or patient for treatment with the gene therapy particle when the level of the tracer signal is above a set threshold. In some embodiments, the tracer is a radioactive, luminescent or a cent signal. In some embodiments, the radioactive tracer element is 18F, 64Cu, 99mTe, 11C, 14C, 1241, 1231, 1311, 150’ 13N and/or 82RbCl.

In yet other embodiments, provided herein are methods and compositions for determining the level of a radiotracer signal in a subject or patient after administration of a thymidine kinase gene therapy construct, and selecting the subject or patient for treatment with the gene therapy construct when the level of the tracer signal is above a set threshold. In some embodiments, the tracer is a radioactive tracer. In other embodiments, the radioactive tracer element is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl. In yet other embodiments, the radioactive tracer element is coupled to a nucleoside or synthetic nucleoside target to form a radioactive . In some embodiments, the nucleoside target is FHBG (9-[4- fluoro(hydroxymethyl)butyl] guanine), FHPG (EMT[3~fluoro~ l roxva— propoxflmethyi,jbguanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l -(2'- deoxy-2'-fluoro-l-B-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoro-S-methyl-l-beta-D-arabinofuranosyluracil), FHOMP (6- ((l -fluorohydroxypropanyloxy)methyl)methylpyrimidine-2,4( l H,3H)—dione), ganciclovir, val-ganciclovir, acyclovir, val-aciVlovir, penciclovir, radiolabeled pyrimidine with oxy(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)— and -(3 -hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovirganciclovir and penciclovir-like side chains. Preferably the ctive target is [18F]FHBG (9- (4-18F-fluoro[hydroxymethyl]butyl)guanine).

Also ed herein are methods comprising: (a) determining the level of [18F]FHBG signal in a subject; and (b) selecting the subject for treatment with a composition wherein the level of FHBG is above a threshold level. In some embodiments, the threshold level is at least about 2.0 SUV (standardized uptake values) or at least 20% above background signal on a PET scan.

Additionally provided herein is a method comprising: (a) determining the level of [18F]FHBG signal in a t; (b) excluding the subject from treatment with a ition wherein the level of FHBG in the t is greater than about 2.0 SUV or greater than about % above background signal on a PET scan; and (c) stering to said subject an anti- cancer agent.

In some embodiments, the ion provides a method for identifying a subject that is susceptible to a cancer treatment, the method sing: a) identifying expression of [18F]FHBG in the subject; b) treating the subject.

Provided herein is a method of ing HSV-TK-FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine), FHPG (9»{I3ufiuomw l ~hydro:<y~2»propoxy]iriethylkiyguanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D- arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi.1ranosyluracil), FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-((l-fluoro hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir , acyclovir, val-aciVlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3- hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-like, ganciclovir-like and penciclovir-like side chains-mediated bystander effect, the method comprising: a) transducing cells with a polynucleotide encoding HSV-TK and a first fluorescent WO 53205 protein; b) transducing the cells with a polynucleotide encoding a second or bioluminescent protein that is optically discernible from the first fluorescent or bioluminescent protein; c) treating the cells with an agent that becomes xic upon being phosphorylated by HSV-TK; and d) measuring the relative amount of expression of the first fluorescent n and the second fluorescent protein. In one embodiment, step d) comprises a Perkin Elmer Plate reader, a fluorimeter; a fluorescent activated cell sorter (FACS); a cellometer; or a spectrophotometer. In another embodiment, step d) comprises measuring fluorescent output of the second fluorescent or inescent protein in vivo in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS The novel es of the invention are set forth with particularity in the appended claims. A better understanding of the es and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the anying drawings of which: Figure 1 illustrates the structure of 9-[4-[18F]Fluoro(hydroxymethyl)butyl]guanine ([18F]FHBG) and its mechanism of inhibition.

Figure 2 is whole body coronal images of [18F]FHBG biodistribution in a healthy human subject at four distinct time periods after 4.53 mCi iv injection of the [18F]FHBG.

Figure 3 is a schematic for a Phase IA clinical trial.

Figure 4 is a schematic for a Phase IB clinical trial.

Figure 5 is a patient’s response to ent with HSV-TK in AAV retroviral vector particle.

Figure 6 measuring the response of a patient to administration of HBG in PET Scan (top panel), CT Scan e panel) and fiJsion of signals (bottom panel).

Figure 7 is a cent image of the biodistribution of the HSV-TK retroviral vector particles in s.

Figure 8 is a comparison of coronal three hour images of 5 mm slices in rats administered Reximmune Cl and C2. The tumor on the left expressed Reximmune-C2 and on the right is Reximmune-Cl.

Figure 9 is a graphical representation of Figure 8, showing the averages of the mean within tumors for one and three hour images. Error bars are standard error of the averages. B12 is the Reximmune-C2 expressing tumor and A9 the Reximmune-Cl expressing tumor. C6 is the native cell line tumor.

DETAILED DESCRIPTION OF THE INVENTION Provided herein are methods and compositions for identifying a patient susceptible to treatment with a gene therapy delivery system. Also provided herein are methods and compositions for identifying subjects or patients that are capable of benefitting from gene therapy treatment. er, provided herein are methods and compositions for fying subjects or patients that express in sufficient quantities a therapeutic protein included in a gene therapy construct. The identification of subjects or ts that are capable of expressing sufficient quantities of a therapeutic protein allows a practitioner to screen and identify patients that can benefit from a particular gene therapy treatment. By doing so, patients and subjects are identified at an early stage that are capable of delivering anti-cancer agents via gene therapy particles to treat, for example, primary and metastatic lesions.

In some embodiments, anti-cancer agents expressed from gene therapy constructs included in viral particles can be administered to patients by enous infusion. In yet other embodiments, anti-cancer agents expressed from gene therapy constructs can be administered to patients via inter-arterial infilsion. In yet other embodiments, the viral particles containing anti- cancer agents can be stered intra-tumoral. In still other embodiments, anti-cancer agents expressed from gene y constructs can be selectively transduced in vitro into target cells.

In yet other embodiments, anti-cancer agents expressed from gene therapy constructs can be targeted to primary and metastatic lesions, y delivering a tumor-killing gene to y and metastatic s while sparing normal cells and tissues. In some embodiments, the ing of gene therapy constructs is ic. In yet other embodiments, the targeting of gene therapy constructs is to a cell-surface or extracellular protein. In some embodiments, the cell- surface or extracellular protein is collagen. In yet other embodiments, the targeting of gene therapy constructs is to a c protein expressed by tumor cells. Such anti-cancer agents provide a powerful tool that can specifically target cancer cells, thereby mitigating the ed side-effects of other known cancer ies.

In some ments, the gene therapy uct is a retrovirus. Retroviruses typically have three common open reading frames, gag, pol, and env, which encode the matrix, gag and nucleocapsid structural ns, encode enzymes including reverse transcriptase, integrase and protease, and encode envelope proteins and transmembrane fusogenic proteins, respectively.

Typically, retroviral vector particles are produced by packaging cell lines that provide the necessary gag, pol, and env gene products in trans. (Miller, et al., Human Gene Therapy, Vol. 1, pgs. 5-14 (1990)). This approach results in the production of retroviral vector particles which transduce mammalian cells, but are incapable of filrther replication after they have integrated into the genome of the cell.

WO 53205 In some ments, the retrovirus comprises at least one therapeutic protein or payload delivered by the gene therapy construct. In some embodiments, the therapeutic protein or payload is an enzyme. In yet other embodiments, the therapeutic protein or payload is thymidine kinase. In still other embodiments, the thymidine kinase is HSV (herpes simplex virus) thymidine kinase. In yet other embodiments, the thymidine kinase is HSV (herpes simplex virus) thymidine -l.

In some embodiments, the HSV-TK gene therapy construct is optimized with respect to maximal gene expression and tumor kill activity both in vitro and in viva including cancer gene therapy. In some embodiments, the HSV-TK gene is codon-optimized. In still other ments, the HSV-TK gene therapy construct is targeted to a specific tumor cell or tissue.

In yet other embodiments, the HSV-TK gene therapy construct is targeted to a cell-surface protein specifically expressed in tumor cells. In still other ments, the HSV-TK gene therapy construct is targeted to a cell-surface protein expressed in tumor tissue or cells. In other embodiments, the HSV-TK gene therapy construct is targeted to collagen.

When sed in vivo in cells, HSV-TK enzymatically cleaves a co-adminstered nucleoside agent, such as ganciclovir, lovir, val-ganciclovir, acyclovir and val-aciclovir, and subsequently transforms the inistered agent into a cytotoxic agent. Mammalian thymidine kinases are insensitive to these co-administered agents. Sensitivity to the cytotoxic agent is therefore only conferred upon tumor cells after expression of the HSV-TK gene.

Ganciclovir is converted by the resulting HSV-TK to the monophosphorylated product, which is then converted to di- and triphosphates by host kinases, leading to cytotoxicity and tumor cell death. Viral thymidine kinase therapy has been previously shown to have promise in the ent of several cancers, including s, hepatoma and melanoma.

HSV-TK also selectively orylates the nucleoside analogue of, for example, 9-[4- 18F-fluoro(hydroxymethyl)butyl]guanine ([18F]FHBG) (FIG. I), which cleaves the radioactive tracer 18F from the FHBG molecule. HSV-TK expression can therefore be closely monitored with positron emission tomography (PET) scans.

Accordingly ed herein are methods and compositions for determining the level of a tracer signal in a subject or patient after administration of a gene y vector, and selecting the subject or patient for treatment with the gene therapy vector when the level of the tracer signal is above a set threshold. In some ments, the tracer is a radioactive, luminescent or a fluorescent signal. In some embodiments, the radioactive tracer element is 18F, 64Cu, 99mTe, 11C, 14C, 1241’ 1231’ 1311’ 150, 13N and/or 82RbCl.

In yet other embodiments, provided herein are methods and compositions for determining the level of a radiotracer signal in a subject or patient after administration of a thymidine kinase gene therapy vector, and selecting the subject or patient for treatment with the gene therapy vector when the level of the tracer signal is above a set threshold. In some embodiments, the tracer is a ctive tracer. In other embodiments, the radioactive tracer t is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123 I, 131I, 15O, 13N and/or 82RbCl. In yet other embodiments, the radioactive tracer element is coupled to a side or synthetic nucleoside target to form a radioactive target. In some embodiments, the nucleoside target is FHBG (9-[4- fluoro(hydroxymethyl)butyl]guanine), FHPG (9%[3 ~fluoro~ l y—Qw propoxyknethyfi)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'- deoxy-2'-fluoro-l-B-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoro-S-methyl-l-beta-D-arabinofuranosyluracil), FHOMP (6- ((l -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4- hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-S -FEP) or 5-(2-)hydroxyethyl)- and 5- (3 xypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir- ganciclovir and penciclovir-like side chains. ably the radioactive target is [18F]FHBG (9- (4-18F-fluoro[hydroxymethyl]butyl)guanine).

Also provided herein are methods comprising: (a) determining the level of [18F]FHBG signal in a subject; and (b) selecting the subject for treatment with a gene therapy composition wherein the level of [18F]FHBG is at least about 2.0 SUV or at least 20% above background on a PET scan.

Additionally provided herein is a method comprising: (a) determining the level of [18F]FHBG signal in a t; (b) including the subject with treatment with a composition wherein the level of FHBG in the subject is greater than about 2.0 SUV on PET scan; and (c) administering to said subject an anti-cancer agent.

In some embodiments, the invention es a method for identifying a subject that is susceptible to a cancer treatment, the method comprising: a) identifying expression of HBG in the t; b) treating the t.

Provided herein is a method of measuring HSV-TK-mediated FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine), FHPG (9~([3wfluoron l nhydroxynzpropoxfimethybguanin c), FGCV (fluoroganciclovir), FPCV penciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D- arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi1ranosyluracil), FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-((l-fluoro hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir, vir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3- 2014/029600 hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains-mediated bystander effect, the method comprising: a) ucing cells with a polynucleotide encoding HSV-TK and a first fluorescent protein; b) transducing the cells with a polynucleotide encoding a second fluorescent or bioluminescent protein that is optically discernible from the first fluorescent or bioluminescent protein; c) treating the cells with an agent that becomes cytotoxic upon being phosphorylated by HSV-TK; and d) ing the ve amount of expression of the first fluorescent protein and the second fluorescent protein. In one embodiment, step d) comprises a Perkin Elmer Plate reader, a fluorimeter; a fluorescent activated cell sorter (FAC S); a cellometer; or a spectrophotometer. In r embodiment, step d) comprises measuring cent output of the second fluorescent or bioluminescent protein in vivo in the subject.

Also provided herein are methods for determining the level of [18F]FHBG signal in a subject and selecting the subject for treatment with a gene therapy composition wherein the level of [18F]FHBG is at least about 2.0 SUV or at least 20% above background on a PET scan.

DEFINITIONS Unless defined ise, all technical and scientific terms used herein have the same g as is commonly understood by one of skill in the art to which the invention(s) belong.

All patents, patent applications, published applications and publications, k sequences, websites and other hed materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the intemet can come and go, but equivalent information can be found by ing the intemet. Reference thereto evidences the availability and public dissemination of such information.

As used herein, “nucleic acid” refers to a polynucleotide containing at least two covalently linked nucleotide or nucleotide analog subunits. A nucleic acid is generally a ibonucleic acid (DNA), a ribonucleic acid (RNA), or an analog of DNA or RNA.

Nucleotide analogs are commercially available and methods of preparing polynucleotides containing such tide analogs are known (Lin et al. (1994) Nucl. Acids Res. 0-5234; Jellinek et al. (1995) Biochemistry 34: 1 1363-1 1372; Pagratis et al. (1997) Nature hnol. :68-73). The nucleic acid is generally single-stranded, double-stranded, or a mixture thereof.

For purposes herein, unless specified otherwise, the nucleic acid is double-stranded, or it is apparent from the context.

As used herein, “DNA” is meant to include all types and sizes ofDNA molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.

As used herein, “nucleotides” include nucleoside mono-, di-, and triphosphates.

Nucleotides also include modified nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.

The term “polynucleotide” as used herein means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single- and double-stranded DNA, as well as single-and double-stranded RNA. The term also includes modified polynucleotides such as methylated or capped polynucleotides.

As used , the term “subject” refers to animals, , insects, and birds into which the large DNA molecules are introduced. Included are higher organisms, such as mammals and birds, including humans, primates, rodents, , pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chicken and others.

As used herein, “administering to a subject” is a procedure by which one or more delivery agents and/or large nucleic acid molecules, together or separately, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent and/or the large nucleic acid molecules.

As used , “delivery vector” or “delivery vehicle” or “therapeutic vector” or “therapeutic system” refers to both viral and non-viral particles that harbor and ort exogenous c acid molecules to a target cell or tissue. Viral vehicles include, but are not limited to, iruses, adenoviruses, lentiviral viruses, herpes viruses and adeno-associated viruses. Non-viral vehicles include, but are not d to, microparticles, nanoparticles, virosomes and liposomes. “Targeted,” as used herein, refers to the use of ligands that are ated with the delivery vehicle and target the e to a cell or tissue. Ligands include, but are not limited to, antibodies, receptors and collagen-binding domains.

As used herein, “delivery,” which is used interchangeably with “transduction,” refers to the s by which exogenous nucleic acid les are transferred into a cell such that they are located inside the cell. Delivery of c acids is a distinct process from expression of c acids.

As used herein, a “multiple cloning site (MCS)” is a nucleic acid region in a plasmid that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these ction enzymes are commercially ble. Use of such enzymes is widely tood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.

As used herein, n of replication” (often termed “ori”), is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be ed if the host cell is yeast.

As used herein, “selectable or screenable markers” confer an identifiable change to a cell permitting easy identification of cells ning an sion vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the ce of the marker allows for its selection, while a negative selectable marker is one in which its presence ts its selection. An example of a positive selectable marker is a drug resistance .

Usually the inclusion of a drug selection marker aids in the cloning and fication of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. In some embodiments, screenable enzymes such as herpes simplex Virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) are utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is e of being expressed simultaneously with the c acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

As used herein, “expression” refers to the process by which nucleic acid is ated into peptides or is transcribed into RNA, which, for example, can be translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression includes, if an appropriate eukaryotic host cell or organism is ed, splicing of the mRNA. For heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.

As used , a “therapeutic course” refers to the periodic or timed administration of the vectors disclosed herein within a defined period of time. Such a period of time is at least one day, at least two days, at least three days, at least five days, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, or at least six months.

Administration could also take place in a chronic , z'.e., for an undefined period of time.

The periodic or timed administration includes once a day, twice a day, three times a day or other set timed administration.

As used herein, the terms “co-administration, 3, nistered in combination with” and their grammatical equivalents or the like are meant to encompass stration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different times. In some embodiments, a therapeutic agent as disclosed in the present application will be co-administered with other agents. These terms ass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present. Thus, in some embodiments, a therapeutic agent and the other agent(s) are administered in a single composition. In some embodiments, a therapeutic agent and the other agent(s) are admixed in the composition. In further embodiments, a therapeutic agent and the other agent(s) are administered at separate times in separate doses.

The term “host cell” denotes, for e, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients for multiple constructs for producing a delivery vector. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein generally refers to a cell which has been ected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be tely identical in logy or in genomic or total DNA complement as the al parent, due to natural, accidental, or deliberate on.

As used herein, “genetic therapy” involves the transfer of heterologous DNA to the certain cells, target cells, of a , particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is uced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product d thereby is produced. In some embodiments, the heterologous DNA, directly or ctly, mediates expression ofDNA that encodes the eutic product. In some embodiments, the heterologous DNA encodes a product, such as a peptide or RNA that mediates, directly or indirectly, expression of a therapeutic t. In some embodiments, genetic therapy is used to deliver a nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. In some embodiments, the introduced c acid encodes a therapeutic compound, such as a growth factor or inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefore, that is not generally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. In some embodiments, the logous DNA encoding the therapeutic product is modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.

As used herein, “heterologous nucleic acid sequence” is generally DNA that encodes RNA and proteins that are not ly produced in vivo by the cell in which it is sed or that mediates or encodes ors that alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes. Any DNA that one of skill in the art would recognize or er as heterologous or n to the cell in which it is sed is herein encompassed by heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of ns, such as antibodies. In some embodiments, antibodies that are encoded by heterologous DNA is secreted or expressed on the e of the cell in which the heterologous DNA has been introduced.

As used herein, the term “thymidine kinase mutant” refers to not only the specific protein described herein (as well as the nucleic acid sequences which encode these ns), but derivatives thereof which may e various structural forms of the primary protein which retain biological actiVity.

As used herein, “unmutated thymidine kinase” refers to a native or wild-type thymidine kinase polypeptide sequence.

As used herein, “suicide gene” refers to a nucleic acid encoding a product, n the product causes cell death by itself or in the present of other compounds.

As used herein, the term “mutated” or “replaced by another nucleotide” means a nucleotide at a certain position is replaced at that position by a nucleotide other than that which occurs in the unmutated or previously mutated sequence. That is, in some instances, specific modifications may be made in different nucleotides. In some embodiments, the replacements are made such that the relevant splice donor and/or acceptor sites are no longer present in a gene.

As used herein, a “polar amino acid” refers to amino acid residues Asp(N), Cys (C), Gln (Q), Gly (G), Ser (S), Thr (T) or Tyr (Y).

As used herein, a olar amino acid” refers to amino acid residues Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), or Val (V).

As used herein, a “basic amino acid” refers to amino acid es Arg (R), His (H), or Lys (K).

As used herein, an c amino acid” refers to amino acid residues Asp (D) or Glu (E).

