NZ751656B2 - Improved thymidine kinase gene - Google Patents
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Abstract
Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases (HSV-TK) are provided, including their use in diagnostic and therapeutic applications. The thymidine kinases may be mutated using conservative mutations, non-conservative mutations, or both. In particular, the mutations of the present invention occur at positions 25, 26, 32 or 33 in comparison to the wild-type HSV1-TK. Also provided are gene therapeutic systems, including viral and retroviral particles. of the present invention occur at positions 25, 26, 32 or 33 in comparison to the wild-type HSV1-TK. Also provided are gene therapeutic systems, including viral and retroviral particles.
Description
IMPROVED THYMIDINE KINASE GENE
CROSS-REFERENCE
This ation is a divisional of New Zealand patent application number 712210, the
entire content of which is incorporated into the present specification by this cross-reference.
New Zealand patent application number 712210 is derived from international patent
application number which claims the t of U.S. Provisional
Application No. 61/784,901, filed March 14, 2013, which applications are orated herein
by reference in their 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 ant
tissue, an ability to invade local and even remote tissues, lack of differentiation, lack of
able symptoms and most significantly, the lack of effective therapy and prevention.
Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not
y defined but mechanisms such as genetic susceptibility, chromosome ge disorders,
viruses, environmental factors and immunologic disorders have all been linked to a malignant cell
growth and transformation. Cancer asses 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. ide, more than 10 n people are diagnosed with cancer every year and it is
estimated that this number will grow to 15 million 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 polynucleotide sequences encoding mutated forms of thymidine
kinase from a human herpes 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 ed to a polynucleotide sequence of SEQ ID NO: 1 or
A polynucleotide sequence encoding a d form of thymidine kinase from a human
herpes simplex virus (HSV-TK), n the encoded HSV-TK is mutated at amino acid residue
, 26, 32, 33, 167, 168 or a combination thereof, wherein the polynucleotide sequence is
mutated compared to a polynucleotide sequence of SEQ ID NO: 3. In one embodiment, the
encoded HSV-TK is mutated at amino acid residues 167, 168, or a combination thereof to a
polar, non-polar, basic or acidic amino acid. In another embodiment, the encoded HSV-TK is
mutated at amino acid residue 167 to a polar, non-polar, basic or acidic amino acid. In yet
another embodiment, the encoded HSV-TK is mutated at amino acid residue 168 to a polar, non-
polar, basic or acidic amino acid. In still another embodiment, the encoded HSV-TK is mutated
at both amino acid residues 167 and 168 to a polar, non-polar, basic or acidic amino acid.
In one embodiment, amino acid e 167 of the encoded HSV-TK is mutated to serine
or phenylalanine. In another embodiment, amino acid residue 168 of the encoded HSV-TK is
mutated to an amino acid selected from the group consisting of: histidine, lysine, cysteine,
serine, and phenylalanine. In still another embodiment, the encoded HSV-TK is mutated at
amino acids 25 and 26. In yet r embodiment, amino acid residues 25 and 26 are mutated
to an amino acid chosen from the group ting of: glycine, serine, and glutamic acid. In
another embodiment, the encoded HSV-TK is d at amino acid residues 32 and 33. In one
ment, the amino acid residues 32 and 33 are mutated to an amino acid chosen from the
group consisting of: glycine, serine, and glutamic acid. In one embodiment, the encoded HSV-
TK is mutated at amino acid residues 25, 26, 32 and 33. In another embodiment, amino acid
residues 25, 26, 32 and 33 are mutated to an amino acid chosen from the group consisting of:
glycine, , and glutamic acid. In still another embodiment, the d HSV-TK comprises
at least one mutation chosen fiom the group consisting of amino acid residues 25, 26, 32 and 33,
and at least one mutation chosen from the group consisting of amino acid residues 167 and 168.
In still other embodiments, the encoded HSV-TK ce fiarther comprises a nuclear
export signal (NES). In another embodiment, the nuclear export signal sequence is inserted at or
’ terminus of the HSV-TK
near the 5 sequence. In another embodiment, the nuclear export signal
ce is LQKKLEELELDG (SEQ ID NO: 24). In one embodiment, the encoded mutant
HSV-TK does not localize exclusively to the nuclear region.
In one embodiment, the encoded modified HSV-TK exhibits a reduced amount of
thymidine kinase activity as compared to ype HSV-TK. In another embodiment, the
ty of the encoded modified HSV-TK is reduced by about 1.5 fold, about 2-fold, about 5-
fold, about d, about 20-fold, about 30-fold, or about 50-fold. In still another embodiment,
the activity of the encoded modified HSV-TK is reduced by about 1.5%, about 2%, about 5%,
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or about 100%.
In one embodiment, the encoded HSV-TK comprises ons at amino acid residues
, 26, 32, 33 and 168. In another embodiment, the encoded HSV-TK comprises mutations
R25G, R26S, R32G, R33S and A168H.
In one embodiment, modified cleotide sequence comprises a nucleic acid
sequence set forth as any one of SEQ ID NOS: 12-22. In still another embodiment, the modified
WO 53258
polynucleotide sequence comprises a nucleic acid sequence set forth as any one of SEQ ID
NOS: 16-22. In one embodiment, the sequence comprises TKl68dmNES (SEQ ID NO: 18). In
still another embodiment, the polynucleotide encodes a modified HSV-TK polypeptide.
In still other embodiments, the polynucleotide further comprises a polynucleotide
sequence coding for a second polypeptide, wherein said second polypeptide is a therapeutic
polypeptide. In still other embodiments, the second therapeutic polypeptide is a second suicide
gene or a growth . In some embodiments, the grth factor is chosen from the group
consisting of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF),
opoietin, G-CSF, GM-CSF, TGF-(x, TGF-B and fibroblast growth factor. In some
embodiments, the second suicide gene is chosen from the group consisting of: a cytosine
deaminase, a VSV-tk, IL-2, nitroreductase (NR), ylesterase, beta-glucuronidase,
cytochrome p450, alactosidase, diphtheria toxin A-chain (DT-A), carboxypeptide G2
(CPGZ), purine nucleoside phosphorylase (PNP), and ytidine kinase (dCK).
In some ments, the polynucleotides fiarther comprises a polynucleotide encoding
for a PiT-2 polypeptide. In still other embodiments, the polynucleotides disclosed herein further
comprises a polynucleotide encoding for a targeting polypeptide. In one embodiment, the
targeting polypeptide binds to an extracellular protein. In another embodiment, the extracellular
protein is collagen.
Also provided herein are methods of killing neoplastic cells in a subject in need thereof,
the method sing administering a therapeutically ive amount of a retroviral particle,
the iral vector encoding an HSV-TK modified peptide as described herein.
In some embodiments, the retroviral particle is administered intravenously,
intramuscularly, subcutaneoustly, intra-arterially, intra-hepatic arterially, intra-thecally, intraperitoneally
and/or intra-tumorally. In other embodiments, the retroviral particle is administered
intra-tumorally or enously. In yet other embodiments, the iral vector particle is
stered intra-arterially.
In other embodiments, at least 1 x 1012 TVP of retroviral vector is administered cumulatively to
the subject in need thereof In still other embodiments, at least 1 x 109 TVP of retroviral vector
is administered at one time to the t in need thereof
In still other embodiments, the prodrug is administered between about 1-2 days after
administration of the retroviral vector particle. In some embodiments, the prodrug is chosen
from the group consisting of ganciclovir, valganciclovir In
, aciclovir, clovir, penciclovir.
some ments, the prodrug is ganciclovir.
Also provided herein are methods for treating cancer in a patient in need f, the
method comprising delivering a therapeutically effective amount of a retroviral vector particle,
the retroviral vector encoding an HSV-TK modified peptide as described herein, followed by
administration of a nucleoside prodrug to the patient in need thereof.