GENE THERAPY Gene therapy involves the transfer of heterologous DNA to certain cells of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is introduced into the selected target cells in a manner such that the logous DNA is expressed and a therapeutic product encoded thereby is produced.

In some embodiments, the heterologous DNA, directly or indirectly, es expression ofDNA that encodes the therapeutic t. In some embodiments, the heterologous DNA encodes a product, such as a peptide or RNA that mediates, directly or indirectly, expression of a therapeutic product. In some embodiments, the introduced nucleic acid encodes a therapeutic compound, such as a grth factor or inhibitor f, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefore, that is not generally produced in the mammalian host or that is not produced in therapeutically effective s or at a therapeutically useful time.

Non-viral and viral methods have been used to deliver heterologous therapeutic DNA into the cell, including viral vector particles derived from retrovirus, irus, adeno- associated viral particles, herpes virus les, vaccinia virus, lentivirus, pox virus, Semliki virus and pseudotyped viruses.

Accordingly, provided herein are viral constructs for gene transfer to cells either in viva, ex vivo or in vitro for gene therapy. Such viral vector les include, but are not limited to retroviral vector particles, adenoviral vector particles, adeno-associated virus particles, herpes virus particles, pseudotyped viruses, lentiviral vector particles, pox virus vector particles, vaccinia virus vector particles and ral vectors. ably, the viral vector particle is a retroviral vector le.

RETROVIRAL CONSTRUCTS In some embodiments, the vector particle employed for gene therapy use is a retroviral vector particle. In still other embodiments, the retroviral vector particle is derived from Moloney Murine Leukemia Virus and is of the LN series of vectors, such as those hereinabove mentioned, and described r in Bender, et al., J. Virol., Vol. 61, pgs. 1639-1649 (1987) and Miller, et al., Biotechniques, Vol. 7, pgs 980-990 (1989). Such vectors, have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term ed" as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.

In some ments, the retroviral vector particle includes a modified envelope, including at least one polynucleotide encoding at least one heterologous polypeptide to be expressed in a desired cell. The heterologous polypeptide may, in one embodiment, be a eutic agent. The therapeutic agent is thymidine kinase, more preferably HSV-TK.

In still other embodiments, therapeutic agents e, but are not limited to, grth factors such as, for example, epidermal grth factor (EGF), vascular elial growth factor (VEGF), erythropoietin, G-CSF, GM-CSF, TGFu, TGF-B, and fibroblast growth factor, cytokines, including, but not limited to, interleukins and tumor necrosis s. Other therapeutic agents include, but are not limited to, anticoagulants, latelet agents, antiinflammatory agents, tumor suppressor proteins, clotting factors, including Factor VII, Factor VIII and Factor IX, protein S, n C, antithrombin III and von Willebrand Factor.

In some embodiments, the polynucleotide encoding the eutic agent is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; the histone promoter; the polIII promoter, the B-actin promoter; inducible ers, such as the MMTV promoter, the metallothionein promoter; heat shock promoters; adenovirus promoters; the albumin promoter; the ApoAI promoter; B l 9 parvovirus promoters; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex Virus thymidine kinase promoter; retroviral LTRs; human growth hormone promoters, and the MxIFN inducible promoter. The promoter also may be the native promoter which controls the polynucleotide encoding the therapeutic agent.

The polynucleotides encoding the modified envelope polypeptide and the therapeutic agent may be placed into an appropriate vector by genetic engineering techniques known to those skilled in the art. When the modified vector is a retroviral vector particle, the polynucleotides encoding the modified envelope polypeptide and the therapeutic agent are placed into an appropriate iral plasmid vector.

The retroviral plasmid vector es one on more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) er bed in Miller, et al., hniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the e, pol III, and B-actin promoters).

Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those d in the art from the teachings ned herein.

WO 53205 In one embodiment, the retroviral plasmid vector, which includes a cleotide encoding the modified envelope and a polynucleotide encoding a therapeutic agent, is employed to transduce a packaging cell line to form a producer cell line, which will generate ious retroviral vector particles. In one embodiment, the packaging cell line is a "pre-packaging" cell line which includes polynucleotides encoding the gag and pol retroviral proteins, but not the pe, or env, n. Such cell lines, upon transduction with the retroviral plasmid vector, generates infectious iral les including the d, or chimeric, envelope and a polynucleotide encoding the therapeutic agent. The vector may transduce the ing cells through any means known in the art. Such means include, but are not limited to, electroporation, and use of liposomes, such as hereinabove described, and CaPO4 precipitation. Such producer cells generate infectious iral vector particles which include the modified envelope, the wild-type retroviral envelope, a polynucleotide encoding the modified, or chimeric, envelope, and a polynucleotide encoding a therapeutic agent.

In another embodiment, there is provided a ing cell which es a nucleic acid sequence encoding a modified chimeric envelope in accordance with the invention, and which may further include nucleic acid sequences encoding the gag and pol proteins. A producer cell for generating viral particles which includes a modified envelope in accordance with the invention is produced by introducing into such packaging cell either a retroviral vector particle or a retroviral plasmid vector, in each case including a polynucleotide encoding a therapeutic agent. The producer cell line thus generates infectious retroviral particles including the modified chimeric envelope and the polynucleotide encoding the therapeutic agent.

TARGETED RETROVIRAL VECTOR RY In some embodiments, ed herein are vector particles having a modified viral surface protein, such as, for example, a modified viral envelope polypeptide, for targeting the vector particle to an extracellular matrix component. The viral surface protein is modified to include a targeting polypeptide including a binding region which binds to, an ellular matrix ent.

In some ments, the targeting polypeptide is ed between two consecutively numbered amino acid residues of the native (i.e., unmodified) receptor binding region of the retroviral envelope. In yet other embodiments, amino acid residues of the receptor binding region may be removed and replaced with the targeting polypeptide.

As an alternative to modifying the receptor binding region, or in addition to the modified or binding region, the retroviral particles may have modifications in other regions of the envelope protein such that other regions of the envelope may include the targeting polypeptide, such as, for example, the secretory signal or "leader" sequence, the hinge region, or the body -l6- 2014/029600 portion. Such modifications may include deletions or substitutions of amino acid residues in the retroviral envelope wherein amino acid residues from s other than the receptor binding region of the envelope are removed and replaced with the targeting polypeptide, or the targeting polypeptide is placed between consecutively numbered amino acid residues of regions other than the receptor binding region of the viral envelope.

In another alternative embodiment, the retroviral envelope, prior to modification thereof to include the targeting polypeptide which binds to the extracellular matrix component, may be an envelope which includes regions of different tropisms. For example, the retroviral envelope may be a y Murine Leukemia Virus envelope which includes a gp70 protein having an ecotropic portion and an amphotropic and/or xenotropic portion.

In general, the targeting polypeptide includes a binding region which binds to an extracellular matrix component, including, but not limited to, collagen (including collagen Type I and collagen Type IV), laminin, f1bronectin, elastin, aminoglycans, proteoglycans, and ces which bind to fibronectin, such as arginine-glycine-aspartic acid, or RGD, sequences.

Binding regions which may be included in the targeting polypeptide include, but are not limited to, polypeptide domains which are functional domains within von rand Factor or derivatives thereof, wherein such polypeptide domains bind to collagen. In one embodiment, the binding region is a polypeptide having the following structural formula: Trp-Arg-Glu-Pro-Ser— t-Ala-Leu-Ser. (SEQ ID NO: 25).

In addition to the binding region, the targeting ptide may fiarther include linker sequences of one or more amino acid residues, placed at the N-terminal and/or C-terminal of the binding region, whereby such s increase rotational flexibility and/or minimize steric hindrance of the modified envelope polypeptide.

HSV-TK Thymidine kinase is a salvage pathway enzyme which phosphorylates l nucleoside substrates as well as nucleoside analogues. Generally, ine kinase is used therapeutically by administration of a nucleoside analogue such as ganciclovir or vir to a cell expressing ine kinase, wherein the ine kinase phosphorylates the nucleoside analogue, creating a toxic t capable of killing the cell.

Polynucleotide sequences encoding ous thymidine kinase as used herein may be prepared from a wide variety of thymidine kinases. In some embodiments, the thymidine kinase mutant is derived from Herpesvz’rz’dae thymidine kinase including, for example, both e herpes viruses, and non-primate herpes viruses such as avian herpes viruses. Representative examples of suitable herpes viruses include, for example, Herpes Simplex Virus (HSV) Type 1, Herpes Simplex Virus Type 2, Varicella zoster Virus, marmoset herpes virus, feline herpes virus type 1, pseudorabies virus, equine herpes virus type 1, bovine herpes virus type 1, turkey herpes virus, Marek's disease virus, herpes virus saimir and Epstein-Barr virus.

IMPROVEMENTS TO TK GENE Disclosed herein, in some embodiments, is a polynucleotide sequence ng HSV- TK. In some embodiments, the polynucleotide sequence encodes a wild-type HSV-TK amino acid sequence. In some embodiments, the polynucleotide ce s a mutated HSV-TK amino acid ce.

Exemplary procedures that may be used in preparation of an optimized polynucleotide ce ed herein include, but are not limited to: codon optimization; correction of splice sites, removal of yrimidine tracts and excess GC content; addition of single Kozak sequence, removal of unwanted Kozak sequences; inclusion of restriction sites for ning into retroviral or other vectors; removal of nuclear localization sequences or addition of nuclear export sequences; addition of mutation sequences; addition of double stop codon sequences; addition of tags, s and fiJsion sequences; ation of sequence file for submission to gene synthesis company; subcloning of synthesized gene into retroviral vectors; inclusion of fluorescent protein genes into retroviral vectors; inclusion of selectable marker genes into iral vectors; preparation of Maxiprep plasmid DNA; transfection of retroviral er or other cells; lab, pilot or GMP scale production of retrovirus; transduction of target cells with retrovirus; GCV or analogus pro-drug mediated cell kill assay; Hypoxanthine/Aminopterin/ Thymidine (HAT) selection assay; selectable marker drug selection procedure to produce retroviral uced cell lines; fluorescent microscopy and photography to detect and document retroviral transduced target cells; quantitative fluorescent detection of retroviral transduced target cells; Western protein expression assay; other procedures and assays as needed for HSV- TK analysis; or a combination thereof. Protocols for such methods are described herein, are cially available or are described in the public literature and databases.

In some embodiments, described herein is a method of obtaining an improved HSV-TK sequence. In some embodiments, the method comprises: a) correction and/or removal of splice sites; and/or b) adjustment to a single Kozak ce. ally, in some embodiments, the method fithher comprises inclusion of restriction sites for sub-cloning of the HSV-TK sequence.

Optionally, or in addition, in some embodiments, the method further comprises removal of nuclear localization sequences.

Provided herein is a polynucleotide sequence encoding a mutated form of viral thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK is mutated at amino acid residue 25, 26, 32, 33, 167, 168, or a combination thereof, wherein the polynucleotide sequence is mutated compared to a cleotide sequence of SEQ ID NO: 1 or 3. In such sequences, 1,2, 3,4, 5, 6, 7, 8, 9, 10, ll, 12, 13, or 14 mutations may be made. ations may be conservative or non-conservative mutations. A mutation may be made such that the encoded amino acid is modified to a polar, non-polar, basic or acidic amino acid.

Provided herein is a polynucleotide sequence encoding a mutated form of thymidine kinase from human x virus (HSV-TK), wherein the encoded HSV-TK es a nuclear export sequence. Provided herein is a polynucleotide sequence encoding a mutated form of thymidine kinase from human simplex virus (HSV-TK), where the encoded HSV-TK is improved in on compared to wild-type HSV-TK and comprises A168H dmNES (Q system = QMV enhancer properly fused to LTR promoter regions), where NES refers to a r export sequence. In one embodiment, a mutant HSV-TKA168HdmNES is a mutant HSV-TK gene for inclusion in Reximmune-C2. In one embodiment, the NES is derived from MAP Kinase Kinase (MAPKK). In yet another embodiment, the polynucleotide sequence for NES is CTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC (SEQ ID NO: 23). In other embodiments, the NES polypeptide sequence is LQKKLEELELDG (SEQ ID NO: 24).

In some embodiments, disclosed herein are mutations to a polynucleotide ce encoding Human x Virus Thymidine Kinase (HSV-TK) wherein mutations are not made to the polypeptide sequence of wildtype HSV-TK.

Nucleotide positions are referred to by reference to a position in SEQ ID NO: 1 ype (wt) HSVl-TK tide sequence) or SEQ ID NO: 3 (HSV-TK in Reximmune-C HSV-TK; SR39 mutant and 26S Mutation of the HSV-TK nuclear localization signal (NLS)).

] In one embodiment, a Sac I-Kpn I ction sites bounding the clonable double stranded oligonucleotides of the mutant HSV-TK SR39 mutant region is provided. See, for example, SEQ ID NOS: 6 and 7, where the Sac I and Kpn I sites are shown on the left and right, respectively. Bold, underlining illustrates the sites where mutations may be made. SEQ ID NOS: 8 and 9 illustrate an exemplary sequence after cutting with Sac I and Kpn I. Exemplary forward and reverse primers that may be used to make the mutations are shown as SEQ ID NOS: and 11.

Exemplary optimized HSV-TK polynucleotide sequences are provided, for example, as SEQ ID NOS: 12-22.

However, when such references are made, the invention is not intended to be limited to the exact sequence as set out in SEQ ID NO: 1 or 3, but includes variants and derivatives thereof Thus, identification of nucleotide ons in other thymidine kinase sequences are contemplated (z'.e., identification of tides at positions which the skilled person would consider to correspond to positions recited in SEQ ID NO: 1 or 3).

In some embodiments, nucleotides are replaced by taking note of the genetic code such that a codon is changed to a different codon which codes for the same amino acid residue. In some embodiments, nucleotides are replaced within coding regions of a HSV-TK encoding nucleic acid sequence, yet the nucleic acid sequence maintains wild type HSV-TK protein expression.

In such embodiments, 5/21 codons contain “C or G” in third position (24%); 0/21 codons contain “C” in third position (0 %); 5/21 codons contain “G” in third position (24%); and 16/21 codons n “A or T” in third position (76%).

In yet other ments, 16/21 codons contain “C or G” in third on (76%); 11/21 codons n “C” in third position (52 %); 5/21 codons contain “G” in third position (24%); and 5/21 codons contain “A or T” in third position (24%).

In some embodiments, the following rare codons are not used or are avoided in the coding region of a polynucleotide encoding HSV-TK, or a variant thereof: GCG for alanine; CGA or CGT for arginine; TTA or CTA for leucine; CCG for proline; TCG for serine; ACG for threonine; and GTA for valine.

In some embodiments, altering codons as described herein results in about 2%, about %, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater percentage increase in ty.

In some embodiments, disclosed herein is a nucleic acid ce ng a ine kinase wherein at least one nucleotide corresponding to a splice donor site is replaced by another nucleotide. In fiarther embodiments, the nucleotides of the splice acceptor sites are not altered. In some embodiments, at least one nucleotide corresponding to a splice acceptor site is replaced by another nucleotide.

In some embodiments, disclosed herein is a nucleic acid sequence encoding a thymidine kinase wherein at least one of the tides corresponding to splice donor site nucleotides at positions 329 and 330 of a polynucleotide ce (e.g., SEQ ID NO: 1 or 3) is replaced by another nucleotide. In some embodiments, both of the tides at positions 327 and 555 are replaced by other nucleotides. For example, on 327 may be mutated to an amino acid residue selected from: G to A. Altemately, or in addition, position 555 may be mutated to an amino acid e selected from: G to A. In one embodiment, the modified HSV-TK has a polynucleotide sequence of SEQ ID NO: 18, in which HSV-TK was improved in the following ways: HSV-TK NESdmNLS Al68H, CO & SC NES = nuclear export sequence from MAP Kinase Kinase (MAPKK) dmNLS = double mutated HSV-TK Nuclear Localization Sequence CO = codon optimized SC = splice donor/acceptor site corrected at 327 and 555, Underlined sequence SEQ ID NO: 18 gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC CACAGCAACGGCAGCACCGCaCTGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG AGATGCCCACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCAC CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC TGGACCAEGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATtACaAT GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaCACATCGGCGGCGAGGCCGGCAGC AGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTCGACCGgCACCCaATCGCaCACCTGCTGT GCTACCCgGCaGCaCGCTACCTGATGGGCtcCATGACaCCaCAEGCCGTGCTGGCCTTCGTGGC CCTGATCCCaCCaACaCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC CGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAGAGCAAC GCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCCaGAGCTGCTGG CaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG CtCCATGCACGTGTTCATCCTGGACTACGACCAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTG CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACaACaCCCGGCAGCATCCCaACaATCTGCG ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT Tgtca In some embodiments, disclosed herein is a nucleic acid sequence encoding a thymidine kinase wherein at least one of the nucleotides ponding to splice acceptor site nucleotides at positions 554 and 555, or at least one of the nucleotides corresponding to splice acceptor site nucleotides at positions 662 and 663, or at least one of the nucleotides corresponding to splice acceptor sites at positions 541 and 542 of the Wild type sequence is replaced by another nucleotide. For example, position 541 may be d to an amino acid raflmflmmmmmeAPmMmmewmmWMMMmmmMMMWmmmhfid from: G to A. Position 554 may be mutated to an amino acid residue selected from: G to A.

Position 555 may be mutated to an amino acid residue selected from: G to A. Position 662 may be d to an amino acid residue selected from: G to A. Position 663 may be mutated to an amino acid e selected from: G to A.

A Kozak sequence flanks the AUG start codon Within mRNA and influences the ition of the start codon by eukaryotic ribosomes. In some embodiments, a polynucleotide sequence encoding HSV-TK ses no more than one Kozak sequence. In some embodiments, the Kozak sequence is upstream of the coding portion of the DNA ce. In some embodiments, the Kozak sequence of a polynucleotide encoding HSV-TK is modified to produce a Kozak sequence with a higher ncy of translation initiation in a mammalian cell.

In some embodiments, modification of the Kozak sequence does not produce an amino acid substitution in the d HSV-TK polypeptide product. In some embodiments, modification of the Kozak sequence s in at least one amino acid substitution in the encoded HSV-TK polypeptide product. In one ment, the modified HSV-TK has a polynucleotide sequence of SEQ ID NO: 18 or 22.

In some ments, the polynucleotide sequence encoding HSV-TK comprises at least5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon substitutions. In some embodiments, the polynucleotide sequence encoding HSV-TK comprises 2H1east5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon substitutions, n the codon substitutions comprise the substitution of a codon having a higher frequency of usage in a mammalian cell than the wild type codon at that position.

However, in some embodiments, less favored codons may be chosen for individual amino acids depending upon the particular situation.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprising at least5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon subsfitufionshaslessflnu1about6596,6696,6796,6896,6996,7096,7196,7296,7396,7496,7596, 7696,7796,7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,919& 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or 3 wherein the sequence identity is ined over the full length of the coding sequence using a global alignment method. In some embodiments, the corresponding encoded polypeptide sequence has atleast75 96,7696,7796,7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV- TK amino acid ce, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at least5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon substitutions, n the codon substitutions comprise the substitution of a codon having the highest ncy of usage in a mammalian cell for the wild type codon at that position. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,939& 94%, 95%, 96%, 97%, 98%, 99%, or 100% ce identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at ,10,l5,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon substitutions, wherein the substituted codons have a frequency of usage greater than or equal to aboutOJ,011,012,013,014,015,016,017,018,019,02,021,022,023,024,025, 7,028,029,03,031,032,033,034,035(ndnghmzhiynneentmdnnmnsthe conespondnngencodedIXflypepfidesequencehasatleast7596,7696,7796,7896,7996,8096, 8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,9396,9496,9596,9696, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO:2or4 In some embodiments, the polynucleotide sequence encoding HSV-TK comprises less than about 45, 40, 35, 30, 25, 20 or fewer codons, wherein the codons have a frequency of usage bssdmnaboutOl,011,012,013,014,015,016,017,018,019,02,02l,022,023,024 or 0.25. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 96,7696,7796,7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9l96, 9296,9396,9496,9596,9696,9796,9896,9996,or10096sequenceidenfiuzu)aIISVCIliannno acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at least7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296, 93%, 94%, 95% or more of codons haVing a frequency of usage greater than or equal to about 01,011,012,013,014,015,016,017,018,019,02,02l,022,023,024,025,026, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, or higher. In some embodiments, the conespondnngencodedIXflypepfidesequencehasatleast7596,7696,7796,7896,7996,8096, 8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,9396,9496,9596,9696, 97%, 98%, 99%, or 100% sequence ty to a HSV-TK amino acid sequence, e.g., SEQ ID NO:2or4 In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at least3596,3696,3796,3896,3996,4096,4196,4296,4396,4496,4596,4696,4796,4896,4996, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more of codons haVing the highest ncy of usage in a ian cell. In some ments, the corresponding encoded polypeptkk:sequenceliasatleast75 96,7696,7796,7896,7996,8096,8l96,8296,8396, 596,8696,8796,8896,8996,9096,9196,9296,9396,9496,9596,9696,9796,9896,9996,or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

] In some embodiments, the polynucleotide sequence encoding HSV-TK comprises less thmnabouu2l96,2096,l996,1896,l796,l696,l596,l496,l396,l296,1196,10960rlessof codons haVing a frequency ofusage less than about 0.1, 0.1 l, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polynuc1eotide sequence comprises less than about21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% or less of codons having a frequency e less than about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25 in a mammalian cell. In some embodiments, the corresponding d polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynuc1eotide sequence encoding HSV-TK comprises codon tutions, wherein at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the codons have been changed as ed to the wild type sequence. In some embodiments, the polynuc1eotide sequence encoding HSV-TK comprises codon substitutions, n at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more ofthe codons have been changed to a codon haVing a higher frequency of usage in a ian cell as compared to the wild type sequence. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence ty to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynuc1eotide sequence encoding HSV-TK comprises codon substitutions, wherein at least 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the codons have been changed to a codon haVing the highest frequency of usage in a mammalian cell as compared to the wild type sequence. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

The Viral thymidine kinase gene from the selected herpesvirus may be readily isolated and d as described below, in order to construct nucleic acid les encoding a thymidine kinase enzyme comprising one or more mutations which increases biological actiVity of the thymidine kinase, as compared to unmutated wild-type thymidine kinase. The biological activity of a thymidine kinase may be readily determined utilizing any of the assays known in the art, including for example, determination of the rate of side analogue uptake or determination of the rate of nucleoside or nucleoside analogue phosphorylation. In addition, thymidine kinase mutants may be readily selected which are characterized by other biological properties, such as thermostability and n stability.

In some embodiments, the polynucleotide sequence encoding HSV-TK is d to remove or modify a predicted signal sequence. In some embodiments, the polynucleotide is modified to remove or modify a nuclear localization sequence (NLS). In some embodiments, the polynucleotide is modified to remove the nuclear localization sequence. In some embodiments, the polynucleotide is modified to modify the NLS so that if no longer fianctions to localize HSV-TK exclusively to the nucleus.

In some embodiments, a HSV-TK polypeptide sequence is mutated at amino acid residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167.

In another e, the sequence is mutated at amino acid e 168. In another example, the sequence is mutated at amino acid residues 167 and 168. Amino acid residue 167 may be mutated to serine or phenylalanine. Amino acid residue 168 may be mutated to ine, lysine, cysteine, serine or phenylalanine. In some embodiments, a HSV-TK polypeptide sequence is mutated at amino acid residues 25 and/or 26. In amino acid residues 25 and/or 26 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. In some embodiments, the HSV-TK polypeptide sequence is mutated at amino acid residues 32 and/or 33. Amino acid residues 32 and/or 33 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. In some embodiments, the HSV-TK ptide is mutated at amino acid residues 25, 26, 32, and/or 33. Amino acid residues 25, 26, 32, and/or 33, may be d to an amino acid chosen from the group ting of: glycine, serine, and glutamate. Amino acid residue modifications may be made in comparison to a polypeptide sequence of SEQ ID NOS: 2 or 4.

In accordance with the present ion, mutant thymidine kinase enzymes which are encoded by the above-described nucleic acid molecules are provided, as well as vectors which are capable of expressing such molecules. In some ments, expression vectors are provided comprising a er operably linked to a nucleic acid molecule of the present invention. In some embodiments, the vector is a Viral vector capable of directing the expression of a nucleic acid molecule. Representative examples of such viral vectors e herpes simplex viral vectors, iral vectors, adenovirus-associated viral vectors, pox vectors, parvoviral vectors, virus vectors and iral vectors. In some embodiments, viral vectors are provided which are capable of directing the expression of a nucleic acid molecule which encodes a thymidine kinase enzyme comprising one or more ons, at least one of the mutations encoding an amino acid substitution which increases a biological activity of thymidine kinase, as ed to ted (z'.e. , wild-type) thymidine kinase.

In some ments, a nucleic acid molecule provided herein encodes a thymidine kinase enzyme capable of phosphorylating a nucleoside analogue at a level at least 10% greater than the level of phosphorylation of the nucleoside analogue by a ype thymidine kinase enzyme. In some ments, the thymidine kinase enzyme is capable of orylating a nucleoside analogue at a level at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% greater than the level of phosphorylation of the nucleoside analogue by a wild-type ine kinase enzyme. Representative examples of suitable nucleoside analogues include gancyclovir, acyclovir, famciclovir, buciclovir, penciclovir, valciclovir, trifluorothymidine, 1-[2-deoxy, 2- fluoro, beta-D-arabino furanosyl]iodouracil, ara-A, araT 1-beta-D-arabinofuranoxyl thymine, -ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2, 5'-dideoxyuridine, idoxuridine, AZT, AIU, dideoxycytidine and AraC. In some ments, the improved TK mutant lacks thymidine kinase activity.

] In some embodiments, the KIII value thymidine kinase activity of a disclosed HSV-TK mutant is at least 2.5 um. In some embodiments, the KIII value of a disclosed HSV-TK mutant is at least 5 um, at least 10 um, at least 15 um, at least 20 um, at least 25 um, at least 30 um, at least 40 um, at least 50 um, at least 60 um, at least 70 um, at least 80 um, at least 90 um, at least 100 um, at least 150 um, at least 200 um, at least 250 um, at least 300 um, at least 400 um, at least 500 um, at least 600 um, at least 700 um, at least 800 um, at least 900 um, or at least 1000 um. In some embodiments, the percent KIn of a disclosed HSV-TK mutant compared to wild- type HSV-TK is at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500%.

Within one embodiment of the present invention, truncated derivatives of HSV-TK mutants are provided. For example, site-directed mutagenesis may be readily performed in order to delete the N—terminal 45 amino acids of a thymidine kinase mutant, thereby constructing a truncated form of the mutant which retains its biological activity.

Mutations in tide sequences constructed for expression of derivatives of ine kinase mutants should preserve the reading frame phase of the coding sequences.

Furthermore, the mutations will preferably not create complementary s that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the or mRNA. Such derivatives may be readily constructed using a wide variety of techniques, including those discussed above.

In some embodiments, a HSV-TK polypeptide ce is mutated at amino acid residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167.

In another example, the sequence is mutated at amino acid residue 168. In r example, the sequence is mutated at amino acid residues 167 and 168. Amino acid residue 167 may be mutated to serine or phenylalanine. Amino acid residue 168 may be d to histidine, lysine, cysteine, serine or phenylalanine. In some embodiments, a HSV-TK polypeptide sequence is mutated at amino acid residues 25 and/or 26. In amino acid residues 25 and/or 26 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. In some embodiments, the HSV-TK polypeptide sequence is d at amino acid es 32 and/or 33. Amino acid residues 32 and/or 33 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. In some embodiments, the HSV-TK polypeptide is mutated at amino acid residues 25, 26, 32, and/or 33. Amino acid residues 25, 26, 32, and/or 33, may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. Amino acid residue modifications may be made in ison to a polypeptide sequence of SEQ ID NOS: 2 or 4.

In accordance with the t invention, mutant thymidine kinase s which are encoded by the above-described c acid molecules are provided, as well as vectors which are capable of expressing such molecules. In some embodiments, expression vectors are provided comprising a promoter operably linked to a nucleic acid molecule of the present ion. In some embodiments, the vector is a viral vector capable of directing the expression of a nucleic acid molecule. Representative examples of such Viral vectors include herpes simplex Viral vectors, adenoviral vectors, adenovirus-associated Viral vectors, pox vectors, parvoviral s, baculovirus vectors and retroviral vectors. In some embodiments, Viral vectors are provided which are capable of directing the expression of a nucleic acid molecule which s a thymidine kinase enzyme comprising one or more mutations, at least one of the ons encoding an amino acid substitution which increases a biological activity of thymidine kinase, as compared to unmutated (z'.e. , wild-type) thymidine kinase.

In some embodiments, a c acid molecule provided herein encodes a thymidine kinase enzyme capable of phosphorylating a nucleoside analogue at a level at least 10% greater than the level of phosphorylation of the nucleoside ue by a wild-type thymidine kinase enzyme. In some ments, the thymidine kinase enzyme is capable of phosphorylating a nucleoside analogue at a level at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% greater than the level of phosphorylation of the nucleoside analogue by a wild-type thymidine kinase enzyme. Representative es of suitable nucleoside analogues include gancyclovir, vir, lovir, buciclovir, penciclovir, valciclovir, trifluorothymidine, l-[2-deoxy, 2- fluoro, beta-D-arabino furanosyl]iodouracil, ara-A, araT l-beta-D-arabinofuranoxyl thymine, -ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2, eoxyuridine, idoxuridine, AZT, AIU, dideoxycytidine and AraC. In some embodiments, the improved TK mutant lacks thymidine kinase activity.

Within one embodiment of the present invention, truncated derivatives of thymidine kinase s are ed. For example, irected mutagenesis may be readily performed in order to delete the N—terminal 45 amino acids of a ine kinase mutant, thereby constructing a truncated form of the mutant which retains its biological activity.

Nhuafionsninudeofidesequencesconfirudedlbrexpmmsnniofdefiyafivesof thymidine kinase mutants should preserve the g frame phase of the coding sequences.

Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA. Such derivatives may be readily constructed using a wide variety of ques, including those discussed above.

Using the methods described herein, the inventors determined that the majority of the ates for zed HSV-TK genes appeared to be compatible with a retroviral expression system and produce biologically useful retroviral titers.

Furthermore, the optimized HSV-TK genes which incorporated most of these optimizations (SEQ ID NO: 18) exhibited ug GCV enzyme activity and selectivity for their y to kill cancer cells following retroviral transduction delivery. The mutant HSV-TK gene Al68H, which was codon optimized and splice corrected appeared to have the highest GCV mediated cancer kill activity (SEQ ID NOs: l2, 16, 18, or 22). The same n of this HSV-TK gene Al68H and mutated at amino acids 159-161 from LIF to IFL exhibited GCV mediated cancer cell kill activity.

The mutant HSV-TK gene A167F (SEQ ID NOs: l3 ,l7, or 19) which was codon optimized and splice corrected had very high GCV mediated cancer kill activity following retroviral transduction delivery, but more surprisingly had NO thymidine kinase activity as determined by expressing this gene following retroviral transduction delivery in 3T3 TK(-) cells selected with HAT medium. To our knowledge, this is the most GCV selective HSV-TK synthetic gene product for GCV activation which has no Thymidine activity ( HAT assay) ever evaluated biologically.

] The double mutant HSV-TK gene Al67F + Al68H (SEQ ID NO: 14) unexpectedly ablates both GCV and Thymidine enzyme activity by exhibiting very little GCV mediated cancer kill activity and very little thymidine activity ( HAT assay), The present inventors identified that it is possible to produce onal HSV-TK fusions of genes such as bacterial ne deaminase, yeast cytosine deaminase, neomycin phosphotransferase and include linker sequences and retain HSV-TK GCV mediated cancer cell killing activity.

] In one embodiment, a codon optimized HSV-TK gene with GCV-mediated cancer killing actiVity may be made which retains one or more nuclear localization sequences which is not fused to one or more other therapeutic genes. onal modifications to and/or evaluations of an optimized HSV-TK gene described herein may include one or more of the following: removal ofknown nuclear localization sequences within HSV-TK; increased pro-drug GCV enzyme actiVity and selectivity for their y to kill cancer cells, evaluate the use of more tags, fusion proteins and linkers of HSV-TK to other genes and ns, co-expression of HSV-TK optimized genes with other optimized suicide and cancer killer genes in cancer cells, include optimized HSV-TK genes in a Reximmune-C type iral vector system; production and testing of a Reximmune-C type GMP product, or any combination thereof.

In one embodiment, a polynucleotide sequence described herein comprises a nuclear export signal. For e, a polynucleotide sequence may comprise TKl68dmNES.

In another embodiment, a retroviral vector for use in the methods described herein comprises one or more splice site modifications.

In another embodiment, a retroviral vector for use in the methods described herein ses HSV-TK m (SEQ ID NO: 13).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK A168Hsm (SEQ ID NO: 12).

In another embodiment, a retroviral vector for use in the methods bed herein comprises HSV-TK Al67de (SEQ ID NO: 17).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK Al68dm (SEQ ID NO: 16).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK A167de and an NES (SEQ ID NO: 19).

In another embodiment, a retroviral vector for use in the methods bed herein comprises HSV-TK A168Hdm and an NES (SEQ ID NO: 18). In such an embodiment, the sequence comprises HSV-TK A168H.

In another ment, a retroviral vector for use in the methods described herein comprises a HSV-TK, n such vector ses an upgraded substrate binding domain and a mNLS/NES set.

WO 53205 In another embodiment, a retroviral vector for use in the methods described herein comprises a HSV-TK, wherein the vector comprises a selectable marker, a glowing gene and/or one or more kill genes.

In another embodiment, a retroviral vector for use in the methods described herein comprises at least two modifications.

Using the methods described herein, the inventors determined that the majority of the optimized HSV-TK genes appeared to be compatible with a retroviral expression system and produce ically useful retroviral titers.

The mutant HSV-TK gene A167F (SEQ ID NOs: l3 ,l7, or 19) which was codon zed and splice corrected had very high GCV mediated cancer kill activity following retroviral transduction delivery, but more singly had no thymidine kinase activity as determined by expressing this gene ing retroviral uction delivery in 3T3 TK(-) cells selected with HAT medium. This is highly GCV selective HSV-TK synthetic gene.

The double mutant HSV-TK gene Al67F + Al68H (SEQ ID NO: 14) exhibited very little GCV mediated cancer kill activity and very little thymidine activity; thus, a proper double mutant may have surprising null properties.

The present ors identified that it is possible to produce fianctional HSV-TK fusions of genes such as bacterial cytosine deaminase, yeast cytosine deaminase, neomycin phosphotransferase and include linker sequences and retain HSV-TK GCV mediated cancer cell killing activity.

In one embodiment, a fully codon optimized HSV-TK gene with GCV-mediated cancer killing activity may be made which retains one or more r localization sequences which is not fused to one or more other therapeutic genes.

Additional modifications to and/or evaluations of an optimized HSV-TH gene described herein may include one or more of the following: removal ofknown nuclear localization sequences within HSV-TK; increased pro-drug GCV enzyme activity and selectivity for their y to kill cancer cells, evaluate the use of more tags, fusion proteins and s of HSV-TK to other genes and proteins, co-expression of HSV-TK optimized genes with other optimized suicide and cancer killer genes in cancer cells, include optimized HSV-TK genes in a Reximmune-C iral vector system; production and testing of a une-C GMP product, or any combination thereof.

The therapeutic vectors may be administered alone or in conjunction with other therapeutic treatments or active . Examples of other active agents that may be used include, but are not d to, chemotherapeutic agents, anti-inflammatory agents, protease inhibitors, such as HIV protease inhibitors, side analogs, such as AZT. In some embodiments, the s of treatment fiarther comprise administering to the subject a chemotherapeutic agent, a biologic agent, or radiotherapy prior to, contemporaneously with, or subsequent to the administration of the therapeutic viral particles. One of skill in the art will appreciate that the retroviral particles described herein may be administered either by the same route as the one or more agents (6.g. the retroviral vector and the agent are both administered intravenously) or by different routes (6.g. , the retroviral vector is administered intravenously and the one or more agents are stered orally).

The dosage of the therapeutic viral particles lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range ing upon the dosage form ed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture . A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z'.e., the concentration of the test compound which achieves a half- maximal infection or a half-maximal tion) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by RT-qPCR or ddPCR methods.

An effective amount or eutically effective of the retroviral particles disclosed herein to be administered to a subject in need of treatment may be determined in a variety of ways. By way of example, the amount may be based on viral titer or efficacy in an animal model. Alternatively the dosing regimes used in clinical trials may be used as general guidelines.

In some embodiments, the daily dose may be administered in a single dose or in portions at various hours of the day. In some embodiments, a higher dosage may be required and may be reduced over time when the optimal initial response is ed. In some embodiments, treatment may be uous for days, weeks, or years, or may be at intervals with ening rest periods. In some embodiments, the dosage is ed in accordance with other treatments the individual may be receiving. However, the method of treatment is in no way limited to a particular concentration or range of the retroviral particle and may be varied for each dual being treated and for each derivative used.

Individualization of dosage may be required to e the maximum effect for a given individual. In some embodiments, the dosage administered to an individual being treated varies depending on the individual’s age, severity or stage of the disease and response to the course of treatment. In some embodiments, clinical parameters for determining dosage include, but are not d to, tumor size, alteration in the level of tumor markers used in al testing for particular malignancies. In some embodiments, the treating physician determines the therapeutically effective amount to be used for a given dual. In some embodiments, the therapies disclosed herein are administered as often as necessary and for the period of time judged necessary by the treating physician.

The therapeutic vectors, ing but not limited to the therapeutic iral particles that are cally to the cell or system of interest, may be ically or ally (locally) delivered to a subject in need of treatment. For example, the therapeutic vectors may be systemically administered enously. Alternatively, the therapeutic s may also be administered intra-arterially. The therapeutic vectors may also be administered topically, intravenously, intra-arterially, intra-tumorally, intracolonically, intratracheally, intraperitoneally, intranasally, intravascularly, intrathecally, intracranially, intramarrowly, intrapleurally, intradermally, subcutaneously, intramuscularly, intraocularly, intraosseously and/or intrasynovially or sterotactically. A combination of delivery modes may also be used, for example, a patient may e the therapeutic vectors both systemically and regionally (locally) to improve tumor responses with treatment of the therapeutic vectors.

In some embodiments, multiple therapeutic courses (e.g., first and second therapeutic course) are administered to a subject in need of treatment. In some embodiments, the first and/or second therapeutic course is administered intravenously. In other embodiments, the first and/or second therapeutic course is administered via intra-arterial infusion, including but not limited to infilsion through the hepatic artery, cerebral artery, coronary artery, pulmonary artery, iliac artery, celiac trunk, gastric artery, splenic artery, renal artery, gonadal artery, subclavian artery, vertebral artery, axilary artery, brachial artery, radial , ulnar , carotid artery, femoral artery, or mesenteric artery and/or superior mesenteric artery. Intra-arterial infusion may be accomplished using endovascular procedures, aneous procedures or open surgical approaches. In some ments, the first and second therapeutic course may be administered tially. In yet other embodiments, the first and second therapeutic course may be administered simultaneously. In still other embodiments, the optional third therapeutic course may be stered sequentially or simultaneously with the first and second therapeutic courses.