Also provided herein are methods of increasing HSV-TK ganciclovir, valganciclovir ,
aciclovir, clovir, penciclovir-mediated killing of neoplastic cells in a subject, the method
comprising delivering a therapeutically effective amount of a iral vector particle
comprising an HSV-TK to the subject in ction with a gap junction intracellular
communication (GJIC)—increasing treatment. In some embodiments, the GJIC-increasing
treatment comprises delivering a polynucleotide sequence encoding at least one gap junction
subunit. In other embodiments, the gap on subunit is connexin 43, connexin 30, or
connexin 26. In yet other embodiments, the gap junction subunit is a gap junction subunit
modified to prevent anslational ations. In still other embodiments, the GJIC-
increasing treatment comprises ring a polynucleotide sequence encoding E-cadherin. In
still other embodiments, the GJIC-increasing treatment comprises delivering to the subject a
compound from the group consisting of: gemcitabine; cAMP; a retinoic acid; a carotenoid; a
glucocorticoid, a flavanoid, apigenin, or lovastatin. In yet other embodiments, the GJIC-
increasing treatment comprises proteasome inhibition. In one ment, the proteasome
inhibition comprises administration ofN—Acetyl-Leu-Leu-Nle-CHO (ALLN) and/or
chloroquine. In other embodiments, the ncreasing treatment comprises radiation or
electrical ent. .
Also provided herein are methods of killing a cell, the method comprising: a) introducing
into the cell a polynucleotide sequence according to any one of claims 1-26; b) ng or
initiating the cell to express the expressed thymidine kinase or variant thereof; an c) ting
the cell with an agent that is converted by thymidine kinase to a cytotoxic agent.
In one embodiment, a polynucleotide sequence encodes a mutated form of thymidine
kinase from a human herpes simplex virus (HSV-TK) sing 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more modifications. In another embodiment, a polynucleotide sequence encodes a mutated form
of thymidine kinase from a human herpes simplex virus (HSV-TK) comprises 2, 3, 4, 5, 6, 7, 8,
9, 10 or more modifications. In another embodiment, a polynucleotide sequence s a
mutated form of thymidine kinase from a human herpes simplex virus (HSV-TK) comprises 3,
4, 5, 6, 7, 8, 9, 10 or more modifications. In r ment, a polynucleotide sequence
encodes a d form of thymidine kinase from a human herpes simplex virus (HSV-TK)
comprises 4, 5, 6, 7, 8, 9, 10 or more ations. In another embodiment, a polynucleotide
sequence encodes a mutated form of thymidine kinase from a human herpes simplex virus
(HSV-TK) comprises 5, 6, 7, 8, 9, 10 or more modifications.
In one ment, the encoded HSV-TK may be mutated at amino acid residues 167,
168, or a combination thereof to a polar, non-polar, basic or acidic amino acid. For example, the
encoded HSV-TK may be mutated at amino acid e 167 to a polar, non-polar, basic or
acidic amino acid. In another example, the encoded HSV-TK may be mutated at amino acid
residue 168 to a polar, non-polar, basic or acidic amino acid. In another example, the encoded
HSV-TK may be mutated at both amino acid residues 167 and 168 to a polar, lar, basic or
acidic amino acid.
In another ment, amino acid residue 167 of the d HSV-TK may be
mutated to serine or phenylalanine.
In another embodiment, amino acid residue 168 of the encoded HSV-TK may be
mutated to an amino acid selected from the group consisting of: histidine, lysine, cysteine,
serine, and phenylalanine.
In another embodiment, the encoded HSV-TK may be mutated at amino acids 25 and 26.
For example, amino acid residues 25 and 26 may be d to an amino acid chosen from the
group consisting of: glycine, serine, and glutamic acid.
In another embodiment, the encoded HSV-TK may be mutated at amino acid residues 32
and 33. For example, amino acid residues 32 and 33 may be mutated to an amino acid chosen
from the group consisting of: glycine, serine, and glutamic acid.
In another embodiment, the encoded HSV-TK may be mutated at amino acid es
, 26, 32 and 33. For example, amino acid residues 25, 26, 32 and 33 may be mutated to an
amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.
In another embodiment, the encoded mutant HSV-TK does not localize exclusively to
the nuclear region.
In another embodiment, the encoded modified HSV-TK ts a reduced amount of
ine kinase actiVity as compared to Wild-type .
In another embodiment, the thymidine kinase actiVity of the encoded modified HSV-TK
may be d by about 1.5 fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about
-fold, or about 50-fold.
In another embodiment,the thymidine kinase actiVity of the encoded modified HSV-TK
may be reduced by about 1.5%, about 2%, about 5%, about 10%, about 20%, about 30%, about
40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.
In another embodiment, the thymidine kinase actiVity of the encoded modified HSV-TK
may be increased by about 1.5 fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold,
about 30-fold, or about 50-fold.
In another embodiment, the thymidine kinase activity of the encoded modified HSV-TK
may be increased by about 1.5%, about 2%, about 5%, about 10%, about 20%, about 30%, about
40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.
Provided herein is polynucleotide sequence as described above, where the encoded HSV-
TK comprises the mutation A167F, A168H or both.
A polynucleotide ce described herein may further comprise a polynucleotide
sequence coding for a second polypeptide, where said second polypeptide is a therapeutic
polypeptide. The therapeutic polypeptide may, in some instances, be a suicide gene. Suicide
genes include, but are not limited to, a cytosine deaminase, a VSV-tk, IL-2, nitroreductase (NR),
carboxylesterase, beta-glucuronidase, cytochrome p450, beta-galactosidase, diphtheria toxin A-
chain (DT-A), carboxypeptide G2 , purine nucleoside phosphorylase (PNP), guanylate
kinase, and ytidine kinase (dCK).
In one embodiment, a modified polynucleotide sequence bed herein may comprise
a nucleic acid sequence set forth as any one of SEQ ID NOS: 12-24.
In another embodiment, a modified polynucleotide sequence described herein may
comprise a nucleic acid sequence set forth as any one of SEQ ID NOS: 22-24.
In one embodiment, a polynucleotide sequence bed herein comprises a nuclear
export signal. For example, a polynucleotide sequence may comprise HSV-TKA168HdmNES
(SEQ ID NO: 18).
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
comprises HSV-TK A167Fsm, wherein ‘sm’ refers to the single mutation pair 26S
(SEQ ID NO: 13) .
In another embodiment, a iral vector for use in the s described herein
comprises HSV-TK m (SEQ ID NO: 12).
In another ment, a retroviral vector for use in the methods described herein
comprises HSV-TK A167de, wherein ‘dm’ refers to the double mutation pair R25G-R26S,
R32G-R33S (SEQ ID NO: 17).
In another embodiment, a retroviral vector for use in the methods described herein
comprises HSV-TK m (SEQ ID NO: 16).
In another embodiment, a retroviral vector for use in the methods described herein
comprises HSV-TK A167de and a nuclear export sequence d from mitogen-activated
protein kinase kinase, an example of which is SEQ ID NO: 19.
In r embodiment, a retroviral vector for use in the methods described herein
comprises HSV-TK Al68Hdm and an NES (SEQ ID NO: 18). In such an embodiment, the
ce ses HSV-TK Al68H.
In another embodiment, a retroviral vector for use in the methods described herein
comprises a HSV-TK, wherein such vector comprises an upgraded substrate binding domain and
a mNLS/NES set. Examples of this exemplary embodiment include SEQ ID NOS: l8 and 19.
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 two modifications.
In another embodiment, a retroviral particle comprises a PiT-2 polynucleotide sequence
and the retroviral particle specifically binds to a PiT-2 receptor on the surface of the target cells,
thereby ng for uptake of the retroviral particle into the cell.
In another ment, a retroviral vector for use in the methods described herein
comprises a HSV-TK, wherein the amino acid sequence encoded by the polynucleotide
sequence comprises TKl68dmNES.
ed herein is a method of increasing FHBG (9-[4-fluoro
(hydroxymethyl)butyl]guanine), FHPG (9u{[3~fluorou l uhydroxyulZmpropoxy]methyl)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-methylbeta-D-arabinofuranosyluracil), FHOMP -fluoro
hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir,
valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled dine with 4-hydroxy
(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3-
hydroxypropyl)-substituted pyrimidine tives bearing hydroxypropyl, acyclovir-,
ganciclovir- and penciclovir-like side -mediated g of neoplastic cells in a subject, the
method comprising delivering a eutically effective amount of vector particles encoding
HSV-TK to the subject in conjunction with a gap junction ellular communication (GJIC)—
increasing treatment.
In one embodiment, the HSV-TK used in such methods may be encoded by any of the
polynucleotide sequences described herein.