In some embodiments, the therapeutic vectors disclosed herein may be administered in conjunction with a sequential or concurrently administered therapeutic course(s) in high doses on a cumulative basis. For example, in some embodiments, a patient in need thereofmay be systemically administered, e.g., intravenously stered, with a first therapeutic course of at least I x 109 TVP, at least I x 1010 TVP, at least I x 1011 TVP, at least I x 1012 TVP, at least I x 1013 TVP, at least I x 1014 TVP, at least I x 1015 TVP, at least I x 1016 TVP, at least I x 1017 TVP, at least I x 1018 TVP, at least I x 1019 TVP, at least I x 1020 TVP, at least I x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis. The first therapeutic course may be systemically administered. atively, the first therapeutic course may be administered in a localized manner, e.g., intra-arterially, for example a patient in need thereofmay be administered via intra-arterial infilsion with at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis.

In yet other embodiments, a subject in need thereofmay receive a ation, either sequentially or concurrently, of systemic and intra-arterial infilsions administration of high doses of delivery vector. For example, a patient in need fmay be first systemically administered with at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a tive basis, ed by an onal therapeutic course of intra-arterial infilsion, e.g., hepatic arterial infusion, administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a cumulative basis. In still another embodiment, a patient in need thereofmay receive a combination of intra-arterial infilsion and ic administration of delivery vector in high doses. For example, a patient in need thereofmay be first be administered via intra-arterial infusion with at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis, followed by an additional therapeutic course of systemically administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a tive basis. The therapeutic courses may also be administered simultaneously, z'.e., a therapeutic course of high doses of delivery vector, for example, at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVRatbwtlleoTVRatbwtlxINITVPormkmmlxlOnTVPddwmyvmmnona 2014/029600 cumulative basis, together with a therapeutic course of arterial infusion, e.g., hepatic arterial infusion, administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a cumulative basis.

In still other ments, a t in need thereofmay additionally receive, either sequentially or concurrently with the first and second therapeutic courses, additional therapeutic courses (e.g, third therapeutic course, fourth therapeutic course, fifth therapeutic course) of cumulative dose of delivery vector, for example, at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis.

In some embodiments, the subject in need of treatment is administered systemically (e.g., intravenously) a dose of at least 1 x 1011 TVP, followed by the stration Via intra- arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1011 TVP. In other embodiments, the patient in need of treatment may be administered systemically (e.g., intravenously) a cumulative dose of at least 1 x 1012 TVP, followed by the administration Via intra-arterial infilsion (e.g., c-arterial infusion) of a dose of at least 1 x 1012 TVP. In one embodiment, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least 1 x 1013 TVP, followed by the administration Via intra-arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1013 TVP. In yet other ments, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least 1 x 1014 TVP, concurrently with the administration Via intra- arterial infusion (e.g., c-arterial infusion) of a dose of at least 1 x 1014 TVP. In still other embodiments, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least 1 x 1015 TVP, together with the stration Via arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1015 TVP. In yet other embodiments, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least 1 x 1016 TVP, concurrently with the administration Via intra- arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1016 TVP. In still other embodiments, the patient in need of treatment may be administered systemically (e.g., enously) a dose of at least 1 x 10137TVP, together with the administration Via intra-arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1017 TVP.

A subject in need of treatment may also be administered, either systemically or localized (for example intra-arterial on, such as hepatic arterial infusion) a therapeutic course of delivery vector for a defined period of time. In some embodiments, the period of time may be at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least 2 months, at least three months, at least four months, at least five months, at least six , at least seven months, at least eight months, at least nine months, at least ten months, at least eleven , at least one year, at least two years, at least three years, at least four years, or at least five years. Administration could also take place in a chronic , z'.e., for an ed or indefinite period of time.

Administration of the therapeutic vector may also occur in a periodic manner, 6.g. at least once a day, at least twice a day, at least three times a day, at least four times a day, at least five times a day. Periodic administration of the delivery vector may be dependent upon the time of delivery vector as well as the mode of administration. For example, parenteral administration may take place only once a day over an extended period of time, whereas oral administration of the delivery vector may take place more than once a day wherein administration of the delivery vector takes place over a shorter period of time.

In one embodiment, the subject is d to rest 1 to 2 days between the first therapeutic course and second eutic course. In some embodiments, the subject is allowed to rest 2 to 4 days between the first therapeutic course and second therapeutic course. In other ments, the subject is allowed to rest at least 2 days between the first and second therapeutic course. In yet other embodiments, the subject is allowed to rest at least 4 days between the first and second therapeutic course. In still other embodiments, the subject is allowed to rest at least 6 days between the first and second therapeutic course. In some embodiments, the subject is allowed to rest at least 1 week between the first and second therapeutic course. In yet other embodiments, the subject is allowed to rest at least 2 weeks between the first and second eutic course. In one embodiment, the subject is allowed to rest at least one month between the first and second therapeutic . In some embodiments, the subject is allowed to rest at least 1-7 days between the second therapeutic course and the optional third therapeutic course. In yet other embodiments, the subject is allowed to rest at least 1-2 weeks between the second therapeutic course and the optional third therapeutic course.

DIAGNOSING A PATIENT THAT IS TIBLE TO THYMIDINE KINASE GENE THERAPYTREATMENT Imaging tests, ing the use of radioactive tracers, contrast imaging technology and other imaging technology can be used to identify ts that are susceptible to gene therapy treatment, including thymidine kinase gene therapy treatment, and thus more likely to benefit from such therapeutic measures.

In a red embodiment, positron emission tomography (PET) scans are used to identify patients capable of ucing retroviral vector particles containing thymidine kinase constructs for sion in vivo. A PET scan produces 3-dimensional images of fianctional processes in the body by ing pairs of gamma rays emitted indirectly by ctive tracers placed on a biological active molecule. PET scans detect energy emitted by positively charged particles (positrons).

Patients administered a retroviral vector particle containing thymidine kinase polynucleotide are co-administered a radiotracer agent capable of being d by expressed thymidine kinase. An e is [18F]FHBG [18F]fiuoro xymethyl)butyl]guanine), which is a high affinity substrate for HSV-TK enzyme, with relative low affinity for mammalian TK enzymes. See Yaghoubi and Gambhir, Nat. Protocol 1:3069-75 (2006); Green et al., J. Nucl. Med. 45:1560-70 (2004). [18F]FHBG is phosphorylated by HSVl-TK or HSVl-sr39TK, which is then trapped within cells expressing thymdine kinase enzyme. Cleavage of this substrate in vivo in patients administered iral s containing a thymidine kinase polynucleotide, including the mutated and/or optimized thymidine kinase constructs bed herein, thus indicates efficient transduction of the retroviral vector particles by the subjects and patients, and thus an initial of patient or subject tibility to thymidine kinase-mediated gene therapy.

Alternatively, other methods for mearuing viral TK activity include chemical exchange saturation transfer magnetic resonance imaging with 5-methyl-5,6-dihydrothymidine and related compounds.

Accordingly, in some embodiments disclosed herein, provided are methods and compositions for detecting thymidine kinase expression in patients administered a retroviral viral particle containing a polynucleotide encoding a thymidine kinase protein. In some ments, the thymidine kinase is derived from vz'rz'dae thymidine kinase. In some embodiments, the thymidine kinase is HSV-TK. In other embodiments, the thymidine kinase is HSV-TKl. In still other embodiments, the thymidine kinase is an optimized version of HSV- In some embodiments, the HSV-TK gene is codon optimized for efficient expression and/or transduction. In other embodiments, the amino us of the thymidine kinase is altered to remove or ate the nuclear localization sequence (NLS) of the viral thymidine kinase sequence. In other embodiments, the thymdine kinase nucleotide sequence es a nuclear export sequence (NES) attached to the amino terminus. In some ments, the nuclear export sequence is LQKKLEELELDG (SEQ ID NO: 24).

] In yet other embodiments, the thymidine kinase coding sequence is mutated to increase substrate g of the expressed thymidine kinase n. In still other ments, the thymidine kinase coding sequence es an Al 68H mutation.

Other examples of substrates targeted by thymidine kinase, including HSV-TK protein, include: FHPG (9v(_[3~flum‘o~lhydro};y~2—pmpo><y]mctltyflguamine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-arabinofi.1ranosyl)-5 -iodouracil), FEAU (fluoroethyl- l -beta-D-arabinofuranosyluracil), FMAU (fluoromethylbeta-D- arabinofuranosyluracil), FHOMP -fluorohydroxypropanyloxy)methyl) pryrimidine-2,4(lH,3H)-dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy-3 -(hydroxymethyl)butyl side chain at N-l (HHGFEP) or 5-(2-)hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-ganciclovir and penciclovir-like side chains.

Examples of radiotracers that can be used to determine if a therapeutic protein, such as thymidine kinase, is expressed in an individual treated with the thymidine kinase gene therapy vectors described herein may also include 18F, 64Cu, 99mTe, 11C, 14C, 124I, I, 131I, 15O, 13N and/or 82RbCl.

Clinical trials for 9—[438F—fluoro~.'§-{hydroxymethyDbutyl]guanine (FHBG) for PET can be found on the following website: www.clinicaltrials.gov. Methods of measuring FHBG with PET in clinical use can be found in clinical trials NCT0087l702, NCT00185 848 and NCT01082926.

Briefly patients will receive a dose of therapeutic drug product on Day 1. On Day 3 to 6, preferably day 4, or at a time period after receiving the dose of therapeutic drug product encoding a modified HSV-TK as disclosed herein, they will be infiJsed with [18F]FHBG intravenously and imaged by PET scan 1-5 hours later, preferably 0.5, 1.0, 1.5, 2, 2.5, 3.0, 3.5 or 4.0 hours later or other riate time after administration for ng, for lation in the tumor sites where HSV-l TK is shown to be expressed. Patients that show uptake of the FHBG will be enrolled in the trial; those that do not will be excluded as disclosed herein. The amount ofFHBG will be ined and based on previous studies. Additional protocols for FHBG/PET may be found, for example, in references 15-39 below.

WO 53205 Accordingly provided herein is are methods and compositions for measuring a tagged substrate of thymidine kinase, including HSV-TK, including FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine), FHPG (gflffiufiuorow 1 ~hydro:<y~2»propoxy}iriethylkiyguanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D- arabinofuranosyl)iodouracil), FEAU -S-ethyl-l-beta-D-arabinofi1ranosyluracil), FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP -fluoro hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3- hydroxypropyl)-substituted pyrimidine derivatives g 2,3-dihydroxypropyl, acyclovirganciclovir and penciclovir-like side chains, the method comprising: a) transducing cells with a polynucleotide encoding HSV-TK; b) treating the cells with a substrate of HSV-TK attached to a radioactive tracer; and c) measuring the relative amount of radioactive signal present in target tissue. In one embodiment, step c) comprises measuring the output of the radioactive tracer in vivo in the subject using PET (positron emission tomography) scanning.

Also provided herein is are methods and compositions for identifying a patient or subject capable of benefitting from gene y treatment, comprising measuring a tagged substrate of thymidine kinase, including HSV-TK, including FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine), FHPG (9ufi3~fluorou l uhydroxyulZmpropoxylmethyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D- arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi1ranosyluracil), FMAU (fluoro-S-methyl- 1 D-arabinofuranosyluracil), FHOMP (6-((l-fluoro hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, lovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3- hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side . In some embodiments, the method comprises: a) administering a gene therapy retroviral particle comprising an HSV-TK polynucleotide and transducing cells with the polynucleotide encoding HSV-thymidine kinase; b) treating the cells with a substrate of HSV-TK attached to a radioactive tracer; c) measuring the relative amount of radioactive signal present in target tissue; and d) identifying patients where the level of radioactively-labelled HSV-TK ate is above a set threshold. In one ment, step c) comprises measuring the output of the radioactive tracer in vivo in the subject using PET (positron emission tomography) scanning. In some embodiments, patients e of benefitting from a gene y protocol include patients or ts exhibiting a level above a set threshold on a PET scan. In some embodiments, the level of ctive HSV-TK substrate is at least about 2.0 SUV or at least 20% above background on a PET scan. In some embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at least 20% above background on a PET scan. In yet other embodiments, the level of radioactive HSV-TK substrate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV or more, or at least 10% above ound, at least 20% above background, at least 30% above background, at least 40% above background or at least 50% above background or more on a PET scan.

In some embodiments, ed herein are methods and compositions for identifying a patient or subject in need of treatment for benign or metastatic s and capable of tting from gene therapy treatment. In some embodiments, the method for identifying patients capable of benefitting from gene therapy for the treatment of benign or metastatic lesions include ing a tagged substrate of thymidine kinase, including , including FHBG (9-[4- fluoro(hydroxymethyl)butyl]guanine), FHPG (9~([3mfluoron 1 nhydroxyn2~ propoxyjmeihyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'- deoxy-2'-fluoroB-D-arabinofi1ranosyl)iodouracil), FEAU (fluoroethylbeta-D- arabinofuranosyluracil), FMAU methylbeta-D-arabinofuranosyluracil), FHOMP (6- ((1 -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4(1H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, lovir, radiolabeled pyrimidine with 4- hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-5 -FEP) or 5-(2-)hydroxyethyl)- and 5- (3 -hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains after administration of the gene therapy treatment.

In some embodiments, the method comprises: a) administering a gene y retroviral particle comprising an HSV-TK polynucleotide and transducing cells with the polynucleotide encoding HSV-thymidine kinase; b) treating the cells with a substrate ofHSV-TK attached to a radioactive tracer; c) measuring the relative amount of radioactive signal present in target tissue; d) identifying patients where the level of ctively-labelled HSV-TK substrate is above a set threshold; and e) treatment said patient or subject with the gene therapy retroviral particle. In one embodiment, step c) comprises measuring the output of the ctive tracer in vivo in the subject using PET (positron emission tomography) scanning. In some embodiments, patients capable of benefitting from a gene therapy protocol include patients or subej cts exhibiting a level above a set threshold on a PET scan. In some embodiments, the level of HBG signal is at least about 2.0 SUV or at least 20% above background on a PET scan. In some embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at least % above background on a PET scan. In yet other embodiments, the level of ctive HSV- TK substrate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV or more, or at least 10% above background, at least 20% above background, at least 30% above background, at least 40% above background or at least 50% above background or more on a PET scan.

Also provided herein are methods sing: (a) determining the level of [18F]FHBG signal in a subject; and (b) ing the subject for treatment with a composition wherein the level of FHBG is at least about 2.0 SUV or at least 20% above background on a PET scan. In some embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at least 20% above background on a PET scan. In yet other embodiments, the level of radioactive HSV-TK ate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV or more, or at least 10% above background, at least 20% above background, at least 30% above background, at least 40% above background or at least 50% above background or more on a PET scan.

Additionally provided herein is a method comprising: (a) ining the level of [18F]FHBG signal in a subject; (b) excluding the subject from treatment with a composition n the level of FHBG in the subject is greater than about 2.0 SUV or at least above 20% above background on a PET scan; and (c) administering to said subject an anti-cancer agent. In some embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at least 20% above background on a PET scan. In yet other embodiments, the level of radioactive HSV-TK substrate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV or more, or at least 10% above background, at least 20% above background, at least 30% above background, at least 40% above background or at least 50% above background or more on a PET scan.

In some embodiments, the invention provides a method for identifying a subject that is susceptible to a cancer treatment, the method comprising: a) identifying expression of [18F]FHBG in the subject; b) treating the subject.

Also provided herein are compositions and methods of ing -mediated FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9~([3~fiuoro—l oxy—2— propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU - deoxy-2'-fluoroB-D-arabinofi1ranosyl)iodouracil), FEAU (fluoroethylbeta-D- arabinofuranosyluracil), FMAU (fluoromethylbeta-D-arabinofuranosyluracil), FHOMP (6- ((1 -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4(1H,3H)—dione), lovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4- hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-5 -FEP) or 5-(2-)hydroxyethyl)- and 5- (3 -hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains phosphorylation using a fluorescent imaging system. In some embodiments, the method comprises: a) transducing cells with a polynucleotide encoding HSV-TK and a first fluorescent protein; b) transducing the cells with a polynucleotide ng a second fluorescent or bioluminescent protein that is optically discernible from the first fluorescent or bioluminescent protein; c) treating the cells with an agent that becomes cytotoxic upon being phosphorylated by ; and d) measuring the relative amount of expression of the first fluorescent protein and the second fluorescent protein. In one embodiment, step d) comprises a Perkin Elmer Plate reader, a eter; a fluorescent activated cell sorter (FAC S); a cellometer; or a spectrophotometer. In another embodiment, step d) comprises measuring fluorescent output of the second fluorescent or bioluminescent protein in vivo in the subject using a fluorescent or inescent imaging system.

THYMIDINE KINASE STIC USES In some embodiments, disclosed herein is a method of selecting a t for y, or for excluding a patient from therapy. In one embodiment, the thymidine kinase gene therapy.

In other embodiments, the thymidine kinase is herpes simplex virus thymidine kinase (HSV- TK). In yet other embodiments, the thymidine kinase is HSV-TKl.

As described herein, HBG and other HSV-TK labeled substrates may be used as a marker for selection or exclusion of subjects for gene therapy. For example, cells sing HSV-TK after administration of a iral vector particle comprising a cleotide encoding HSV-TK will ively phosphorylate the nucleoside analogue 9-[4-fluoro (hydroxymethyl) butyl]guanine ([18F]FHBG). See, e.g., Yaghoubi and Gambhir, Nat. Protocols 1:3069-75 (2006). [18F]FHBG imaging above a certain threshold can then be used to fy HSV-TK positive cells and to select or exclude a t for gene therapy.

Accordingly, in one embodiment, a subject is administered a gene therapy composition, wherein the gene therapy composition s an HSV-TK polypeptide. The subject is stered a labeled nucleoside analog HSV-TK substrate after a predetermined period of time, and monitored until background of the labeled substrate is reached in the subject.

The label activity is measured, and compared against a scan detecting lesions in the subject. If the imaging activity: 1) is above a set old; and 2) correlates with the lesion location in the subject, then the subject is a candidate for HSV-TK gene therapy.

In some embodiments, the nucleoside analog is FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine), FHPG (9mfi3nfluorom l “hydroxywl«propoxyjmethyflguanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D- arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi1ranosyluracil), FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-((l-fluoro hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, lovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-l (HHGFEP) or 5-(2-)hydroxyethyl)- and 5-(3- hydroxypropyl)-substituted pyrimidine derivatives g 2,3-dihydroxypropyl, acyclovir-, lovir- and penciclovir-like side chains. In some embodiments, the label is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl. ably, the labeled nucleoside analog HSV-TK substrate is [18F]FHBG ((9-[4-18F-fluoro(hydroxymethyl)butyl]guanine).

In some embodiments, the nce pattern in a subject determines the length of time delay for measuring background and determining, for example, [18F]FHBG imaging ty. In humans, for example, [18F]FHBG ound rapidly decreases from most tissues outside the lower abdomen, as seen in As seen, between 7.6 and 42.6 minutes after administration of [18F]FHBG, levels are high in the liver, kidneys and bladder. This is in contrast to the heart and lungs, for example, which show virtually no background [18F]FHBG signal after administration. After 45.3 minutes to 80.3 minutes, [18F]FHBG levels have decreased significantly in the liver and kidneys, with persistent high signal in the bladder. From 83.3 minutes to 155.6 minutes, levels in the liver and s and decreased even further, with maintenance of high signal levels in the bladder. Accordingly, depending upon the organ and subject individual, some time may be required for background levels to se in order to measure HSV-TK gene expression. No time may be needed for measurement in organ systems outside of the lower abdomen region, as seen in the heart and lungs. In these organ systems, a sufficient threshold for gene therapy suitability may be, for example, at least above 1.0 SUV, at least above 1.5 SUV, at least above 2.0 SUV, at least above 2.5 SUV, at least above 3.0 SUV, at least above 3.5 SUV or at least above 4.0 SUV.

In contrast, some delay may be needed in order to image signals above background levels in, for example, the liver and kidneys. See Yaghoubi et al. Nat. Protocols at vol. 1, p. 3073. Because of the background signals in these organ systems, a sufficient threshold for determination of suitability for gene therapy treatment may be, for example, at least 10% above background, at least 15% above background, at least 20% above background, at least 25% above background, at least 30% above background, at least 35% above background, at least 40% above background, at least 45% above background, at least 50% above background, at least 55% above background, at least 60% above background, at least 65% above background, at least 70% above background, at least 75% above background, at least 80% above ound, at least 85% above ound, at least 90% above ound, at least 95% above background or at least 100% or more above ound when measured after a predetermined amount of time. For example, as seen in background signals in the liver are considerably less after at least 1 to 1-1/2 hours after administration of [18F]FHBG. Accordingly, [18F]FHBG signal measurements in the liver should not be taken until after [18F]FHBG signal levels have decreased to ound, approximately 1 to 1-1/2 hours, depending upon clearance rate in each individual subject.

In some organ systems, HSV-TK gene expression may not be measurable, for e, in the bladder, where high background signal levels are maintained over time.

In other embodiments, a ratio of administered [18F]FHBG to measured [18F]FHBG signal is ed to determine if a subject should be included or ed from a gene therapy protocol. For example, if a subject that is ed with, for example, 500 MBq of [18F]FHBG and exceeds the threshold of, for example, 50 MBq of [18F]FHBG signal, the subject is capable of producing a therapeutically ive amount of phosphorylated lovir, or a derivative thereof, from a construct described herein to be therapeutic, indicating that the subject may respond in a gene therapy ion. In such an embodiment, the subject is a ate for treatment with a gene therapy construct described herein.

In other embodiments, the subject is injected with 100-750 MBq or 100-600 MBq or 100-500 MBq or 200-500 MBq or 200-400 MBq, or 2.0 to 15.5 MBq/kg or 2.0 to 12.0 MBq/kg or 2.0 to 10.0 MBq/kg or 2.0 to 7.5 MBq/kg of [18F]FHBG and s the threshold of, for example, 10-100 MBq or 10-90 MBq or 10-80 MBq or 10-70 MBq or 10-60 MBq or 20-50 MBq or 20-40 MBq of [18F]FHBG signal. In some embodiments, the subjected is injection with 200-500 MBq of [18F]FHBG and exceeds the threshold of, for example, 20-50 MBq of [18F]FHBG signal. In some embodiments, the ratio of [18F]FHBG signal injected to [18F]FHBG signal measured is 2:1, 5:1, 10:1, 20:1 30:1, 40:1 or 50:1. In some embodiments, the ratio of [18F]FHBG signal injected to [18F]FHBG signal measured is from about 2:1 to about 50:1, from about 2:1 to about 40:1, from about 5:1 to about 30:1, from about 5:1 to about 20:1, from about :1 to about 10:1 or about 10:1.

In another embodiment, if a subject produces ientphosphorylated FHBG to generate a signal of greater than 2.0 SUV or at least 20% above background on PET scan, the subject is likely to produce a therapeutically effective amount of TK from a construct described herein and the t is likely respond in a gene therapy situation. In some embodiments, the subject may be selected for combination therapy with another anti-cancer agent or treatment described herein.

In other embodiments, the subject produces suff1cientphosphorylated FHBG to generate a signal of greater than about 1.5 SUV, greater than about 2.0 SUV, greater than about 2.5 SUV, greater than about 3.0 SUV, r than about 4.0 SUV or greater than about 5.0 SUV. In yet other embodiments, the subject generates a signal of at least 10% above ound, at least 20% above background, at least 30% above ound, at least 40% above background or at least 50% or more above background.

CANCERS Non-limiting examples of cancers can include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related s, AIDS-related lymphoma, anal cancer, appendix , astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, al astrocytoma/malignant , ependymoma, oblastoma, supratentorial primitive neuroectodermal tumors, Visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma ofunknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical , childhood cancers, chronic lymphocytic ia, chronic myelogenous leukemia, chronic roliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal , Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, n lymphoma, aryngeal cancer, intraocular ma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral caVity cancer, liposarcoma, liver , lung cancers, such as non-small cell and small cell lung , lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma, oblastoma, melanomas, mesothelioma, metastatic us neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin ma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal caVity cancer, yroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelVis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland , sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unkown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

In other embodiments, the s to be treated are chosen from the group consisting of primary hepatocellular carcinoma, atic breast carcinoma to liver, metastatic pancreatic cancer to liver, atic gastric cancer to liver, metastatic esophageal cancer to liver, metastatic lung cancer to liver, metastatic melanoma to liver, metastatic ovarian carcinoma to liver and metastatic kidney cancer to liver.

FORMULATIONS ceutical compositions comprising a therapeutic vector can be formulated in any conventional manner by mixing a ed amount of the therapeutic vector with one or more physiologically acceptable rs or excipients. For example, the therapeutic vector may be suspended in a carrier such as PBS (phosphate buffered saline). The active compounds can be administered by any appropriate route, for example, , parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

In some embodiments, the therapeutic vector and physiologically acceptable salts and solvates are formulated for administration by inhalation or insufflation (either through the mouth or the nose) or for oral, buccal, parenteral or rectal administration. In some embodiments, for administration by inhalation, the therapeutic vector is delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other le gas. In some embodiments, a pressurized aerosol dosage unit or a valve to deliver a d amount. In some embodiments, capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator are formulated containing a powder mix of a eutic compound and a suitable powder base such as lactose or starch.

In some embodiments, the pharmaceutical itions are formulated for oral administration as tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., atinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); ants (e.g, magnesium te, talc or silica); disintegrants (e.g, potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). In some embodiments, the tablets are coated by methods well known in the art.

In some embodiments, liquid preparations for oral administration are in the form of, for example, solutions, syrups or suspensions, or they are ated as a dry product for constitution with water or other suitable vehicle before use. In some embodiments, such liquid preparations are prepared by tional means with pharmaceutically able additives such as suspending agents (e.g., ol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous es (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- 2014/029600 hydroxybenzoates or sorbic acid). In some embodiments, the preparations also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. In some embodiments, pharmaceutical compositions are formulated oral administration to give controlled release of the active compound. In some embodiments, the pharmaceutical compositions are formulated for buccal in the form of tablets or lozenges formulated in conventional .

In some ments, the therapeutic vector is formulated for parenteral administration by injection, e.g., by bolus injection, or continuous infusion. In some embodiments, formulations for injection are in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative. In some embodiments, the compositions are ated as sions, solutions or emulsions in oily or aqueous vehicles. In some embodiments, the formulations comprise formulatory agents such as ding, stabilizing and/or dispersing agents. atively, in some embodiments, the active ingredient is in powder lyophilized form for constitution with a suitable vehicle, e. g., sterile pyrogen-free water, before use.

] In some embodiments, the therapeutic vector is formulated as a depot preparation. In some embodiments, such long acting formulations are administered by implantation (for example, subcutaneously, intramuscularly or ly into or in close proximity to a tumor) or by intramuscular injection. Thus, for example, in some embodiments, the therapeutic compounds are formulated with suitable polymeric or hydrophobic als (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for e, as a sparingly soluble salt.

In some embodiments, the active agents are formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemal or intraspinal application. In some embodiments, such solutions, particularly those intended for ophthalmic use, are formulated as 0.0l%-lO% isotonic solutions, pH about 5-9, with appropriate salts. In some embodiments, the compounds are formulated as aerosols for topical application, such as by inhalation.

The concentration of active compound in the drug composition will depend on tion, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

In some embodiments, the compositions are ted in a pack or dispenser device which comprise one or more unit dosage forms containing the active ingredient. In some embodiments, the pack may comprises metal or plastic foil, such as a r pack. In some embodiments, the pack or dispenser device is accompanied by ctions for administration.

In some embodiments, the active agents are packaged as articles of cture containing packaging al, an agent provided herein, and a label that indicates the disorder for which the agent is provided.

ANIMAL MODELS In some ments, the retroviral vector particles, hereinabove described are administered to an animal in vivo as part of an animal model for the study of the effectiveness of a gene therapy treatment. In some embodiments, the iral vector particles are administered in varying doses to different animals of the same species. The animals then are evaluated for in viva expression of the desired therapeutic or diagnostic agent. In some embodiments, from the data obtained from such evaluations, a person of ordinary skill in the art determines the amount of retroviral vector particles to be administered to a human t.

KITS Also provided are kits or drug delivery systems comprising the compositions for use in the methods described herein. All the essential materials and ts required for administration of the retroviral particles disclosed herein may be assembled in a kit (6.g. packaging cell construct or cell line, cytokine expression vector). The components of the kit may be provided in a y of formulations as described above. The one or more therapeutic retroviral particles may be formulated with one or more agents (e.g., a chemotherapeutic agent) into a single pharmaceutically acceptable composition or separate pharmaceutically acceptable compositions.

The components of these kits or drug delivery systems may also be ed in dried or lyophilized forms. When reagents or components are provided as a dried form, titution generally is by the addition of a suitable solvent, which may also be provided in another container means.

Container means of the kits may generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the at least one substance can be .

The kits disclosed herein may also comprise instructions regarding the dosage and or stration information for the retroviral particle. Instructions can include instructions for practicing any of the methods described herein including treatment methods. Instructions can additionally e indications of a satisfactory clinical endpoint or any adverse symptoms that may occur, or additional information required by regulatory agencies such as the Food and Drug Administration for use on a human subject.

The ctions may be on “printed matter,” e.g., on paper or cardboard within or affixed to the kit, or on a label affixed to the kit or packaging material, or attached to a vial or tube containing a ent of the kit. Instructions may additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD- ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM, IC tip and hybrids of these such as magnetic/optical storage media.

In some embodiments, the kits or drug delivery systems include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic ners into which the desired vials are retained. Irrespective of the number or type of ners, the kits may also comprise, or be ed with, an instrument for assisting with the inj ection/administration or placement of the ultimate complex composition within the body of a subject. Such an instrument may be an applicator, inhalant, syringe, pipette, forceps, measured spoon, eye r or any such medically ed delivery vehicle.

Packages and kits can fiarther include a label specifying, for example, a t description, mode of administration and/or indication of treatment. Packages provided herein can include any of the compositions as bed herein. The package can further include a label for treating one or more diseases and/or conditions.

The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc). The label or packaging insert can include riate written instructions. Kits, therefore, can additionally include labels or instructions for using the kit components in any method described herein. A kit can include a compound in a pack, or dispenser together with instructions for administering the compound in a method described herein.

EXAMPLES In order that those in the art may be better able to practice the compositions and s described herein, the following examples are provided for ration purposes.

Example 1: al Trial.

A dose escalation trial was conducted to evaluate the , pharmacokinetics, and codynamics of Reximmune-C2 (Thymidine Kinase and GM-CSF Genes) in refractory subjects with primary hepatocellular carcinoma or tumors metastatic to the liver.

Background and Rationale Reximmune-C2 is comprised of a genetic delivery platform containing an internal d that encodes for therapeutic ns of interest. The genetic delivery platform has been dosed in over 280 subjects worldwide; approximately 270 subjects were d with the vector containing dnGl as a payload (Rexin-G) and 16 subjects with thymidine kinase (vTK) and the immune stimulator Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) as a d (Reximmune-C). The genetic delivery platform is a highly engineered non-recombinant Mouse Moloney Viral vector (MoMLV). Previously, a Phase 1 dose escalation trial was performed investigating the combination of Rexin-G and Reximmune-C in subjects with refractory y or metastatic solid tumors (Genevieve Trial). This ed Phase I clinical trial (entitled Genevieve 2 Trial) is an extension of a trial undertaken investigating Reximmune-C2 alone — without the Rexin-G — utilizing an improved form of thymidine kinase in a thymidine kinase plus GM-CSF combination.

In the original Genevieve trial, sixteen t were recruited over 3 dose levels with the mean exposure in the highest dose group being 8.0x 1010 cfus (# of pts = 7) and the longest duration 6 cycles (range of cycles 3-6). For Part A of the study, treatment consisted of a previously determined safe and effective (optimal) dose of G, and escalating doses of Reximmune-C. Specifically, Rexin-G, 2 x 1011 cfu, on Days 1, 3, 5, 8, 10 and 12, Reximmune- C, 1.0, 2.0 or 3.0 x 1010 Cfil on Day 3 (Dose Levels 1, II, 111 respectively), and valacyclovir at 1 gm p.o. three times a day on Days 6-19, as one cycle. For the Part B part of the study, subjects who had no toxicity or in whom toxicity had resolved to Grade 1 or less could receive additional cycles of therapy up to a total of 6 treatment cycles.

There were no dose-limiting toxicities at any dose level. ted adverse events were reported for the 16 subjects in the study, but the number of events was low (in most cases 1 or 2 occurrences per preferred term), and most were Grade 1 or 2. Related non-serious adverse events occurred in 2 subjects and both were Grade 2. Four subjects experienced serious adverse events, all of which were deemed not related to the study drug.

The rationale for continuation of this Phase 1 trial is that: (l) ine kinase itself could prove to be an effective ncer agent particularly in subjects whose tumors trate a bystander effect; (2) administration of the genetic delivery rm to date to an international group of subjects has demonstrated a very high degree of safety; and (3) biodistribution in animals suggests a high biodistribution to the liver. Moreover, the addition of GM-CSF could contribute to an logical effect and enhanced tumor cell kill h tumor associated antigens h recruitment of the riate immune cells.

The biodistribution of the viral particles is t to the liver, followed by spleen, then lung — this is the rationale for focusing initially on hepatocellular tumors where the dose intensity should be the t. There is also a high clinical unmet need for effective anticancer agents for these cancers.

It is understood that the embodiments disclosed herein are not limited to the particular methods and components and other processes described as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates ise. Thus, for example, a reference to a "protein" is a reference to one or more proteins, and includes equivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention s. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and s employed in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature and serve to provide a r understanding of certain aspects of the present invention.

Example 2: TK diagnostic assay for gene therapy ations.

Animal and human studies have usly shown the y of measuring vTK expression by PET imaging using [18F]-FHBG. These imaging tools will be utilized as a personalized surrogate test in accessing appropriate dosing and exposure and used in the IB portion to determine which subjects have the best opportunity to benefit from the drug candidates.

This clinical trial is divided into two phases: Phase IA in which Reximmune-C2 was stered as a single intravenous dose on three out of five days and the presence of the HSV- TK-m2 expression potentially monitored by HBG PET scanning after 3-8 days (Schematic for Phase IA is illustrated in . Valganciclovir (the oral form of ganciclovir) dosing is initiated on day 8 for 5 days irrespective of the PET scan s. An approximately one week drug holiday s. Each cycle will be of three weeks duration.

There will be three patients in the first and subsequent cohorts until a patient experiences Dose Limiting Toxicity (DLT) or two instances ofNCI-CTC Grade 2 toxicities attributed to the study drug (except nausea/vomiting, fatigue, anorexia, alopecia, or anemia). If there are no DLTs, patients will move to the next dose level. If there is a DLT, the cohort will be expanded to 6 patients and the dose level will not be exceeded if 2 or more patients t DLTs.

Once the Maximum Administered Dose (MAD) is reached, a modified Fibonacci le will be followed starting with the cohort dose which had no DLTs and continuing until dose-limiting toxicities are observed in two patients at a dose level. Once the Recommended Phase 2 Dose (RP2D) is defined, 6-12 patients will be recruited.

Phase IB is designed to explore the activity of Reximmune-C2 in patients of a defined tumor type and stage based on the Phase IA data and who are HBG scan positive day three to six after one dose (RP2D) of Reximmune-C2. If the scan is positive, the patient is accepted into the Phase IB ent phase of the protocol and the RP2D is given as three doses within 5 days, followed by 5 days of valganciclovir beginning on day 8 of that phase, followed by a one week drug holiday. Each cycle is of three week duration. Patients who have a negative [18F]FHBG PET scan after one single dose of Reximmune-C2 will be dosed with 5 days of valganciclovir and will not continue in the study.

The patient DLT will be defined as the occurrence of any of the following events which is attributed to une-C2 and occurring during the first cycle (3 weeks) of drug administration: Grade 4 neutropenia (i.e., te neutrophil count (ANC) < 500 cells/mm3) for 7 or more consecutive days or febrile neutropenia (i.e., fever 2 38.50 C with an ANC < 1000 cells/mm3); Grade 4 ocytopenia (< 25,000 cells/mm3 or bleeding episode requiring platelet transfusion); Grade 3 or greater nausea and/or vomiting despite the use of adequate/maximal medical intervention and/or prophylaxis; Any Grade 3 or greater non- hematological ty (except Grade 3 injection site reaction, alopecia, fatigue); Retreatment delay of more than 3 weeks due to delayed recovery from a toxicity related to treatment with Reximmune-C2; and Grade 3 or greater hypersensitivity reaction despite the riate use of premedications (by Common ty Criteria defined as “symptomatic bronchospasm, requiring parenteral medications(s), with or without urticaria; allergy-related edema- dema”).

Reximmune-C2 is infused intravenously over 15-60 minutes (depending on the dose) via an infusion pump. Reximmune-C2 is ed in 30 ml vials stored at -80 °Ci 10 0C.

In this Phase I trial, the safety, pharmacokinetics, and pharmacodynamics of escalating doses of Reximmune-C2 will be investigated. The maximum tolerated dose will be identified and a recommended Phase 2 dose will be defined for Reximmune C2. Any antitumor ty and clinical responses to Reximmune-C2 treatment will be described.

The starting dose in this trial is based on: human clinical safety ence with the related vector platform drug products Rexin-G and une-C and the results of the 21 day rat GLP toxicology study for Reximmune-C2.

Objectives ] The primary objective of the study is to determine the maximum tolerated dose (MTD), dose limiting toxicity (DLT), safety, and a recommended Phase 2 dose (RP2D) of Reximmune- C2 stered over a three week cycle consisting of a series of three doses given intraveneously within five days in week 1, ed by 5 daily doses of valganciclovir in week 2 in patients enrolled in this study who have been diagnosed with advanced primary or metastatic tumors to the liver.

Secondary objectives include: (i) evaluation of the plasma cokinetics of Reximmune-CZ; (ii) ment of the surrogate of HSV-TK-m2 n expression from Reximmune-C2 via serial [18F]FHBG PET and/or SPECT imaging; (iii) description and ment of any preliminary evidence of anti-tumor activity of Reximmune-CZ; and (iv) to provide clinical research testing for antibodies to retrovector gp70 env, replication-competent retrovirus in peripheral blood lymphocytes ; vector integration into genomic DNA of PBLs, and circulating hGM-CSF protein.

Methods Study Design: Parallel group, open label dose tion, three-center clinical trial.

Stratification: None.

Therapy: une-C2 will be administered as an intravenous infilsion to separate patients. In Phase IA — investigating Reximmune-C2 - the dose will be escalated among cohorts of patients until DLT is observed. At the RP2D, onal patients will be recruited. In Phase IB patients will be pre-screened by [18F]FHBG PET for expression of the HSV-TK-m2. Those that express HSV-TK-m2 will receive additional doses of Reximmune-C2. Patients will not be pre-medicated unless hypersensitivity reactions occur.