The GJIC-increasing treatment may comprise, for example, delivering a polynucleotide
sequence encoding at least one gap on subunit. A gap junction subunit may be, for
example, connexin 43, connexin 30, or connexin 26. The gap on subunit may be a gap
junction subunit modified to prevent posttranslational modifications.
In one embodiment, the GJIC-increasing treatment comprises delivering a
polynucleotide sequence encoding E-cadherin.
In another embodiment, the GJIC-increasing treatment comprises delivering to the
subject a compound from the group consisting of: gemcitabine; cAMP; a retinoic acid; a
carotenoid; a glucocorticoid, a flavanoid, apigenin, or lovastatin.
In another embodiment, the GJIC-increasing ent comprises some inhibition.
Proteasome inhibition may comprise administration ofN—Acetyl-Leu-Leu-Nle-CHO (ALLN)
and/or chloroquine.
In another embodiment, the ncreasing treatment comprises radiation or
photodynamic treatment, including coadministration with oxidative agents and agents that
activate MAP kinases.
In another embodiment, the GJIC-increasing treatment comprises electrical treatment.
ed herein is a method of killing a cell, the method sing: (a) introducing into
the cell a polynucleotide sequence described herein; (b) allowing or ting the cell to express
the expressed thymidine kinase or t thereof; and (c) contacting the cell with an agent that
is converted by thymidine kinase to a cytotoxic agent.
Provided herein is a method of increasing thymidine kinase bystander effect, the method
comprising delivering a sequence ng a gap junction subunit in ction with a
iral vector particle encoding HSV-TK. In some embodiments, the retroviral les may
be targeted to a cell or system of interest. In some embodiments, the retroviral targeting method
may comprise the incorporation of a factor that recognizes or binds to the cell or system of
st. In some embodiments, the retroviral targeting method may comprise the incorporation
of targeting proteins, including binding to proteins or receptors on the e of the cell of
system of interest, including antibodies, receptor binding proteins or proteins that bind to
cellular components, including but not limited to collagen. In some embodiments the targeting
protein may comprise proteins that bind to collagen, ing but not limited to peptides,
proteins and/or protein domains that include a collagen binding domain.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended
claims. A better understanding of the features and ages 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 ed, and the accompanying
drawings of which:
Figure l exemplifies how HSV-TK splice site l avoids an inactivated form of
HSV-TK. PCR analysis of T-cell lines and primary T cells transduced with HSV-TK s
with (HuT SF/Tk/mut) or without (HuT GlTkl SvNa) splice site removal.
Figure 2 provides an exemplary schematic for a Phase IA clinical trial with a
composition described herein.
Figure 3 provides an provides an ary schematic for a Phase IB clinical trial with a
composition described herein.
Figure 4 provides an provides an exemplary schedule of events for Phase IA clinical trial
for cohorts l to 3.
Figure 5 provides an provides an exemplary schedule of events for Phase IA clinical trial
for cohorts 4 and above.
Figure 6 provides an es an exemplary schematic for a Phase IB al trial with a
composition described .
Figure 7 provides an provides an exemplary schematic for a Schedule A of clinical trial
for treatment with a composition described herein.
Figure 8 provides an provides an exemplary schematic for a Schedule B of clinical trial
for treatment with a composition bed herein.
Figure 9 provides an illustration of a PiT-2 transmembrane molecule. The box
represents the approximate location of an Anti-PiT-2 Western antibody binding site.
Figure 10 provides an illustration of a PiT-2 transmembrane molecule. The box
represents the approximate location of an Anti-PiT-2 IHC antibody binding site.
Figure 11 provides exemplary Reximmune constructs with s HSV-TK
modifications. Figure llA: GM-CSF Minus, HSV-TK l67sm. Figure llB: GM-CSF Minus,
HSV-TK l68sm. Figure 110 GM-CSF Minus, HSV-TK l67dm. Figure llD: GM-CSF
Minus, HSV-TK l68dm. Figure llE: GM-CSF Minus, HSV-TK l67dm + NES. Figure llF:
GM-CSF Minus, HSV-TK l68dm + NES.
Figure 12: K, Protein Detection By Western is for the retroviral vectors
shown in Figure 11. Viral DNA was ected into 293T Vector Producer Cells, the cells were
lysed, HSV-TK proteins were detected with an anti-HSV-TK antibody. All of the HSV-TK viral
vectors were found to express high levels of HSV-TK protein.
Figure 13 provides exemplary retroviral vectors. Figure 13A: RexRed-TK A168H.
Figure 13B: RexRed-TK l67-dm. Figure 13C: RexRed-TK 168 dm.
Figure 14 provides onal exemplary retroviral vectors where a ular form of
codon optimation was employed.
Figure 14A: RexRed-TK l67-dm + NES. Figure 14B: RexRed-TK l68-dm + NES.
Figure 14C: -TK l67-dm + NES JCO. Figure 14D: RexRed-TK l68-dm + NES JCO.
JCO = justified codon optimization.
Figure 15 provides additional exemplary retroviral vectors. Figure 15A: -TK
Al68F. Figure 15B: RexRed-TK Al68F (GCV specific). Figure 15C: Rex-Hygro-R-TK Al68F
containing the hygromycine resistance gene.
Figure 16 provides additional exemplary retroviral s. Figure 16A: Rex-Hygro-R-
TK Al67F. Figure 16B: Q-PiT-2 is a vector containing a viral receptor gene that binds to a PiT-
2 receptor on the surface of target cells.
Figure 17 provides additional exemplary retroviral vectors. Figure 17A: Original
une-C. Figure 17B. Reximmune-C containing an e with a mTK39 (HSV-
TKSR39) kill gene with neomycin resistance gene (NeoR) and selectable marker inserted.
Figure 18 provides an exemplary of Reximmune-C + a mutated bacterial cytidine
deaminase (mBCD) kill gene.
Figure 19 provides an ary of Reximmune-C + a mutated yeast ne deaminase
(mYCD) kill gene.
Figure 20 illustrates one example of a RexRed Super TK which includes a g gene
(RFP) and a kill gene that contains the identified sequences at the noted positions.
Figure 21 provides an illustration of retroviral s having an updated substrate
binding domain and +/- mNLS and/or +/-NES set, highlighting the sequence differences
between Reximmune-Cl or 2, SR-39 and the Wildtype HSV-TK gene, and having installed a
second therapeutic gene in place of the RFP gene between the LTR and SV40 promoters
Figure 22 illustrates RexRed Super TK Al67F which includes a glowing gene (RFP) and
a kill gene that contains the noted sequences at positions 159-161 and 167-169.
Figure 23 provides exemplary retroviral vectors that are Reximmune-C multicolor clones
of LNCE A375 transduced cells. Figure 23A: LNC-EGFP which contains an enhanced green
fluorescent protein as a glowing gene. Figure 23B: RexRed which contains a red fluorescent
protein as a glowing gene.
Figure 24 es exemplary vectors that a glowing gene only or a hygromycin
resistance gene selectable marker only.
Figure 25: Tk-GCV kill results in parent and PiTCHO-Kl lines. The graphs illustrate
the data for a single RxC2-transduction protocol. The same batch of RxC2 was used for all
experiments (titer imately 5E+lO total virus particles per milliliter (TVP) as determined
by reverse transcriptase in tandem with quantitative polymerase chain reaction (RT-qPCR)).
Figure 25A: GCV kill of RxC2-transduced CHO-Kl parent line after 4 days in GCV (4 doses).
Figure 25B: GCV kill of RxC2-transduced PiTCHO-Kl after 4 days in GCV (4 doses).
Figure 26: Tk-GCV kill in parent and PiTCHO-Kl following a Triple RxC2-
transduction ol. Figure 26A: GCV kill of RxC2-triple transduced CHO-Kl parent on day
9 (10% plate, 5 doses GCV). Figure 26B: GCV kill of riple transduced PiTCHO-Kl
on day 9 (10% plate, 5 doses GCV).
Figure 27: illustrates TK-GCV kill after triple transduction with Reximmune-C2 (HSV-
HdmNES) (SEQ ID NO: 18) in a MIA-PaCa-2 human pancreatic carconima cell line.