Statistical Methods: Descriptive statistics will be used for statistical analysis.

Sample Size Determination: Precise sample size cannot be defined, as it is dependent on the observed toxicity. For each schedule, cohorts of three to six subjects will be d at each dose level until the MTD is defined. Once the MTD is identified, this dose level will be expanded to a maximum of 12 patients who will be treated to better define the tolerability and pharmacokinetics of the dose and schedule. It is expected that 45-70 subjects will be enrolled, with 33 to 46 in the IA n.

Enrollment Criteria Subjects must meet all of the following inclusion criteria to be eligible for randomization into the study: 1. Diagnosis of histologically documented, ed stage, primary or metastatic adult solid tumors in the liver that are refractory to standard therapy or for which no curative standard therapy exists. 2. Evidence of radiographically measurable or evaluable disease. 3. All acute toxic effects of any prior radiotherapy, herapy, or surgical ures must have resolved to National Cancer Institute (NCI) Common Toxicity Criteria (CTC)(Version 4.0) Grade < 1. 4. Age must be > 18 years. 5. Last dose of antineoplastic therapy except for hormonal therapy must be > 21 days.

External beam radiotherapy must have been < 25% bone marrow-containing skeleton. 6. Patients may be Hepatitis B and C positive. (Patients may continue their antiviral medications). 7. Patients may have intracranial metastases of any number if they have been brain irradiated and stable for 6 weeks. Patients may be taking eizure medicines but must not be on steroids. 8. Kamofsky performance status must be 2 70. 9. Life expectancy of at least 3 months. 10. Patients must be able to travel to St. Luke’s Medical Center for the PET scans. ll. Required baseline tory data include: Absolute neutrophil count 3 _ 9 2 mm [SI units 10 /L] (ANC) Platelets 2 75,000/mm3 [SI units 10%] Hemoglobin 2 8.0 gm/dL [SI units ] Serum Creatinine S 1.5 x laboratory upper limit of normal (L-ULN) Bilirubin S 2.0 mg/dL Alkaline phosphatase S 5 x L-ULN AST, ALT S 5 x L-ULN LDH S 5 x L-ULN Pregnancy test (females of Negative within 7 days of ng Protocol childbearing potential) 12. Signed informed consent indicating that they are aware of the neoplastic nature of their disease and have been informed of the ures to be followed, the experimental nature of the therapy, atives, potential benefits, side s, risks, and discomforts. 13. Willing and able to comply with scheduled visits, treatment plan, and laboratory tests.

The presence of any of the following will exclude a subject from study enrollment 1. Concurrent therapy with any anticancer therapy including any other investigational agent. ] 2. Known intracranial edema or a CVA within 6 weeks of screening. 3. Pregnant or breast-feeding women. Female subjects must agree to use effective contraception, must be surgically sterile, or must be nopausal. Male ts must agree to use effective contraception or be surgically sterile. The definition of effective contraception will be based on the judgment of the Investigator or a designated associate. All at-risk female subjects must have a negative pregnancy test within 7 days prior to the start of study treatment. 4. Clinically significant cardiac disease (New York Heart ation, Class III or IV). 5. Dementia or altered mental status that would prohibit informed consent. 6. Other severe, acute, or chronic medical or psychiatric condition or laboratory abnormality that may increase the risk associated with study participation or study drug administration or may interfere with the interpretation of study results and, in the judgment of the Principal Investigator, would make the subject inappropriate for this study. 7. Known side effects to antivirals in the ganciclovir class. 8. Patients who are known to be HIV positive. 9. t must not be taking steroids at the time of screening.

Rationalefor the Starting Dose and Schedule Reximmune-C has been dosed in 16 ts over a range of 1.0, 2.0 or 3.0 x1010 cfu (Dose Levels 1, II, 111 respectively on day 3 of the . There were no dose-limiting toxicities at any dose level. Unrelated adverse events were reported for the 16 patients in the study, but the number of events was low (in most cases 1 or 2 occurrences per preferred term), and most were Grade 1 or 2. Related ious adverse events occurred in 2 patients and both were Grade 2. Four patients experienced serious e events, all of which were deemed not related to the study drug. The trial was closed prior to ining the l dose and schedule of Reximmune-C. In this trial, the new Genevieve-2 Trial, initial dosing will be based on the 21 day toxicology and the -ml study. Future dosing will proceed using total viral particles (TVP) which is a more accurate measure of titer than cfu per mL.

The schedule is based on the rationale that Reximmune-C2 exposure will not transduce all of the tumor cells. Therefore, patients will be dosed three times in a cycle over a period of 5 days.

The time between exposure to GDS and the expression of HSV-TK-m2 (and hGM- CSF) is estimated to be 48 to 72 hours. Therefore, 72 hours after the third dose of Reximmune- C2, valganciclovir will be initiated. The dose (which will be adjusted for renal on) will be given at conventional antiviral dose levels. Due to the potential toxicity of valganciclovir and the published observations that 5 days of ganciclovir should be sufficient to kill the majority of cells ning HSV-TK-m2, 5 days of therapy was chosen. Due to the potential toxicity of both Reximmune-C2 and valganciclovir, this will be followed by an approximately 9 day drug holiday. The hGM-CSF may be at sufficient concentrations at the time of valganciclovir addition to influence the presentation of any tumor associated antigens (TAAs) that may appear during tumor cell apoptosis.

Plasma samples will be taken after the first and third doses in Cycle One and after the first dose in Cycle Two for pharmacokinetics.

] As bution is primarily to the liver, toxicities will be carefillly monitored there and e of the implications, the bone marrow.

This clinical protocol calls for the administration of Reximmune-C2 via intravenous infusion to patients with advanced malignancies, either primary hepatocellular or tumors metastatic to the liver. There will be two parts: Phase IA (dose tion 3 doses/week every three weeks) and Phase IB (pre-screening after one dose of Reximmune-C2 and an HBG scan). If the PET scan is positive, the patient will continue on study. If the PET scan is negative, the patient will receive 5 days of valganciclovir and will not continue in the trial. For Phase IA, dose tion will follow an accelerated titration design, incorporating three patients per dose level until either one instance of DLT or two instances CTC Grade 2 toxicities attributed to the study drug (except nausea/vomiting, fatigue, ia, alopecia or anemia) are observed. Thereafter, dosing in the clinical protocol will follow a modified Fibonacci schedule until dose-limiting toxicities are achieved.

Trial Design This is a Phase 1, open-label, four center, scalating trial. The dose will be increased until DLT is observed, and the MTD is defined.

Reximmune-C2 will be administered as an IV infusion over 15-60 minutes. It is anticipated that 33-70 patients will be treated during the course of the study.

For Phase IA, the dose of Reximmune-C2 will be escalated from 6.0x1011 TVP. In the accelerated dose escalation phase, cohorts of three patients will be enrolled at each dose level.

The dose escalation increment will be 100% until a DLT or two CTC Grade 2 or r toxicities are observed. When the rated dose escalation ends, the dose escalation for a new t in the standard dose escalation will follow a modified Fibonacci scheme (i.e., dose WO 53205 increments of 67%, 50%, 40%, 33% and 25%). A minimum of three patients per dose level will be enrolled. For Phase 1B, the dose of Reximmune-C2 will be the RP2D. DLT will be assessed.

If a DLT is observed in 3 2 out of six patients at a dose level, there will be no further dose escalation; this dose level will define the maximum administered dose (MAD).

The dose just below the MAD will be considered the MTD. Once the MTD is defined, this dose level can be expanded to a maximum of twelve patients to fiarther characterize the pharmacokinetic and pharmacodynamic parameters and suitability as a recommended dose for Phase 2 clinical studies.

Treatment of Patients Only qualified personnel who are familiar with procedures that minimize undue exposure to themselves and to the environment should undertake the preparation, handling, and safe disposal of rapeutic agents in an riate environment.

Reximmune C2 is a y Murine replication incompetent retrovector particle containing the genes encoding for a HSV-TK-m2 and hGM-CSF. The drug t contains DMEM (low glucose), RD-Retrovector Particles, L-glutamine, Sodium pyruvate, human serum albumin, n-butyric acid, Pulmozyme®, magnesium and other excipients.

Drug t is available in one vial size: 30 mL type 1 clear glass vials with a 20 mm finish (containing 25 mL of 31 .0x1010 TVP). The vials are closed with 20 mm Teflon coated serum stoppers and 20 mm flip-off red flip tops.

] Reximmune-C2 will be administered intravenously by infusion pump over 15 s up to a volume of 100 mL, from >100 mL to 200mL over 30 minutes, from >200 mL to 300 mL over 45 minutes, and from >300 mL to 400 mL over 60 minutes. Volumes over 400 mL will be administered at a rate determined by the Investigator and the Gleneagles Medical Monitor. Once the MTD has been identified for the schedule, the time of administration may be changed, if indicated (and as agreed between the Investigator and the Gleneagles Medical Monitor).

Valganciclovir is administered orally, and should be taken with food. Serum creatinine or creatinine clearance levels should be monitored lly. Dosage adjustment is required based on creatinine clearance as shown in the Table below. Valganciclovir dosing may begin on day 7 to 9 of the cycle but must be given for 5 consecutive days.

Creatinine clearance can be calculated from serum creatinine by the following a: For males = {(140 — age[years]) x (body weight [kg])}/ {(72) x (0.011 x serum nine [micromol/L])} For females = 0.85 x male value. 2014/029600 Table I. Valganciclovir Dosing for Renally Impaired Patients Cr CL (ml/min) Dose Day 1 Dose Days 2 - 5 260 ml/min 900 mg (two 450 mg tablets) 900 mg (two 450 mg bid tablets) qday 40-59 ml/min 450mg bid 450mg qday -39 ml/min 450mg 450 mg Day 3 and Day 5 -24 ml/min 450mg 450 mg Day 4 The purpose of the Phase 1 study is to establish the MTD, DLT, safety and a RP2D of the investigational agent. Toxic effects are thus the primary study endpoint and will be assessed continuously. Response information will be obtained if patients have disease that can readily be measured and re-assessed. These assessments will be made with every cycle. rmore, a se must be noted between two examinations at least 6 weeks apart in order to be documented as a confirmed response to therapy. 0 Evaluable for toxicity - All patients will be evaluable for toxicity if they receive any study drug. 0 ble for se - All patients who have received at least a single cycle of treatment and had tumor re-assessment will be considered evaluable for response. In addition, those patients who develop early ssive disease will also be considered evaluable for response. Patients on therapy for at least two cycles of treatment will have their response evaluated.

] The determination of antitumor efficacy will be based on objective tumor ments made according to the Immune-Related Response Criteria (irRC) system of evaluation and treatment ons by the Investigator will be based on these assessments.

Given the presence of the GM-CSF transgene in Reximmune-C2 and the possibility of an immune response contributing to the tumor effect, the Immune response Criteria will be utilized for clinical response. The reasons for using The immune Response Criteria vs RECIST 1.1 are as follows: (1) the appearance of measurable anti-tumor ty may take longer for immune therapies than for cytotoxic therapies; (2) responses to immune therapy occur after conventional PD; (3) discontinuation of immune therapy may not be appropriate in some cases, unless PD is confirmed (as is usually done for response); (4) allowance for “clinically insufficient” PD (e.g. small new lesions in the presence of other responsive lesions) is recommended; and (5) durable SD may represent antitumor activity.

The comparisons between RECIST 1.1 and the Immune-Related se Criteria are listed below: Table 11. Comparison ofWHO RECIST and Immune-Related se Criteria WHO irRC New measurable lesions Always represent PD Incorporated into tumor burden (i.e., 2 5 X 5 mm) New, nonmeasurable lesions Always represent PD Do not define progression (but (i.e., < 5 X 5 mm) preclude irCR) Non-index lesions Changes contribute to defining Contribute to defining irCR BOR of CR, PR, SD, and PD (complete disappearance CR earance of all lesions in Disappearance of all lesions in two consecutive observations not two consecutive observations not less than 4 wk apart less than 4 wk apart PR 2 50% decrease in SPD of all 2 50% se in tumor burden index lesions compared with compared with baseline in two baseline in two ations at observations at least 4 wk apart least 4 wk apart, in absence of new lesions or unequivocal progression of non-index lesions SD 50% decrease in SPD compared 50% decrease in tumor burden with baseline cannot be compared with baseline cannot be established nor 25% increase established nor 25% increase compared with nadir, in absence compared with nadir ofnew lesions or unequivocal progression of dex lesions PD At least 25% increase in SPD At least 25% increase in tumor compared with nadir and/or burden compared with nadir (at unequivocal progression of non- any single time point) in two indeX s and/or appearance consecutive observations at least ofnew lesions (any any single 4 wk apart time point) Timing and Type of Assessments All ne imaging-based tumor assessments are to be performed within 14 days prior to the start of treatment. For the purposes of this study, all patients’ tumor ments should be re-evaluated starting 9 weeks after initiation of treatment and every 6 weeks thereafter (e. g., Week 9, Week 15, Week 21, etc.) for both Phase IA and Phase IB. All patients with responding tumors (irCR or irPR) must have the response confirmed no less than 6 weeks after the first documentation of response. All patients with tumor progression must have progression confirmed no less than 6 weeks after the first documentation of progression.

The same method of assessment and the same que should be used to characterize each identified and reported lesion at baseline and during follow-up. g-based evaluation is preferred to evaluation by clinical examination when both methods have been used to assess the antitumor effect of treatment. All measurements should be recorded in metric notation.

CT and CT/PET are the methods for tumor assessments. Conventional CT should be performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT should be performed using a 5 mm uous truction algorithm. This applies to the chest, abdomen, and pelvis.

Chest CT will used for assessment of pulmonary lesions.

Clinical lesions will only be considered measurable when they are superficial (e. g., skin nodules, palpable lymph nodes). In the case of skin lesions, ntation by color photography including a ruler to estimate the size of the lesion is recommended. [18F]FHBG PET-CT scans will be obtained after the patient receives the first three doses of ReXimmune-C2 (cycle 1) in Phase IA and after the screening dose of Reximmune-C2 in Phase IB. In Phase IA additional [18F]FHBG PET-CT scans can be obtained in subsequent cycles at the tion of the Investigator and with approval of the Medical Monitor.

Ultrasound should not be used to measure tumor lesions that are clinically not easily accessible for objective response evaluation, e. g., visceral lesions. It is a possible alternative to clinical measurements of superficial palpable nodes, SC lesions, and d nodules.

Ultrasound might also be useful to confirm the complete disappearance of superficial lesions usually ed by clinical ation.

Endoscopy, laparoscopy, and radionuclide scan should not be used for se ment.

All patients’ files and radiological images must be available for source verification and may be submitted for extramural review for final assessment of mor ty. ability of Tumor Lesions At baseline, tumor lesions will be categorized by the igator as measurable or non-measurable by the criteria as described below: 0 Measurable: Lesions that can be accurately measured in at least one dimension (longest diameter to be recorded) as 2 20 mm with conventional techniques or as 2 10 mm with spiral CT scan. Clinical s will only be considered measurable when they are superficial (e. g., skin nodules, palpable lymph nodes). 0 Non-Measurable: All other s, including small lesions (longest diameter < 20 mm with conventional techniques or < 10 mm with spiral CT scan) and bone lesions, leptomeningeal disease, ascites, pleural or pericardial effusions, lymphangitis of the skin or lung, abdominal masses that are not ed and followed by imaging techniques, cystic lesions, previously irradiated lesions, and disease documented by indirect evidence only (e. g., by laboratory tests such as alkaline phosphatase).

NOTE: Cytology and histology: If measurable disease is restricted to a solitary lesion, its neoplastic nature should be confirmed by cytology/histology.

] Response to therapy may also be assessed by independent, central, radiologic blinded review.

Recording Tumor Measurements All measurable lesions up to a maximum of 10 lesions, representative of all involved organs, should be identified as target lesions and measured and recorded at baseline and at the stipulated intervals during treatment. Target lesions should be selected on the basis of their size (lesion with the longest diameters) and their suitability for accurate repetitive measurements (either by imaging techniques or clinically).

The longest diameter will be recorded for each target lesion. The sum of the longest diameter for all target lesions will be calculated and ed as the baseline. The sum of the longest diameters is to be used as reference to fiarther characterize the objective tumor response of the measurable dimension of the disease during treatment. All measurements should be recorded in metric notation in centimeters.

All other lesions (or sites of disease) should be fied as non-target lesions and should also be recorded at baseline. Measurements are not required and these lesions should be followed as “present” or “absent.” Definitions of Tumor Response Immune-Related se Criteria criteria will be followed for assessment of tumor ] Determination of Overall Response by Immune-Related Response Criteria Target Lesions for Solid Tumors 0 Complete response (irCR) is defined as the disappearance of all lesions (whether measurable or not, and no new lesions); confirmation by a repeat, consecutive assessment no less than 6 weeks from the date first documented. 0 l response (irPR) is defined as a > 50% decrease in tumor burden relative to baseline ed by a consecutive assessment at least 6 weeks after the first documentation. 0 Progressive disease (irPD) is defined as a > 25% increase in tumor burden ve to nadir (minimum ed tumor burden) confirmed by a repeat, consecutive assessment no less than 6 weeks from the date first nted s recorded since the treatment started, or the appearance of one or more new lesions. 0 Stable e (irSD) is defined as not meeting the ia for irCR or irPR, in absence of irPD.

Non-Target Lesions for Solid Tumors The cytological confirmation of the neoplastic origin of any effusion that s or worsens during treatment when the measurable tumor has met criteria for response or irSD is ory to differentiate between response or irSD and irPD.

Confirmation of Tumor Response To be assigned a status of irPR or irCR, changes in tumor measurements in ts with ding tumors must be confirmed by repeat studies that should be performed 3 6 weeks after the criteria for response are first met. In the case of irSD, follow-up measurements must have met the irSD criteria at least once after study entry at a minimum al of 6 weeks.

When both target and non-target lesions are present, individual assessments will be recorded separately. The overall assessment of response will involve all ters as depicted in Table 111.

The best overall response is the best response ed from the start of the treatment until disease progression/recurrence (taking as a reference for tumor progression the smallest measurements recorded since the treatment started). The patient’s best response assignment will depend on the achievement of both ement and confirmation criteria.

Patients will be defined as being not ble (NE) for response if there is no post-randomization oncologic assessment. These patients will be counted as failures in the analysis of tumor se data.

Clinical Efficacy Assessment: Performance Status.

Patients will be graded according to the Kamofsky performance status scale as described in Table IV.

Table IV. Karnofsky Performance Status Criteria Normal, no complaints, no evidence of e Able to carry on normal activity, minor signs or symptoms of disease Normal activity with effort, some signs or ms of disease Care for self. Unable to carry on normal activities or to do active work Requires occasional assistance, but is able to care for most of his/her needs 50 Requlres erable ass1stance and frequent medlcal care. . . . 4O Disabled, requires special care and assistance Severely disabled, hospitalization is indicated although death not imminent Hospitalization necessary, very sick, active supportive treatment necessary Moribund, fatal processes progressing rapidly Death -6l- Tumor Marker Response Method of ment While not a fillly validated measure of efficacy in many malignancies, serial determinations of tumor markers may allow evaluation of an easily performed, inexpensive, quantitative, clinical tool as a potential additional means for following the course of the illness during therapy.

A tumor marker se or increase will not be assessed as an ive measure of e. In particular, a rising tumor marker value will not be considered in the definition of tumor progression, but should prompt a repeat radiographic evaluation to document whether or not radiographic tumor progression has occurred.

Calculated Endpoint Definitions ] Survival is defined as the time from date of first study drug treatment to date of death.

In the absence of confirmation of death, survival time will be ed at the last date of follow- Tumor se rate is defined as the proportion of patients who have any evidence of objective irCR or irPR.