GCV kill of RxC2-triple transduced MIA-PaCa2, 25% of initial cells reseeded, day 8, with
various concentrations of GCV.
Figure 28 Illustrates TK-GCV kill after triple transduction of PiTMIA-PaCa-2 cells
with Reximmune-C2. GCV kill of RxC2-triple transduced PiTMIA-PaCa2, 25% of initial
cells, day 8with various concentrations of GCV.
Figure 29: Graphic results from a bystander in vitro assay where human melanoma A375
Hygro TK clones were treated with 20 mM GCV.
Figure 30: Graphic results from a der in vitro assay where C6-Hygro-TK clones
were treated with 20 mM GCV.
Figure 31 is a graph depicting the percentage of GCV kill after une-C2 triple
transduction of various cancer cell lines..
Figure 32 rates a graph of RxC2-tranduced CHO-Kl cell lines after four days in
GCV.
Figure 33 illustrates a graph of RxC2-tranduced HA-CHO-Kl cell lines after four
days in GCV.
Figure 34 illustrates immuno histochemistry (IHC) ofHSV-TK sub cell Localization in
293T cells Transient Transfection, 24 hour Primary AB (Santa Cruz) with RexCl HSV-TK (left
panel) and RexC2 HSV-TK (right panel)
DETAILED DESCRIPTION OF THE INVENTION
HSV-TK gene therapeutic products are available, but are non-optimal with t to
maximal gene expression and tumor kill activity both in vitro and in viva including cancer gene
therapy.
Disclosed herein for the first time is an optimization of codons within HSV-TK genes to
produce improved suicide genes with enhanced pro-drug activation mance in the context
2014/029814
of a viral or psuedoviral gene delivery system. The optimized gene delivery system insures both
l HSV-TK pro-drug enzyme activity and production of high titers of viral particles.
Thus, disclosed herein is the optimization of candidate optimized HSV-TK genes
prepared using both bioinformatics software and custom analysis by the present inventors
utilizing knowledge of the functions and limitations of the genes and viral vector system.
The ing zation steps represent exemplary methods that were utilized by the
t inventors to arrive at the embodiments described herein. Software ed codon
zation may be utilized to remove rare and low use codons to improve HSV-TK protein
expression. The GC content within the newly codon optimized gene may be adjusted to avoid
gene synthesis and other ms.
Known splice or and splice donor sequences within HSV-TK may be
removed.
Tracts of poly-pyrimidines, particularly those introduced by codon optimization
which may be involved in splicing may be removed.
One single strong Kozak translation initiation sequence may be included in front
of the start codon (ATG) while possible Kozak sequences within HSV-TK open reading frame
may be removed. Some of these sequences may have been introduced by codon optimization
and it would be understood that modifications may need to be made in multiple ions to
optimize a gene for improved tumoricidal activity.
Nuclear Localization Sequences (NLSs) within HSV-TK may be removed to
export expressed HSV-TK wherein the expressed HSV-TK protein is not localized exclusively
to the nucleus, but instead accumulates in the cytoplasm.
Restriction sites flanking HSV-TK gene making it possible to clone the gene into
many locations in the disclosed retroviral vectors may be added, while excluding these same
restriction sites within the HSV-TK gene itself.
A double stop codon at end of HSV-TK gene may be included to insure complete
termination of HSV-TK translation.
Mutations near the substrate g domain at amino acid locations 159-161
within the HSV-TK gene may be evaluated.
Mutants in the substrate binding domain at amino acid location 167 within the
HSV-TK gene may be evaluated for increased enzyme activity s the ug nucleoside
analogue, such as gangciclovir and similar pro-drugs, as well as selectivity for their ability to kill
cancer cells.
Mutants in the substrate binding domain at amino acid location 168 within the
HSV-TK gene may be evaluated for increased ug GCV enzyme activity and selectivity for
their ability to kill cancer cells.
] Mutants in the substrate binding domain at amino acid location 167 + 168 within
the HSV-TK gene may be evaluated for increased pro-drug GCV enzyme activity and selectivity
for their ability to kill cancer cells.
The use of tags, fusion proteins and linkers of HSV-TK to other genes and
proteins may be evaluated.
Further methods of optimization may also be considered for use in the methods
described herein. Once a gene is optimized in this way, its gene sequence can be sent to a gene
synthesis company for custom gene synthesis.
DEFINITIONS
Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as is ly understood by one of skill
in the art to which the invention(s) belong. All patents, patent applications, published
ations and publications, GenBank sequences, websites and other published 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 ation can be found by searching the intemet. nce
thereto evidences the availability and public dissemination of such information.
As used , “nucleic acid” refers to a
polynucleotide containing at least two covalently linked nucleotide or nucleotide analog
subunits. A c acid is generally a ibonucleic acid (DNA), a ribonucleic acid (RNA),
or an analog of DNA or RNA. Nucleotide s are commercially available and methods of
preparing polynucleotides containing such nucleotide analogs are known (Lin et al. (1994) Nucl.
Acids Res. 22:5220-5234; Jellinek et al. (1995) Biochemistry 34: 1 1363-1 1372; is et al.
(1997) Nature Biotechnol. 15 :68-73). The nucleic acid is generally single-stranded, double-
stranded, or a mixture thereof. For purposes herein, unless specified ise, the c 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 ing modified nucleotides and nucleotide analogs.
2014/029814
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 cleotides.
As used herein, the term “subject” refers to animals, , insects, and birds into
which the large DNA molecules are introduced. Included are higher organisms, such as
s and birds, including humans, primates, rodents, cattle, 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 tely, 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 c acid molecules.
[001 19] As used herein, ery vector” or “delivery vehicle” or “therapeutic ” or
peutic system” refers to both viral and non-viral particles that harbor and transport
exogenous nucleic acid molecules to a target cell or tissue. Viral vehicles include, but are not
limited to, retroviruses, adenoviruses, iral viruses, herpes s and adeno-associated
viruses. Non-viral vehicles include, but are not limited to, articles, nanoparticles,
virosomes and liposomes. “Targeted,” as used herein, refers to the use of ligands that are
associated with the delivery vehicle and target the vehicle 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 process by which exogenous nucleic acid les are transferred into a cell such
that they are d inside the cell. Delivery of nucleic acids is a distinct process from
expression of nucleic acids.
As used herein, a “multiple g site (MCS)” is a c acid region in a
plasmid that contains multiple restriction enzyme sites, any of which can be used in ction
with standard recombinant technology to digest the vector. “Restriction enzyme digestion”
refers to catalytic cleavage of a nucleic acid molecule with an enzyme that fianctions only at
specific locations in a nucleic acid molecule. Many of these restriction enzymes are
cially available. Use of such enzymes is widely understood 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.
2014/029814
As used herein, “origin of replication” (often termed “ori”), is a c nucleic
acid ce at which ation is initiated. Alternatively an autonomously replicating
sequence (ARS) can be employed if the host cell is yeast.
As used , “selectable or screenable markers” confer an identifiable change
to a cell permitting easy identification of cells containing an expression vector. Generally, a
selectable marker is one that confers a ty that allows for selection. A positive selectable
marker is one in which the presence of the marker allows for its selection, while a negative
selectable marker is one in which its presence prevents its selection. An example of a positive
selectable marker is a drug ance marker.
Usually the ion of a drug selection marker aids in the cloning and
fication of transformants, for example, genes that confer ance 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
ments, screenable enzymes such as herpes x 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 capable 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.
The term “transfection” is used to refer to the uptake of foreign DNA by a cell.
A cell has been “transfected” when exogenous DNA has been introduced inside the cell
membrane. A number of transfection techniques are generally known in the art. See, e.g.,
Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular Cloning: A tory
Manual (1989); Davis et al., Basic Methods in Molecular Biology ; Chu et al., Gene
13: 197 (1981). Such techniques can be used to introduce one or more exogenous DNA
moieties, such as a nucleotide integration vector and other nucleic acid molecules, into suitable
host cells. The term captures chemical, electrical, and viral-mediated transfection procedures.
As used herein, “expression” refers to the process by which nucleic acid is
translated into peptides or is transcribed into RNA, which, for e, can be translated into
peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, sion
includes, if an appropriate eukaryotic host cell or organism is selected, 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 herein, 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 manner, 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 stration.
As used , the terms “co-administration, 3, (Cadministered in combination
with” and their grammatical equivalents or the like are meant to encompass administration 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 sed in the t
application will be co-administered with other agents. These terms encompass 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 ments, a therapeutic agent
and the other agent(s) are stered in a single composition. In some ments, 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” s, for example, microorganisms, yeast cells, insect
cells, and ian cells, that can be, or have been, used as recipients for multiple constructs
for ing 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
transfected with an exogenous DNA sequence. It is understood that the progeny of a single
parental cell may not necessarily be completely identical in morphology or in genomic or total
DNA complement as the original parent, due to l, accidental, or deliberate mutation.
As used herein, “genetic therapy” involves the transfer of heterologous DNA to
the certain cells, target 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 heterologous DNA is expressed and a therapeutic product encoded
thereby is produced. In some ments, the heterologous DNA, directly or indirectly,
mediates expression ofDNA that s the therapeutic product. In some embodiments, the
heterologous DNA s a product, such as a peptide or RNA that mediates, directly or
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2014/029814
indirectly, expression of a therapeutic product. In some embodiments, genetic therapy is used to
deliver a c acid encoding a gene product to e 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
lly produced in the mammalian host or that is not produced in therapeutically effective
amounts or at a therapeutically useful time. In some embodiments, the heterologous 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, ologous nucleic acid sequence” is generally DNA that
encodes RNA and proteins that are not normally produced in vivo by the cell in which it is
expressed or that mediates or encodes mediators that alter expression of nous DNA by
affecting ription, translation, or other regulatable biochemical processes. Any DNA that
one of skill in the art would recognize or consider as heterologous or foreign to the cell in which
it is expressed 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 es, and DNA that s other types of proteins, such
as antibodies. In some ments, antibodies that are encoded by heterologous DNA is
secreted or expressed on the surface of the cell in which the heterologous DNA has been
uced.
As used herein, the term “thymidine kinase ” refers to not only the c
protein described herein (as well as the nucleic acid sequences which encode these proteins), but
derivatives thereof which may include 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,
wherein 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 on by a nucleotide other than that which
occurs in the unmutated or previously mutated sequence. That is, in some instances, specific
ations 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. See, e.g., Figure l.
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 “non-polar amino acid” refers to amino acid residues Ala (A),
Ile (1), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), or Val (V).
As used herein, a “basic amino acid” refers to amino acid residues Arg (R), His
(H), or Lys (K).
] As used herein, an “acidic amino acid” refers to amino acid residues Asp (D) or
Glu (E).
IMPROVED HSV-TK
Thymidine kinase is a salvage pathway enzyme which phosphorylates natural
nucleoside substrates as well as nucleoside analogues. Generally, viral thymidine kinase is
exploited therapeutically by administration of a nucleoside analogue such as ganciclovir or
acyclovir to a cell expressing Viral thymidine kinase, wherein the Viral thymidine kinase
phosphorylates the nucleoside analogue, creating a toxic product capable of killing the cell.
Polynucleotide sequences encoding viral thymidine kinase of the present
invention may be ed from a wide variety of Viral ine kinases. In some
ments, the Viral thymidine kinase mutant is derived from Herpesvirz'dae ine kinase
including, for example, both primate herpes viruses, and non-primate herpes viruses such as
avian herpes viruses. Representative examples of suitable herpes viruses include, for e,
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.
Herpes s may be y obtained from commercial sources such as the
American Type Culture tion ”, Rockville, Md.). viruses may also be
isolated from naturally ing courses (e.g., an infected animal).
IMPROVEMENTS TO TK GENE
Disclosed herein, in some embodiments, is a polynucleotide sequence encoding
HSV-TK. In some ments, the polynucleotide sequence encodes a wild-type HSV-TK
amino acid sequence. In some embodiments, the polynucleotide sequence encodes a mutated
HSV-TK amino acid sequence.
Exemplary ures that may be used in preparation of an optimized
polynucleotide sequence provided herein include, but are not limited to: codon optimization;
correction of splice sites, removal of poly-pyrimidine tracts and excess GC content; addition of
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WO 53258
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, linkers and fusion sequences; preparation of sequence file for
submission to gene sis company; subcloning of synthesized gene into retroviral vectors;
ion of cent protein genes into retroviral vectors; inclusion of selectable marker
genes into retroviral vectors; preparation of Maxiprep d DNA; transfection of retroviral
producer or other cells; lab, pilot or GMP scale production of retrovirus; transduction of target
cells with retrovirus; GCV or analogus ug mediated cell kill assay;
Hypoxanthine/Aminopterin/ Thymidine (HAT) selection assay; able marker drug ion
procedure to e iral transduced 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 commercially available or are described in the public literature and
ses.
In some embodiments, described herein is a method of obtaining an improved
HSV-TK ce. In some embodiments, the method comprises: a) correction and/or removal
of splice sites; and/or b) adjustment to a single Kozak sequence. Optionally, in some
embodiments, the method further 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 cleotide sequence ng a mutated form of
thymidine kinase from human simplex virus (HSV-TK), n 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 polynucleotide sequence of SEQ ID NO: 1 or
3. In such sequences, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, 13, or 14 mutations may be made.
Provided herein is a polynucleotide sequence encoding a mutated form of
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 polynucleotide sequence of SEQ ID NO: 1.
In such sequences, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, 13, or 14 mutations may be made.
Provided herein is a polynucleotide sequence encoding a mutated form of
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 ation thereof, wherein the
polynucleotide sequence is mutated compared to a polynucleotide sequence of SEQ ID NO: 3.
In such sequences, 1,2, 3,4, 5, 6, 7, 8, 9, 10, ll, 12, 13, or 14 mutations may be made.
Modifications may be conservative or non-conservative mutations. A mutation
may be made such that the d amino acid is modified to a polar, non-polar, basic or acidic
amino acid.
Provided herein is a polynucleotide sequence encoding a d form of viral
thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK includes
a nuclear export sequence. Provided herein is a cleotide sequence encoding a mutated
form of thymidine kinase from human simplex virus (HSV-TK), where the encoded HSV-TK is
improved in function compared to wild-type HSV-TK and comprises A168H dmNES (Q
system-QMV er properly fused to LTR promoter regions), where NES refers to a nuclear
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 cleotide 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
sequence encoding Human Simplex Virus Thymidine Kinase (HSV-TK) wherein mutations are
not made to the polypeptide ce of wildtype .
tide positions are referred to by reference to a position in SEQ ID NO: 1
(wildtype (wt) HSVl-TK nucleotide sequence) or SEQ ID NO: 3 (HSV-TK in Reximmune-C
HSV-TK; SR39 mutant and R25G-R26S Mutation of the HSV-TK nuclear localization signal
(NLS)).
In one ment, a Sac I-Kpn I restriction sites bounding the le 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 zed HSV-TK polynucleotide sequences are provided, for
example, as SEQ ID NOS: l2-24.
However, when such references are made, the ion is not intended to be
limited to the exact sequence as set out in SEQ ID NO: 1 or 3, but includes ts and
derivatives thereof. Thus, identification of nucleotide locations in other thymidine kinase
sequences are contemplated (i.e., identification of nucleotides at positions which the d
person would consider to correspond to positions recited in SEQ ID NO: 1 or 3).
In some embodiments, tides are replaced by taking note ofthe c code
such that a codon is d to a different codon which codes for the same amino acid residue.
In some embodiments, nucleotides are replaced within coding s of a HSV-TK encoding
nucleic acid ce, yet the nucleic acid sequence maintains wild type HSV-TK protein
expression.
In some embodiments, codons are mutated to such that the encoded HSV-TK
ts increased activity. In some embodiments, the codon GCT is used to represent alanine.