TTP is defined as the time from treatment to first conf1rmed documentation of tumor ssion or to death due to any cause. For patients who do not have objective evidence of tumor progression and who are either d from study treatment or are given mor treatment other than the study treatment, TTP will be censored. A tumor marker increase meeting criteria for tumor marker progression does not constitute te objective evidence of tumor progression. However, such a tumor marker increase should prompt a repeat raphic evaluation to document whether or not objective tumor progression has occurred.

TTF is defined as the time from treatment to first conf1rmed documentation of tumor progression, or to off-treatment date, or to death due to any cause, whichever comes first.

Patients who are still on treatment at the time of the analysis and patients who are removed from therapy by their physicians during an objective response and who, at the off-treatment date, have no evidence for objective tumor progression will not be considered to have experienced treatment failure, unless the withdrawal is due to the occurrence of a medical event. For these patients, TTF will be censored at the off-study date. Censoring for TTF will also be performed in those patients who are given mor treatment, other than the study treatment, before the first of ive tumor progression, off-study date, or death. A tumor marker increase meeting criteria for tumor marker progression does not constitute adequate objective evidence of treatment failure. However, such a tumor marker increase should prompt a repeat radiographic evaluation to document whether or not objective tumor progression (and thus treatment failure) has occurred.

Time to first definitive performance status worsening is the time from treatment until the last time the performance status was no worse than at ne or to death, due to any cause, in the absence of us documentation of definitive confimed performance status worsening.

For patients who do not have definitive mance status worsening and who are either removed from study or are given mor treatment other than the study treatment, definitive performance status worsening will be censored.

Time to first definitive weight loss is defined as the time from treatment until the last time the percent weight decrease from baseline was < 5% or to death due to any cause in the absence of us documentation of definitive weight loss. For patients who do not have definitive weight loss and who are either removed from study or are given antitumor treatment other than study treatment, definitive weight loss will be censored.

Additional evaluations of the data may include best objective response, confirmed and unconfirmed objective response rate, duration of study ent, time to first ence ofnew lesions, time to tumor response, stable disease at 24 weeks, and rate of progression free survival at 24 weeks. Data may be evaluated by RECIST 1.1 criteria, if needed. ent Administration Assessment For both Phase IA and IB: dose intensity is defined as the total ycle times the number ofweeks n start of treatment and last treatment plus 13 days.

Percent relative dose intensity is defined as the proportion of the actual dose intensity divided by the planned dose intensity for that same period of time.

ABBREVIATIONS ALT Alanine aminotransferase ANC Absolute neutrophil count AST Aspartate aminotransferase AUC Area under the plasma concentration-time curve BSA Body surface area (mg/m2) CL Systemic plasma clearance Cmax Peak plasma concentration CR te response CRF Case report form CT Computerized tomography CTC Common Toxicity Criteria DLT Dose Limiting Toxicities WO 53205 EOI End of infusion FDA Food and Drug Administration G-CSF Granulocyte-colony stimulating factor (filgrastim, Neupogen®) GCP Good clinical practice GM-CSF Granulocyte-macrophage colony-stimulating factor (sargramostim, Leukine®) HIV Human Immunodeficiency Virus HR Hazard ratio IEC Independent Ethics Committee i.p. Intraperitoneal IRB utional Review Board IV Intravenous, intravenously LD10 or Dose that is lethal to 10% or 50% of animals Lactate dehydrogenase VIaximum Administered Dose VIagnetic resonance imaging VIaximum tolerated dose \Iational Cancer Institute \Iot evaluable for tumor response \10 Observed Adverse Effect Level \Ion-complete response \Ion-progressive disease Peripheral Blood Mononuclear Cells Propylene Glycol: hor® EL: Ethanol Progressive disease Partial response SAER-S Serious Adverse Event Report-Study Subcutaneous, aneously Stable disease Dose that is severely toxic to 10% of s Time to Progression Time to e Half-life Time ofmaximum plasma concentration V SS Steady state volume of distribution While preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.

Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosed embodiments. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments. It is intended that the following claims define the scope of the embodiments and that methods and structures within the scope of these claims and their equivalents be covered thereby. nces 1. Lentivirus-based 2-transfected atic cancer cells for deep in vivo imaging of metastatic disease. Yu Z, Zhou J, Hoffman RM., Methods Mol Biol. 2012;872:69- 83.doi:lO.l007/978617792_5. 2. Color-coded real-time subcellular fluorescence imaging of the interaction between cancer and host cells in live mice.Yamauchi K, Tome Y, Yamamoto N, Hayashi K, Kimura H, Tsuchiya H, Tomita K, Bouvet M, Hoffman RM.Anticancer Res. 2012 Jan;32(l):39-43. 3. Lentivirus-based DsRedtransfected pancreatic cancer cells for deep in vivo imaging of metastatic disease.Zhou J, Yu Z, Zhao S, Hu L, Zheng J, Yang D, Bouvet M, Hoffman RM.J Surg Res. 2009 Nov;lS7(l):63-70. doi: lO.lOl6/j.jss.2008.08.027. Epub 2008 Oct 9. 4. Fluorescent LYVE-l antibody to image dynamically lymphatic trafficking of cancer cells in vivo.McElroy M, i K, Garmy-Susini B, Kaushal S, Vamer JA, Moossa AR, n RM, Bouvet M.J Surg Res. 2009 Jan;15 l(l):68-73. doi: lO.lOl6/j.jss.2007. .

Epub 2008 Jan 18. ] 5. Lentiviral reporter constructs for fluorescence tracking of the temporospatial pattern of Smad3 signaling.Stuelten CH, Kamaraju AK, Wakefield LM, Roberts techniques. 2007 Sep;43(3):289-90, 292, 294. 6. lular imaging in the live mouse.Hoffman RM, Yang M.Nat Protoc. 2006;1(2):775-82. 7. In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the ion of liver metastases.Bouvet M, Tsuji K, Yang M, Jiang P, Moossa AR, Hoffman RM.Cancer Res. 2006 Dec l;66(23): 1 1293-7. 8. Dual-color imaging of r-cytoplasmic dynamics, ity, and proliferation of cancer cells in the portal vein area.Tsuji K, Yamauchi K, Yang M, Jiang P, Bouvet M, Endo H, Kanai Y, Yamashita K, Moossa AR, Hoffman RM.Cancer Res. 2006 Jan l;66(l):303-6. ] 9. FL-CTL assay: fluorolysometric ination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells.Chen K, Chen L, Zhao P, Marrero L, Keoshkerian E, Ramsay A, Cui Y.J Immunol Methods. 2005 May;300(1- 2): 100-14. 10. Murine leukemia virus (MLV) replication monitored with fluorescent proteins.Sliva K, Erlwein O, Bittner A, Schnierle BS.Virol J. 2004 Dec 20;1 :14. 11. Real-time whole-body imaging of an orthotopic metastatic te cancer model expressing red fluorescent n.Yang M, Jiang P, Yamamoto N, Li L, Geller J, Moossa AR, Hoffman state. 2005 Mar 1;62(4):374-9. 12. Cellular dynamics visualized in live cells in vitro and in vivo by differential dual- color nuclear-cytoplasmic fluorescent-protein expression.Yamamoto N, Jiang P, Yang M, Xu M, Yamauchi K, Tsuchiya H, Tomita K, Wahl GM, Moossa AR, Hoffman RM.Cancer Res. 2004 Jun 15;64(12):4251-6. 13. In vivo imaging with fluorescent ns: the new cell biology.Hoffman RM.Acta Histochem. 2004;106(2):77-87. 14. Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vz'v0.Yamamoto N, Yang M, Jiang P, Xu M, Tsuchiya H, Tomita K, Moossa AR, Hoffman RM.Clin Exp asis. 0(7):633-8. 15. Yaghoubi S, Barrio JR, m M, Iyer M, Namavari M, Satyamurthy N, Goldman R, Herschman HR, Phelps ME, Gambhir SS. Human pharmacokinetic and dosimetry studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression. J Nucl Med. 2001 Aug;42(8):1225-34. 16. Pafieda A, Collantes M, Beattie SG, Otano I, Snapper J, Timmermans E, Guembe L, Petry H, Lanciego JL, Benito A, Prieto J, Rodriguez-Pena MS, Pefiuelas I, Gonzalez- nolaza G. Adeno-associated virus liver transduction efficiency measured by in vivo HBG positron emission tomography imaging in rodents and nonhuman primates. Hum Gene Ther. 2011 Aug;22(8):999-1009. doi: 10.1089/hum.2010.190. Epub 2011 Apr 6. 17. Johnson M, Karanikolas BD, an SJ, Powell R, Black ME, Wu HM, Czemin J, Huang SC, Wu L. ion of variant HSV1-tk gene expression to determine the sensitivity of HBG PET imaging in a prostate tumor. J Nucl Med. 2009 May;50(5):757-64. doi: .2967/jnumed.108.058438. Epub 2009 Apr 16. 18. Pefiuelas I, Mazzolini G, Boan JF, Sangro B, Marti-Climent J, Ruiz M, Ruiz J, Satyamurthy N, Qian C, Barrio JR, Phelps ME, Richter JA, Gambhir SS, Prieto J. Positron on Tomography Imaging of Adenoviral-Mediated Transgene Expression in Liver Cancer Patients Gastro (2005)128: 1787. 19. Sangro B, Mazzolini G, Ruiz M, Ruiz J, Quiroga J, Herrero I, Qian C, Benito A, Larrache J, Olagiie C, Boan J, Pefiuelas I, Sadaba B, Prieto J. A phase I clinical trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma Can. Gene Ther. (2010) 17: 837—843. 20. Willmann JK, Paulmurugan R, Rodriguez-Porcel M, Stein W, Brinton TJ, Connolly AJ, Nielsen CH, Lutz AM, Lyons J, Ikeno F, Suzuki Y, Rosenberg J, Chen IY, Wu JC, Yeung AC, Yock P, Robbins RC, Gambhir SS. g gene sion in human mesenchymal stem cells: from small to large animals. Radiology. 2009 Jul;252(1):117-27. doi: .1148/radiol.2513081616. Epub 2009 Apr 14. PubMed PMID: 19366903; PubMed Central PMCID: PMC2702468. 21. Yaghoubi SS, Jensen MC, Satyamurthy N, Budhiraja S, Paik D, Czemin J, Gambhir SS. Noninvasive detection of therapeutic cytolytic T cells with [18F]FHBG PET in a patient with glioma. Nat Clin Pract Oncol. 2009 Jan;6(1):53-8. doi: 10.1038/ncponc1278. Epub 2008 Nov 18. PubMed PMID: 19015650; PubMed Central PMCID: PMC3526373. 22. Roelants V, Labar D, de Meester C, Havaux X, Tabilio A, Gambhir SS, Di Ianni M, Bol A, Bertrand L, Vanoverschelde JL. Comparison between adenoviral and retroviral s for the transduction of the thymidine kinase PET er gene in rat mesenchymal stem cells. J Nucl Med. 2008 Nov;49(11):1836-44. doi: 10.2967/jnumed.108.052175. Erratum in: J Nucl Med. 2009 Jan;50(1):17. PubMed PMID: 18984872. 23. Lee SW, Padmanabhan P, Ray P, Gambhir SS, Doyle T, Contag C, Goodman SB, Biswal S. Stem cell-mediated accelerated bone g observed with in vivo molecular and small animal imaging technologies in a model of skeletal injury. J Orthop Res. 2009 Mar;27(3):295-302. doi: 10.1002/jor.20736. PubMed PMID: 18752273. 24. Chin FT, Namavari M, Levi J, Subbarayan M, Ray P, Chen X, Gambhir SS.

Semiautomated radiosynthesis and biological evaluation of [18F]FEAU: a novel PET imaging agent for HSVl-tk/sr39tk er gene expression. Mol Imaging Biol. 2008 Mar-Apr;10(2):82- 91. Epub 2007 Dec 22. PubMed PMID: 18157580. 25. Yaghoubi SS, Gambhir SS. PET g of herpes simplex virus type 1 thymidine kinase (HSVl-tk) or mutant HSVl-sr39tk reporter gene expression in mice and humans using HBG. Nat Protoc. (6):3069-75. PubMed PMID: 17406570. 26. Deroose CM, De A, Loening AM, Chow PL, Ray P, Chatziioannou AF, r SS. Multimodality imaging of tumor xenografts and metastases in mice with combined small- animal PET, small-animal CT, and bioluminescence g. J Nucl Med. 2007 (2):295- 303. PubMed PMID: 17268028; PubMed Central PMCID: PMC3263830. 27. Kim SJ, Doudet DJ, Studenov AR, Nian C, Ruth TJ, Gambhir SS, sh CH.

Quantitative micro positron emission tomography (PET) imaging for the in vivo ination of pancreatic islet graft survival. Nat Med. 2006 Dec;12(12):1423 -8. Epub 2006 Dec 3. PubMed PMID: 17143277. 28. Yaghoubi SS, Couto MA, Chen CC, Polavaram L, Cui G, Sen L, Gambhir SS.

Preclinical safety evaluation of [18F]FHBG: a PET reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr3 9tk's expression. J Nucl Med. 2006 Apr;47(4):706-15. PubMed PMID: 16595506. 29. Xiong Z, Cheng Z, Zhang X, Patel M, Wu JC, Gambhir SS, Chen X. Imaging chemically d adenovirus for targeting tumors expressing integrin alphavbeta3 in living mice with mutant herpes simplex virus type 1 ine kinase PET reporter gene. J Nucl Med. 2006 Jan;47(1):130-9. PubMed PMID: 97. 30. Shu CJ, Guo S, Kim YJ, Shelly SM, Nijagal A, Ray P, Gambhir SS, Radu CG, Witte ON. Visualization of a primary anti-tumor immune se by positron emission tomography. Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17412-7. Epub 2005 Nov 17.

PubMed PMID: 16293690; PubMed Central PMCID: PMC1283986. 31. Sen L, Gambhir SS, Furukawa H, Stout DB, Linh Lam A, Laks H, Cui G.

Noninvasive imaging of ex vivo intracoronarily delivered nonviral therapeutic transgene expression in heart. Mol Ther. 2005 Jul;12(1):49-57. PubMed PMID: 15963920. 32. Yaghoubi SS, Barrio JR, Namavari M, urthy N, Phelps ME, man HR, Gambhir SS. Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living ts with positron emission tomography. Cancer Gene Ther. 2005 Mar;12(3):329-39. PubMed PMID: 15592447. 33. Miyagawa M, Anton M, Haubner R, Simoes MV, Stadele C, Erhardt W, Reder S, Lehner T, Wagner B, Noll S, Noll B, Grote M, Gambhir SS, Gansbacher B, Schwaiger M, Bengel FM. PET of cardiac transgene expression: comparison of 2 approaches based on herpesviral thymidine kinase reporter gene. J Nucl Med. 2004 Nov;45(11):1917-23. PubMed PMID: 15534063. 34. Green LA, Nguyen K, Berenji B, Iyer M, Bauer E, Barrio JR, Namavari M, Satyamurthy N, Gambhir SS. A tracer kinetic model for [18F]FHBG for tating herpes x virus type 1 thymidine kinase reporter gene sion in living animals using PET. J Nucl Med. 2004 Sep;45(9):1560-70. PubMed PMID: 15347725. 35. Wu JC, Chen IY, Wang Y, Tseng JR, Chhabra A, Salek M, Min JJ, Fishbein MC, Crystal R, Gambhir SS. Molecular imaging of the kinetics of ar endothelial growth factor 2014/029600 gene expression in ic myocardium. Circulation. 2004 Aug 10;110(6):685-91. PubMed PMID: 15302807. 36. Su H, Forbes A, Gambhir SS, Braun J. tation of cell number by a positron emission tomography reporter gene strategy. Mol Imaging Biol. 2004 May-Jun;6(3): 139-48.

PubMed PMID: 15193248. 37. Chen IY, Wu JC, Min JJ, Sundaresan G, Lewis X, Liang Q, Herschman HR, Gambhir SS. Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene ry. Circulation. 2004 Mar 23;109(11):1415-20. Epub 2004 Mar 8. PubMed PMID: 15007006. 38. Sundaresan G, Paulmurugan R, Berger F, Stiles B, Nagayama Y, Wu H, Gambhir SS. MicroPET g of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene. Gene Ther. 2004 Apr;l l(7):609-l8. PubMed PMID: 14724687. 39. Green LA, Yap CS, Nguyen K, Barrio JR, Namavari M, Satyamurthy N, Phelps ME, Sandgren EP, Herschman HR, Gambhir SS. Indirect monitoring of endogenous gene expression by positron emission aphy (PET) imaging of reporter gene expression in transgenic mice. Mol Imaging Biol. 2002 Jan;4(1):7l-8l. PubMed PMID: 50. 40. Ghosh P. Reproducible quantification in PET-CT: Clinical relevance and technological approaches. White Paper Siemens ary 2012). 41. Shankar L.K. et al. Consensus recommendations for the use of 18F-FDG as an indicator of therapeutic response in National Cancer Institute trials. J. Nucl. Med. 2006 June; 47: 1059-66. 42. Bar-Shir A. et al. Transforming thymidine into a ic resonance imaging probe for gene expression. J. Am. Chem. Soc. 2013 Jan; 17-24. 43. Muller U. et al. Synthesis and inical evaluation of a new C-6 alkylated pyrimidine derivative as a PET imaging agent for HSVl-tk gene expression. Am. J. Nucl. Med.

Mol. Imaging 2013; 3:71-84. 44. Mescic A. et al. C-5 hydroxyethyl and hydroxypropyl acyclonucleosides as substrates for thymidine kinase of Herpes simplex virus type 1 (HSV-l TK): Syntheses and biological evaluation. Molecules 2013; 18:5104-24.