In some embodiments, the codon AGA is used to represent arginine. In some embodiments, the
codon AAT is used to represent asparagine. In some embodiments, the codon GAT is used to
represent aspartic acid. In some embodiments, the codon TGT is used to represent cysteine. In
some embodiments, the codon CAG is used to represent glutamine. In some embodiments, the
codon GAA is used to represent glutamic acid. In some embodiments, the codon GGA is used
to represent e. In some embodiments, the codon CAT is used to represent histidine. In
some ments, the codon ATT is used to represent isoleucine. In some embodiments, the
codon CTG is used to represent e. In some embodiments, the codon AAA is used to
represent lysine. In some embodiments, the codon ATG is used to represent methionine. In
some embodiments, the codon TTT is used to represent phenylalanine. In some embodiments,
the codon CCT is used to represent proline. In some embodiments, the codon TCT is used to
represent serine. In some embodiments, the codon ACA is used to represent threonine. In some
embodiments, the codon TGG Is used to represent tryptophan. In some embodiments, the codon
TAT is used to represent tyrosine. In some embodiments, the codon GTG is used to represent
valine. In some embodiments, the codon TGA is used as a stop codon. Exemplary codon
positions for mutation are ed in the following table.
Improved Codon Usage for Designing Human Genes, First Choice
Codon Optimization which Reduces G/C content
n Aspartic Acid (Asp) ( D ) GAT
Cysteine ( Cys) ( C ) TGT
[- Glutaminewmw
7 Glutamic Acid ( Glu) ( E ) GAA
n Glycine ( Gly) ( G) GGA
n ine (His) (H) CAT
Isoleucine ( Ile) (I) ATT
11 Leucine ( Leu) ( L )
12 Lysine ( Lys) ( K)
13 Methonine (Met) (M )
14 Phenylalanine ( Phe ) ( F) TTT
Proline ( Pro ) ( P ) CCT
16 Serine (Ser) ( S ) TCT
17 Threonine ( Thr) ( T ) ACA
18 Tryptophan ( Trp) ( W)
19 ne ( Tyr) (Y ) TAT
Valine (Val ) (V)
21 Stop (Term) ( * ) TGA
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 on (76%).
In yet other embodiments, about 3-7 codons of 21 codons contain “C or G” in the
third position; above 0-3 codons of 21 codons contain “C” in the third position; about 3-7
codons of 21 codons contain “G” in the third position; and about 14-18 codons of 21 codons
contain “A or T” in the third on.
In some embodiments, the codon GCA is used to represent alanine. In some
embodiments, the codon AGG is used to represent arginine. In some embodiments, the codon
AAC is used to represent asparagine. In some embodiments, the codon GAC is used to
represent aspartic acid. In some embodiments, the codon TGC is used to represent cysteine. In
some embodiments, the codon CAA is used to represent glutamine. In some ments, the
codon GAG is used to represent ic acid. In some embodiments, the codon GGC is used
to represent glycine. In some embodiments, the codon CAC is used to represent histidine. In
some embodiments, the codon ATC is used to represent isoleucine. In some embodiments, the
codon CTC is used to represent leucine. In some embodiments, the codon AAG is used to
represent . In some embodiments, the codon ATG is used to represent methionine. In
some embodiments, the codon TTC is used to represent phenylalanine. In some embodiments,
the codon CCA is used to represent proline. In some embodiments, the codon AGC is used to
represent serine. In some embodiments, the codon ACT is used to represent threonine. In some
embodiments, the codon TGG is used to represent tryptophan. In some embodiments, the codon
TAC is used to represent tyrosine. In some embodiments, the codon GTC is used to ent
valine. In some embodiments, TAA is used as a stop codon.
Improved Codon Usage for Designing Human Genes, 2nd Choice
Codon Optimization which Reduces G/C content
Am
11 cm
12 M9
14 W
16 A62
19 ”2
GT2
21 W
In such embodiments, 16/21 codons contain “C or G” in third position (76%);
11/21 codons contain “C” in third on (52 %); 5/21 codons contain “G” in third position
(24%); and 5/21 codons contain “A or T” in third on (24%).
In yet other embodiments, about 14-18 codons of 21 codons contain “C or G” in
the third position; about 9-13 codons of 21 codons contain “C” in the third position; about 3-7
codons of 21 codons contain “G” in the third position; and about 3-7 codons of 21 codons
contain “A or T” in the third position.
In some embodiments, the following rare codons are are avoided if possible,
unless changing the rare codon sequence creates new splice acceptor and/or alternate Kozak
sites or adds an unwanted restriction site or other problematic seqeunce, within the coding
region of a polynucleotide encoding mutant HSV-TK, or a variant thereof: GCG for alanine;
CGA or CGT for arginine; TTA or CTA for e; CCG for e; TCG for serine; ACG for
threonine; and GTA for valine. Rare codons to be avoided if possible are those that have a
codon/a.a./fraction per codon per a.a. less than or equal to 0.12.
Rare Codon
‘44 USU C 0.45
‘50 US“ C 0.54
.30 US$.* 0.47
"1: _.. .24 USS W 1.00
000 L 013 0.00 P 0.23 an H 0 V 0
000 L 0.20 0.0.: P 0.32 0ch H 0.33 000 R 0.13
CCP P 0.23 0P0 a 0,2?
000 L 0,40 0P0 a 0J3 000 R 0.20
P00 I 0.30 P00 T 0.23 PPU 110.4": P00 3 0.15
P00 :1: 0.47 P00 T 0.30 3"}: £10.53 PP: 3 0.24
PUP. I 0.1"! P0 .T 0.23 PPP K 0.03 P0P R 021
P00 M 1.00 m PPS K 0.37 .000 R 021
000V0.10 000A I: F0.. --.1 mL‘lr-IEl 0.46 SSH E 0.15
000 v 0.24 000 P 0.40 mmw5293’- HHUU CI. m “:3. EEC E 0.34
EBA P I2!._ MI La 0.42 GEE-‘1 5 0.225
000' 0' 0.40 Em 0.53 GEE G 0.225
E [Efidcnfa.amffraction per sedan pEr 0.0,]
“ HBED a data frfim the Sauna Usage fiatabaae
In some embodiments, altering codons as described herein results in about 2%,
about 5%, 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 tage increase in activity.
High tage Codon Optimization was found to improve protein expression
but increases GC gene t. Codon optimization was assessed and determined to have the
following characteristics.
Hfiamen: firsfies‘hy LimaWm ‘
V hyflbmth
Fs'e. suem“ s
. base. WsiE-asémss
Awafii‘s mm g’ A3533 i B ‘}
Agar?mm * and i" B ‘
\ H
2: I
anim at: Luci; {L}
{uterine :ef' Lire} {' L 3
{mains :'? Lire} i L}
] Because of the unsatisfactory results obtained with fully ted codon
optimization software programs, customized codon optimization was performed to increase both
n expression and titers obtained. The initial step includes the use of a codon optimizer
program as a first screen in order to set each codon for the correct reading frame to that most
preferred in the subject species, including humans, giving a ‘raw’ codon optimization.
Generally, any desired cloning restriction sites are excluded from use during this stage of the
13100688.
The results are further refined by editing DNA ces in a DNA editor
program, and searching for degenerate codons, such as pyrimidines (e.g., by searching for “Y”
codons). The following operations are then performed, in this order:
Manual search of the sequence for runs of “Y,” generally at least five or more
“Y” sequential runs. These sequences are highlighted in a given sequence, and the DNA editor
program is used to determine a translation that includes the DNA sequence listed in register to
the peptide sequence to insure that changes to codons do not affect the translated protein.
Each codon in a run of 5 or more Y’s is evaluated. When available, the wobble
base of each codon is converted to the most favorable A-G base for the amino acid (usually an
adenine), and the result ed. If the result of the change creates a purine-rich run ending at
’ AG, the changes
or near a 3 are manually reversed. If there is no most favorable A-T base
available for the wobble base or it causes another sequence conflict, the the most favorable C-G
base is used for the wobble base.
If the result is a rare codon (< 10% usage), that codon is moved to the next
available codon in the frame.
If another codon change can ablate the putative acceptor site, changes are made
to revert to the al sequence. If no such alternative change is available, then the original
alteration is implemented.
Once this process is complete, the sequence is examined 5’ to 3’ for alternate
reading frames. At each reading frame, the 5 bases 3’ of the ATG codon are examined for their
suitability as Kozak sequences. If the ATG gives a methionine in the reading frame of the
desired gene, s are limited to ablating the Kozak sequence, first by converting the wobble
base of the “- ” wobble base to the ATG to
a “T” (if possible), then the “-4” wobble base.
In rare cases, it may be desirable to convert the second codon in the g
frame, if ally an “AGN” base (Ser/Arg) to a codon ing in T (for ) or C (for
arginine). The situation is generally not encountered when strictly ng the above algorithm
however, as the “AGN” codons are avoided due to the “AG” sequence pair.
In cases where the alternate reading frame differs from that of the message, AND
the Kozak ce surrounding it fits the consensus “CCACCatgG”, the wobble base of the in-
frame codon is altered to remove the start codon. This generally happens (but not always) as a
result of the codon optimization and/or splice acceptor ablation process.
In-process checks are lly performed to ensure that the peptide sequence is
ged. At the final check stage, if there are too many ‘rare’ codons in use (generally 2 or
more) it may be desirable to prioritize which are used, with preference to changes given to the
longer pyrimidine runs from the ‘raw’ codon optimized ce. Finally, any needed
restriction sites are added, and a last check is performed to insure that the polypeptide is
unchanged from the original sequence before the optimization process is begun and that any
desired restriction sites remain unique to those that are added for cloning purposes.
Splice Site Modification
Introns are generally spliced out ofRNA in order to join exons. A splice donor
site is a site in RNA on the 5' side of the RNA which is removed during the splicing process and
which contains the site which is cut and rejoined to a nucleotide residue within a splice acceptor
site. Thus, a splice donor site is the on between the end of an exon and the start of the
intron. Generally, a splice donor site in RNA is the eotide GU (or a GT dinucleotide in
the corresponding DNA sequence).
A splice acceptor site is a site in RNA on the 3' side of the RNA which is
removed during the splicing process and which contains the site which is cut and ed to a
tide residue within a splice donor site. Thus, a splice acceptor site is the junction between
the end of an intron (typically terminating with the dinucleotide AG) and the start of the
downstream exon.
In some embodiments, disclosed herein is a nucleic acid sequence encoding a
thymidine kinase n at least one nucleotide corresponding to a splice donor site is replaced
by r nucleotide. See, e.g., Figure l (Chalmers et al., Mol. Ther. 4:146-8 (2001)). In
further embodiments, the tides of the splice acceptor sites are not altered. In some
embodiments, at least one nucleotide ponding 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 nucleotides corresponding to splice donor site
nucleotides at positions 329 and 330 of a polynucleotide sequence (e.g., SEQ ID NO: 1 or 3) is
ed by another nucleotide. In some embodiments, both of the nucleotides at positions 327
and 555 are ed by other nucleotides. For example, position 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 residue selected from: G to A. In one embodiment, the modified
HSV-TK has a polynucleotide ce 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 zed
SC = splice donor/acceptor site corrected at 327 and 555,
Underlined sequence
2014/029814
SEQ ID NO: 18
gtCaGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGG
CCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGC
GCaCTGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCC
TGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCT
GGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAG
ACCATCGCCAACATCTACACCACCCAGCACCGCCTGGACCAEGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaCA
CATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTCGACCGgCACCCa
ATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtCCATGACaCCaCAEGCCGTGCTGG
CCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCG
CCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATC
CGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACT
GGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCC
aCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTAC
AACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCGCtCCATGCACGTGTTCATCCTGGACT
ACGACCAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGT
GACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCC
AACTAATAGGGATCCCTCGAGAAGCTTgtca
In some embodiments, disclosed herein is a nucleic acid sequence encoding a
thymidine kinase wherein at least one of the nucleotides corresponding to splice acceptor site
nucleotides at ons 554 and 555, or at least one of the nucleotides ponding 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 mutated to an amino acid
residue selected from: G to A. Position 542 may be mutated to an amino acid residue selected
from: G to A. Position 554 may be mutated to an amino acid residue selected from: G to A.
Position 555 may be d 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.
In some embodiments, at least one of the nucleotides of the wild-type HSV-TK
encoding sequence is replaced as described in Table 1 below.
TABLE 1
Position Mutation
84 843 c —> A
846 C a A
879 C a G
882 C a A
168 885 c HA
171 897 c —>A
378 —m1_ C HA
961 AHT
Position Mutation Position Mutation
420 C—>A 962 G—>C
A Kozak sequence flanks the AUG start codon Within mRNA and influences the
recognition of the start codon by eukaryotic ribosomes. In some embodiments, a polynucleotide
sequence ng HSV-TK comprises no more than one Kozak sequence. In some
embodiments, the Kozak ce is am of the coding portion of the DNA sequence. In
some embodiments, the Kozak sequence of a polynucleotide encoding HSV-TK is modified to
produce a Kozak sequence with a higher efficiency of translation initiation in a mammalian cell.
In some ments, modification of the Kozak sequence does not produce an amino acid
substitution in the encoded HSV-TK polypeptide product. In some embodiments, modification
of the Kozak sequence results in at least one amino acid substitution in the d HSV-TK
polypeptide product. In one embodiment, the modified HSV-TK has a polynucleotide sequence
of SEQ ID NO: 18.
In some embodiments, a polynucleotide sequence encoding HSV-TK comprises a
modification that inserts one or more restriction sites. The optimal site for insertion of one or
more restriction sites may be ined empirically and/or using a computer program to
analyze the sequence. In one non-limiting embodiment, a first restriction site is inserted
' end of
upstream of the Kozak and ATG start site and a second restriction site is inserted at the 3
the sequence. See, for example, SEQ ID NO: 18, underlined section below.
HSVTK NESdmNLS Al68H, CO & SC
NES = nuclear export sequence from MAPKK
dmNLS = double mutated Nuclear Localization Sequence
CO = codon optimized
SC = splice corrected at 327 and 555, previously bed
Kozak Sequence, usly described
Restriction Sites, ined and specified as:
CGC ACCGGT ACGCGT = Not-I, Age-I, and MLU-I)
(GGATCC CTCGAG AAGCTT = SamH-", Xho-I and Pind- )
SEX)IDIVO:18
gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGG
CAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGC
AGCACCGCaCTGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCC
TGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCT
GGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAG
GCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaCA
CATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTCGACCGgCACCCa
ATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtCCATGACaCCaCAaGCCGTGCTGG
CCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCG
CCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATC
CGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACT
GGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCC
aCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTAC
AACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCGCtCCATGCACGTGTTCATCCTGGACT
ACGACCAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGT
GACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCC
AACTAATAGGGATCCCTCGAGAAGCTTgtca
Other splice site modifications are disclosed in the examples below and are
considered for inclusion as a modified TK sequence that can be used in the claimed methods.
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises
at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon
substitutions. In some embodiments, the cleotide sequence encoding HSV-TK comprises
at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon
tutions, wherein the codon substitutions comprise the substitution of a codon having a
higher frequency of usage in a ian 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
sing at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or
more codon substitutions has less than about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID
NO: 1 or 3 wherein the sequence identity is determined over the fill length of the coding
sequence using a global alignment . 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.
In some embodiments, the cleotide sequence ng HSV-TK comprises
at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon
substitutions, wherein the codon substitutions comprise the substitution of a codon having the
highest frequency of usage in a mammalian cell for the wild type codon at that position. In some
ments, the corresponding encoded ptide 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 polynucleotide ce encoding HSV-TK comprises
at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon
substitutions, wherein the substituted codons have a frequency of usage greater than or equal to
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, 0.25,
0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35 or higher. 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.
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 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 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 polynucleotide sequence encoding HSV-TK ses
at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95% or more of codons having a frequency of usage greater than or equal to 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, 0.25, 0.26,
0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, or higher. In some embodiments, the
corresponding encoded ptide 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 polynucleotide ce encoding HSV-TK ses
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% or more of codons haVing
the t frequency of usage in a mammalian cell. 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.
In some embodiments, the polynucleotide sequence encoding HSV-TK ses
less than about2l%, 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 some embodiments, the polynucleotide
sequence comprises less than about2l%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,
11%, 10% or less of codons haVing a frequency ofusage 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 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.
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises
codon substitutions, 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 compared to the wild type
sequence. In some embodiments, the polynucleotide sequence encoding HSV-TK comprises
codon substitutions, wherein 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 mammalian cell as compared to the wild type
sequence. In some embodiments, the ponding encoded ptide 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 ce, e.g., SEQ ID NO: 2 or 4.