VV()2014/153205 SE UENCES SEQ ID NO: 1: wild type HSVl-TK nucleotide ce a:ggcttcgtaccccggccatcaacacgcg SC egchchaccaggctgcgcgt :ctcgcggcc a:agcaaccgacg':acggcg:tgcgccc:cgccggcagcaagaagccacggaag':ccgcccgga gcagaaaatgcccacgctaetgcggg “a,a eagacggtccccacgggatggggaaaaccacc accacgcaachc egg vggcccvggg “cgcgcgacga:atcgtctacgtacccgagccgatga cttactggcgggtgctgggggc :tccgagacaatcgcgaacatctacaccacacaacaccgcc: cgaccaggg ,gagaLa ggggacgcggcgg egg ,aa egacaagcgcccagataacaatg ggca,gch ,aLgccg ,gaccgacgcchLchgc “CC ,Ca,a ecgggggggaggc:gggagc cacatgccccgcccccggccc:caccctcatc:tcgaccgcca :cccatcgccgccctcctg:g C:acccggccgcgcgg ,ach eanggcagcatgaccccccaggccg ,chggch ,chggcc Ceca ,cccgccgacct:gcccggcaccaacatcgtgc :tggggccc eccggaggacagacaca :cgaccgcc:ggccaaacgccagcgccccggcgagcggc:ggaccngcLaLchggc ,gcgaL :cgccgcgt:tacgggc:acttgccaaLacgg egcgg ea “cvgcagvgcggcgggtcg'199099 gaggactggggacagc ecggggacggccgtgccgccccaggg':gccgagccccagagcaacg cacgacccca ,cggggacachLa ,Laccc eg ,Lchggcccccgagttgctggc ccccaacggcgacc eg ,aacg,gLLLgcc ,gggccttggacg':cttggccaaacgcc:ccgt tccatgcachcL ea ,chgga ,Lacgaccaa:cgcccgccggctgccgggacgccctgctgc aacttacctccggga egg eccagacccacgtcaccacccccggc :ccataccgacgatatgcga cctggcgcgcacg ,gcccgggagatgggggaggctaactga SEQ ID NO: 2: wild type HSVl-TK amino acid sequence MASYPGHQ {ASAEiDQAARSRGHSN {{TALR?RRQQ fiAT fiVRP fiQKMPTLL<VYn DGPHGMG {TT TTQLLVALGS { "VY V?: ?MTYWQV. AN YTTQ {LDQGZ SAGDAAVVMTSAQ: TMG ?YAVTDAVLAP I G {A} b QYLMGSMTPQAVPAFVAL" N"VPGAPP 13R {PAK {Q ?G I R<VYG LLAWTVQYLQCGGSW {Ll. DWGQLSGTAVPPQGAEPQSNAGPRP { GDTPETPE{A' fiPPA?NG3L YWVFAWALDVLA<RLQ S HVF: LDYDQS ?AGCaDALLQLTSGMVQTHVTTPGS PT CDPA{TEAR fiMG fiAW SEQ ID NO: 3: HSV-TK in une-C HSV-TK; SR 39 mutant and R25G-R26S Mutation of NLS a:ggcctcgtaccccggccatcaacacgcg SC accaggctgcgcgt :ctcgcggcc atagcaacggatccacggcg:tgcgccc:cgccggcagcaagaagccacggaag':ccgcccgga gcagaaaatgcccacgctaetgcggg “a,a eagacggtccccacgggatggggaaaaccacc accacgcaactgctggtggccc eggg “cgcgcgacga:atcgtctacgtacccgagccgatga ggcgggtgctgggggc :tccgagacaatcgcgaacatctacaccacacaacaccgcc: cgaccaggg ,gagaLa ecggccggggacgcggcgg egg ,aa egacaagcgcccagataacaatg ggca,gch g ,gaccgacgcch,chgc “CC ,Ca,a gggaggCZgggagC cacatgccccgcccccggccc:caccatCttcctcgaccgcca ,cccachchLcaLchg eg C:acccggccgcgcgg ,ach ,anggcagcatgaccccccaggccg ,chggch ,chggcc Ceca ,cccgccgacct:gcccggcaccaacatcgtgc :tggggccc eccggaggacagacaca :cgaccgcc:ggccaaacgccagcgccccggcgagcggc:ggaccngcLaLchggc ,gcgaL :cgccgcgt:tacgggctacttgccaaLacgngcgg ea chgcagvgcggcgggtcg'199099 gaggactggggacagcsLscggggacggccgtgccgccccagggtgccgagccccagagcaacg cgggcccacgacccca,a,cggggacacgttat2taccctgtttcgggcccccgagttgctggc cggcgacc,g,a,aacg,gLLLgcc,gggccttggacg:cttggccaaacgcc:ccgt tccatgcachcLSea,chgga,Lacgaccaa:cgcccgccggctgccgggacgccctgctgc aacttacctccgggaegg,ccagacccacgtcaccacccccggc:ccataccgacgatatgcga cctggcgcgcacg,SSgcccgggagatgggggaggctaactga SEQ ID NO: 4 (amino acid sequence encoded by SEQ ID NO: 3) MASYPGHQHASAEDQAA<S{GHSNGSTALR?RRQQfiATfiV{?fiQ<M?TLLRVYIDGPHGMGKTT TTQLLVALGSRDD VYV?%?MTYWQVPGASHT AN YTTQi<PDQGfi SAGDAAVVMTSAQIT mLu'r'G) ?YAVTDAVPA?{ GGHAGSS{A???ALT ELDR{? AF PCY?AARYLMGSMTPQAVLAFVA "?PTLPGTW"VLGAL?fiD{H D<PA<RQ<?G%{LDLAMPAA {{VYGLLAWTVRYLQCGGSW? GTAVP?QGAE?QSNAG?{?{ GDTLETLE{A?flLLA?WGDLYNVFAWALDVLAKRLQ {VFILDYDQS?AGC?DALLQLTSG VQTiVTTPGS ?T CDLARTEARH GfiAN SEQ ID NO: 5: HSV-TK Sites to mutate are in bold, underlining (HSV-TK nuclear localization sequence, RR, and Substrate Binding Domain, LIF and AAL atggcctcgtaccccggccatcaacacgcgtCtgcgttcgaccaggctgcgcgttctcgc 60 M A S Y P G H Q T A S A F D Q A A R S R ggccatagcaaccgacgtacggcg:tgcgccc:cgccggcagcaagaagccacggaagtc G H S N 3 3 T A L R P 3 3 Q Q E A T E V cgcccggagcagaaaatgcccacgctactgcgggtttatatagacggtccccacgggatg R P E Q K M P T L L R V Y I D G P H G M gggaaaaccaccaCcacgcaactgctggtggccctgggttcgcgcgacgatatcgtctac G K T T T T Q L L V A L G S R D D I V Y gtacccgagccgatgacttactggcgggtgctgggggcttccgagacaatcgcgaacatc V P E P M T Y W R V L G A S E T I A N acacaacaccgcctcgaccagggtgagatatcggccggggacgcggcggtggta Y T T Q H R L D Q G d S A G D A A V V atgacaagcgcccagataacaatgggcatgccttatgccgtgaccgacgccgttctggct M T S A Q I T M G M P Y A V T D A V L A cctcatatcgggggggaggctgggagctcacatgccccgcccccggccctcaccctcatc P H I G G E A G S S T A P P P A L T E l EEggaccgccatcccatcgccgccctcctgtgctacccggccgcgcggtaccttatgggc F D R H P I A A E L C Y P A A R Y L M G agcatgaccccccaggccgtgctggcgttcgtggccctcatcccgccgaccttgcccggc WO 53205 S M T P Q A V L A F V A L I P P T L P G accaacatcgtgcttggggcccttccggaggacagacacatcgaccgcctggccaaacgc T N I V L G A L P Lti D R H I D R L A K R CagcgccccggcgagcggctggaccngcLaLchgchgcgaLchccgcgtttacggg Q R P G E R L D L A M L A A I R R V Y G ctacttgccaaLacgngcgg,aLCLgcagLgcggcgggtcgtggcgggaggactgggga L L A N T V R Y L Q C G G S W R m D W G cagctttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggccca Q L S G T A V P P Q G A E P Q S N A G P cgaccccatatcggggacacg,La,LLacchgLLchggcccccgagttgctggccccc R P H I G D T L F T L F R A P m L L A P aacggcgachgLaLaachgLLLgccnggccttggacgtCttggccaaacgcctccgt N G D L Y N V F A W A L D V L A K R L R tccatgcacgtctttatcctggattacgaccaatcgcccgccggctgccgggacgccctg 1020 S M H V F I L D Y D Q S P A G C R D A L ctgcaacttacctccgggatggtccagacccacgtcaccacccccggc:ccataccgacg 1080 L Q L T S G M V Q T T V T T P G S I P T atatgcgacctggcgcgcacgtttgcccgggagatgggggaggctaactga C D L A R T F A R E M G E A N * SEQ ID NOS: 6 and 7: Sac I-Kpn I (SR39) mutant region GAGCTCACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCC- ETCGAGTGTACGGGGCGGGGGCCGGGAGTGGEAGéAGEAGCTGGCGGTAGGGTAGCGG- Sac " —EEC§T§CTGTGCTACCCGGCCGCGCGGTACC (SfiQ 3 NO: 6) —§§GEAEGACACGATGGGCCGGCGCGCCATGG (SfiQ 3 NO: 7) Kpn " IIII: :IIII Gm- IIIIIIIIIII AG” ' GAGCTC IIIIIIIIIII SEQ ID NOS: 8 and 9: Sac I-Kpn I (SR39) mutant region (cut) CACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCCEECéTE TCGAGTGTACGGGGCGGGGGCCGGGAGTGGEAGéAGEAGCTGGCGGTAGGGTAGCGGéé Sac I (cut) CTGTGCTACCCGGCCGCGCGGTAC (SfiQ 3 NO: 8) GEAEGACACGATGGGCCGGC (SfiQ 3 NO: 9) Kpn 2(Cut) GGTACC IIIIIIIIIII GTAC - 3’ IIIIIIIIIII SEQ ID NOS: 10 and 11: Primers SR39sackpn F1 ’CACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCCEECéTECTG TGCTACCCGGCCGCGCGGTAC 3’ (SfiQ 3 NO: 10) ckpn R1 ’CGCGCGGCCGGGTAGCACAGCATGAAGGCGATGGGATGGCGGTCGAEGAéGAEGGTGAGGGC CGGGGGCGGGGCATGTGAGCT 3’ (SfiQ 3 NO: 11) SEQ ID NO: 12 Gene #3 mHSV-TK CO A168H(LIF...AHL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT CGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 13 Gene #4 mHSV-TK CO TK A167F(LIF...FAL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC CACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG ACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC CGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 14 Gene #5 mHSV-TK CO dual mutant A167F-A168H (LIF...FHL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCTTCCACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 15 Gene #6 mHSV-TK CO MB-IFL A168H(IFL...AHL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCATCTTCCTG GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 16 Gene #1 HSV-TK A168H dmNLS CO SC: Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCAGGATCT GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC CACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 17 Gene #2 HSV-TK A167F dmNLS CO SC: Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCAGGATCT CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC TGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG ACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 18 Gene #3 HSV-TK A168H NESdmNLS CO SC: Length:1221 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC CACAGCAACGGCAGCACCGCACTGCGGCCAGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG AGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGACCACACGGCATGGGCAAGACCAC CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC TGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATTACAAT GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCACCACACATCGGCGGCGAGGCCGGCAGC AGCCACGCACCACCACCAGCACTGACCCTGATCTTCGACCGGCACCCAATCGCACACCTGCTGT GCTACCCGGCAGCACGCTACCTGATGGGCTCCATGACACCACAAGCCGTGCTGGCCTTCGTGGC CCTGATCCCACCAACACTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC ATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCACCACAGGGCGCCGAGCCACAGAGCAAC GCCGGACCACGACCACACATCGGCGACACCCTGTTCACCCTGTTCCGGGCACCAGAGCTGCTGG CACCAAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG GCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG CCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 19 Gene #4 HSV-TK A167F NESdmNLS CO SC: Length:1221 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC CACAGCAACGGCAGCACCGCACTGCGGCCAGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG AGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGACCACACGGCATGGGCAAGACCAC CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC TGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATTACAAT GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCACCACACATCGGCGGCGAGGCCGGCAGC AGCCACGCACCACCACCAGCACTGACCCTGATCTTCGACCGGCACCCAATCTTCGCACTGCTGT GCTACCCGGCAGCACGCTACCTGATGGGCTCCATGACACCACAAGCCGTGCTGGCCTTCGTGGC CCTGATCCCACCAACACTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC ATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCACCACAGGGCGCCGAGCCACAGAGCAAC GCCGGACCACGACCACACATCGGCGACACCCTGTTCACCCTGTTCCGGGCACCAGAGCTGCTGG CACCAAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG CTCCATGCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 20 Gene #5 HSV-TK A168H NESdmNLS JCO SC: Length:1221 GGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA CATTCTAATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC TGGATCAAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT GCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGT GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG AGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG ATCTATGCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 21 Gene #6 HSV-TK A167F LS JCO SC: Length:1221 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA CATTCTAATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC TGGATCAAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT GGGAATGCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTTTCGCACTGCTGT GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG TTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG ATCTATGCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 22 HSV-TK dmNLS Al68H, CO & SC dmNLS = double mutated Nuclear Localization Sequence CO = codon optimized SC = splice corrected at 327 and 555 Kozak Sequence, Underlined gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaCTGCGgCCaGGATCT CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC aCCaCACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTC GACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtccATGA CaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG GACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC aCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTC ACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCtccATGCACGTGTTCATCCTGGACTACGACCAGtc aCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACa ACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTgtca SEQ ID NO: 23 — MAP Kinase Kinase Nuclear Export Polynucleotide Sequence CTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC SEQ ID NO: 24 - MAP Kinase Kinase r Export Polypeptide ce LQKKLfifiLfiLDG SEQ ID NO: 25 — Targeting Moiety WREPSF ALS

Claims (43)

1. Use of a retroviral particle sing an HSV-TK polynucleotide encoding a mutated form of HSV-TK comprising a nuclear export sequence (NES) in the manufacture of a diagnostic for identifying a patient with lesions capable of benefitting from gene therapy, n the identifying comprises: a) administering the retroviral particle comprising the HSV-TK polynucleotide comprising the NES to the patient, wherein the HSV-TK polynucleotide encodes a d form of HSV-TK comprising ons at amino acid residues 32, 33, and 168 of the viral r localization sequence (NLS), wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisting of glycine, serine, glutamic acid, an acidic amino acid, and cysteine, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK; b) administering to the patient a substrate of HSV-TK attached to a radioactive tracer; c) ing the ve amount and location of the radioactive signal present in the patient; and d) determining the on of lesions in the patient, wherein patients with: ctive signals above a certain threshold, and location of the radioactive signal correlating with lesions measured in step c) of the patient, are identified as capable of tting from gene therapy treatment.

2. The use of claim 1, wherein the ate of HSV-TK is chosen from the group consisting of FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9-([3-fluoro- oxy propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'- deoxy-2'-fluoro13-D-arabinofuranosyl)iodouracil), FEAU (fluoroethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoromethyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-((1 - fluorohydroxypropanyloxy)methyl)methylpyrimidine-2,4(1H,3H)-dione), ganciclovir, valganciclovir, acyclovir, valacyclovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-1 (HHGFEP) or 5-(2-)hydroxyethyl)-and 5-(3- hydroxypropy1)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains.

3. The use of claim 1 or claim 2, wherein the substrate of HSV-TK is FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine).

4. The use of any one of claims 1-3, wherein the radioactive tracer is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl.

5. The use of any one of claims 1-4, wherein the radioactive tracer is 18F.

6. The use of claim 1, wherein the HSV-TK substrate is [18F]FHBG 18F-fluoro (hydroxymethyl)butyl]guanine).

7. The use of any one of claims 1-6, n the ctive signal is measured using positron emission tomography (PET) scanning.

8. The use of claim 7, wherein the threshold level is at least above 2.0 SUV (standardized uptake value) or at least 20% above background when the radioactive signal is measured using PET scanning.

9. The use of claim 7, n the threshold level is between 1.0 SUV and 3.0 SUV, or between 20% to 40% above background when the radioactive signal is measured using PET scanning.

10. The use of any one of claims 1-9, wherein the mutation is A168H.

11. The use of any one of claims 1-10, wherein the nuclear export sequence (NES) is located at or near the amino terminus of the expressed d form of HSV-TK.

12. The use of claim 1, wherein the HSV-TK polynucleotide is SEQ ID NO: 18.

13. The use of claim 12, wherein the retroviral particle further comprises a polynucleotide encoding for a targeting protein expressed on the viral envelope.

14. The use of claim 13, wherein the targeting protein binds to collagen, laminin, fibronectin, elastin, glycosaminoglycans, proteoglycans or RGD.

15. The use of claim 14, wherein the targeting protein binds to collagen.

16. The use of claim 15, wherein the ing protein is SEQ ID NO: 25.

17. Use of a retroviral particle comprising an HSV-TK polynucleotide encoding a mutated form of HSV-TK comprising a NES in the manufacture of a medicament for treating a patient in need of treatment for lesions capable of tting from gene therapy treatment, wherein the patient is identified by the following steps a)-e): a) administering the retroviral particle comprising the HSV-TK polynucleotide comprising the NES to the patient, and transducing cells from the patient with the polynucleotide encoding HSV-TK, wherein the HSV-TK polynucleotide s a d form of HSV-TK sing mutations at amino acid residues 32, 33, and 168, wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group ting of glycine, serine, glutamic acid, an acidic amino acid and ne, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK; b) treating the cells with a substrate of HSV-TK attached to a radioactive tracer; c) measuring the relative amount and on of radioactive signal present in target tissue; d) identifying if a level of radioactive signal is above a threshold; e) determining the location of lesions in the patient; and f) re-administering the patient with the retroviral particle comprising the HSV-TK polynucleotide of a) if: (i) the measured radioactive signal in the patient is above the threshold; and (ii) the location of the ed radioactive signal correlates with the location of the lesions measured in e).

18. The use of claim 17, wherein the substrate of HSV-TK is chosen from the group consisting of FHBG fluoro(hydroxymethyl)butyl]guanine), FHPG (9-([3-fluoro- l—hydroxy propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'- 2'-fluoro(3-D-arabinofuranosyl)iodouracil), FEAU (fluoroethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoromethyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-((1 - fluorohydroxypropanyloxy)methyl)methylpyrimidine-2,4(1H,3H)-dione), ganciclovir, valganciclovir, acyclovir, clovir, lovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-1 (HHGFEP) or 5-(2-)hydroxyethyl)-and 5-(3- ypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains.

19. The use of claim 17, wherein the substrate of HSV-TK is FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine).

20. The use of any one of claims 17 to 19, wherein the radioactive tracer is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl.

21. The use of any one of claims 17 to 20, n the radioactive tracer is 18F.

22. The use of claim 21, wherein the HSV-TK substrate is [18F]FHBG (9-[4-18F-fluoro (hydroxymethyl)butyl]guanine).

23. The use of any one of claims 17 to 22, n the radioactive signal is measured using positron emission tomography (PET) scanning.

24. The use of claim 23, wherein the threshold level is at least above 2.0 SUV (standardized uptake value) or at least 20% above background when the radioactive signal is ed using the PET scan.

25. The use of claim 23, wherein the threshold level is between 1.0 SUV and 3.0 SUV when the radioactive signal is measured using the PET scan.

26. The use of claim 24 or claim 25, wherein the gene therapy retroviral particle comprises a second therapeutic polynucleotide.

27. The use of any one of claims 17 to 26, wherein the mutation is A168H.

28. The use of any one of claims 17 to 27, wherein the NES is located at or near the amino terminus of the expressed mutated form of .

29. The use of claim 17, wherein the HSV-TK polynucleotide is SEQ ID NO: 18.

30. The use of claim 29, wherein the retroviral particle further comprises a polynucleotide encoding for a ing protein expressed on the viral envelope.

31. The use of claim 30, wherein the targeting protein binds to collagen, laminin, fibronectin, elastin, glycosaminoglycans, proteoglycans, or RGD.

32. The use of claim 31, wherein the targeting protein binds to collagen.

33. The use of claim 32, wherein the targeting protein is SEQ ID NO: 25.

34. The use of any one of claims 1 to 9, wherein the d form of HSV-TK r comprises mutations at amino acid residues 25 or 26, wherein the amino acid residues correspond to positions 25 and 26 of SEQ ID NO: 2.

35. The use of claim 34, wherein amino acid es 25 or 26 are mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.

36. The use of any one of claims 1 to 9, or 34, wherein amino acid residue 168 is d to an amino acid selected from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.

37. The use of any one of claims 17 to 26, wherein the d form of HSV-TK further comprises mutations at amino acid residues 25 or 26, wherein the amino acid residues correspond to positions 25 and 26 of SEQ ID NO: 2.

38. The use of claim 37, wherein amino acid es 25 or 26 are mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.

39. The use of any one of claims 17 to 26, wherein amino acid residue 168 is mutated to an amino acid selected from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.

40. The use of claim 34, wherein the mutation is A168H.

41. The use of claim 34 or claim 40, n the nuclear export sequence (NES) is located at or near the amino terminus of the expressed d form of HSV-TK.

42. The use of claim 37, wherein the mutation is A168H.

43. The use of claim 37 or claim 42, wherein the nuclear export sequence (NES) is located at or near the amino terminus of the expressed mutated form of HSV-TK.