In some embodiments, the polynucleotide 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 ed to the wild type sequence. In some
embodiments, the corresponding encoded ptide ce 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% ce identity to a HSV-TK amino acid
sequence, e.g., SEQ ID NO: 2 or 4.
Non-Conserved Mutations
The Viral thymidine kinase gene from the selected herpesvirus may be readily
isolated and mutated as described below, in order to construct nucleic acid molecules encoding a
thymidine kinase enzyme comprising one or more ons 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 nucleoside analogue uptake or
determination of the rate of nucleoside or nucleoside analogue phosphorylation. In addition,
thymidine kinase mutants may be readily selected which are terized by other biological
properties, such as thermostability and protein stability.
In some embodiments, the polynucleotide sequence encoding HSV-TK is
modified to remove or modify a predicted signal sequence. In some embodiments, the
polynucleotide is d to remove or modify a nuclear localization sequence (NLS). In some
embodiments, the polynucleotide is d to remove the nuclear localization sequence. In
some embodiments, the cleotide is modified to modify the NLS so that if no longer
functions to localize HSV-TK exclusively to the nucleus.
In some embodiments, a HSV-TK polypeptide sequence is d 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 another example, the
WO 53258
sequence is mutated at amino acid residues 167 and 168. Amino acid e 167 may be
mutated to serine or phenylalanine. Amino acid residue 168 may be mutated to histidine, lysine,
cysteine, serine or alanine. 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 glutamic
acid. 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 ting of: e, serine, and glutamic acid. 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 glutamic acid. 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 invention, 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 embodiments, expression
vectors are provided comprising a promoter 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 include
herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors, pox
vectors, parvoviral vectors, baculovirus vectors and iral vectors. In some embodiments,
viral vectors are provided which are capable of directing the expression of a c acid
le which s a ine kinase enzyme comprising one or more mutations, at least
one of the mutations encoding an amino acid substitution which increases a biological ty of
thymidine kinase, as compared to ted (z'.e. , wild-type) thymidine kinase.
In some embodiments, a nucleic acid molecule provided herein encodes a
thymidine kinase enzyme e of phosphorylating a nucleoside analogue at a level at least
% greater than the level of phosphorylation of the nucleoside analogue by a wild-type
thymidine kinase enzyme. In some embodiments, 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 examples of suitable nucleoside analogues include gancyclovir,
acyclovir, famciclovir, buciclovir, penciclovir, valciclovir, trifluorothymidine, eoxy, 2-
fluoro, beta-D-arabino furanosyl]iodouracil, ara-A, araT 1-beta-D-arabinofuranoxyl thymine,
-ethyl-2'-deoxyuridine, -5'-amino-2, eoxyuridine, idoxuridine, AZT, AIU,
2014/029814
dideoxycytidine and AraC. In some embodiments, the improved TK mutant lacks thymidine
kinase activity.
In some embodiments, the Km value for thymidine kinase activity of a disclosed
HSV-TK mutant is at least 2.5 um. In some embodiments, the KIn value for thymidine kinase
activity of a disclosed HSV-TK mutant is at least 5 um, at least 10 um, at least 15 um, at least
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 t KIn of a
disclosed HSV-TK mutant compared to wild-type HSV-TK is at least 15%, at least 20%, at least
%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, or at
least 500%.
Within one ment 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 ucting
a truncated form of the mutant which retains its ical activity.
Mutations in nucleotide sequences constructed for expression of derivatives of
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 ures, such as loops or hairpins, which would
adversely affect translation of the receptor mRNA. Such tives may be readily constructed
using a wide variety of techniques, including those discussed above.
MODIFIED THYMIDINE KINASE MUTANTS
Using the methods described herein, the inventors determined that the majority of
the candidates for optimized HSV-TK genes appeared to be compatible with a retroviral
expression system and produce biologically useful retroviral titers.
rmore, the optimized HSV-TK genes which orated most of these
optimizations (SEQ ID NO: 18 ted pro-drug GCV enzyme activity and selectivity for their
ability to kill cancer cells following retroviral transduction ry. The mutant HSV-TK gene
A168H, which was codon optimized and splice corrected appeared to have the highest GCV
mediated cancer kill activity (SEQ ID NOs: 12, 16, 18, or 22). The same version of this HSV-
TK gene A168H and mutated at amino acids 159-161 from LIF to IFL exhibited GCV mediated
cancer cell kill activity.
The mutant HSV-TK gene Al67F (SEQ ID NOS: l3 ,17, 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 ing 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 tion 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 ine enzyme ty 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 functional HSV-TK
fusions of genes such as bacterial ne deaminase, yeast cytosine deaminase, neomycin
phosphotransferase and include linker ces 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.
Additional 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 ty and selectivity
for their ability to kill cancer cells, te the use of more tags, fusion proteins and linkers 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 type retroviral vector ; tion and testing of a Reximmune-C type
GMP product, or any combination thereof.
EXEMPLARY POLYNUCLEOTIDE SE UENCES
In one embodiment, a polynucleotide ce described herein comprises a
nuclear export signal. For example, a polynucleotide ce may comprise TKl68dmNES.
In another embodiment, a retroviral vector for use in the s described
herein comprises one or more splice site modifications.
In another embodiment, a retroviral vector for use in the s described
herein comprises HSV-TK Al67Fsm (SEQ ID NO: 13).
In another ment, a retroviral vector for use in the methods described
herein comprises HSV-TK Al68Hsm (SEQ ID NO: 12).
In another ment, a iral vector for use in the methods described
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 Al67de and an NES (SEQ ID NO: 19).
In another embodiment, a retroviral vector for use in the methods described
herein comprises HSV-TK Al68Hdm and an NES (SEQ ID NO: 18). In such an embodiment,
the sequence comprises HSV-TK A168H.
In another embodiment, a retroviral vector for use in the methods described
herein comprises a HSV-TK, wherein such vector comprises an upgraded substrate g
domain and a mNLS/NES set.
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,
fluorescent or bioluminescent 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 ations.
CONSTRUCTION OF THYMIDINE KINASE MUTANTS
ine kinase mutants of the present invention may be constructed using a
wide variety of techniques. For example, mutations may be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked by ction sites ng
ligation to fragments of the native sequence. Following ligation, the resulting reconstructed
sequence s a derivative having the d amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific (or t specific)
mutagenesis procedures may be employed to provide an altered gene having particular codons
altered according to the substitution, deletion, or insertion required. Deletion or truncation
derivatives of ine kinase mutants may also be constructed by ing convenient
restriction endonuclease sites adjacent to the d deletion. Subsequent to restriction,
overhangs may be filled in, and the DNA religated. Exemplary methods of making the
alterations set forth above are disclosed by Sambrook et al. (Molecular g: A Laboratory
Manual, 2Ild Ed., Cold Spring Harbor Laboratory Press, 1989).
Other derivatives of the thymidine kinase mutants disclosed herein include
conjugates of thymidine kinase s along with other ns or polypeptides. This may be
accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins which
may be added to facilitate ation or identification of thymidine kinase mutants (see US.
Pat. No. 4,851,341, see also, Hopp etaZ.,Bi0/Techn010gy 6:1204, 1988.).
IMPROVEMENT OF DIATED KILLING
In some embodiments, the polynucleotide sequence encoding HSV-TK further
comprises a sequence encoding a secondary therapeutic agent or polypeptide. In some
embodiments, secondary therapeutic agent or ptide is a diagnostic or eutic agent or
polypeptide.
In some embodiments, the secondary eutic agent or polypeptide is an
additional “suicide protein” that causes cell death by itself or in the presence of other
compounds. In some embodiments, the second suicide gene is chosen from the group including:
penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, carboxypeptidase A, linamarase
(also referred to as B-glucosidase), the E. coli gpt gene, and the E. coli Deo gene, a cytosine
deaminase, a VSV-tk, IL-2, nitroreductase (NR), ylesterase, beta-glucuronidase,
cytochrome p450, beta-galactosidase, diphtheria toxin A-chain (DT-A), carboxypeptide G2
(CPG2), purine nucleoside phosphorylase (PNP), and deoxycytidine kinase (dCK).
In some embodiments, the second suicide protein converts a prodrug into a toxic
compound. As used herein, “prodrug” means any compound useful in the methods disclosed
herein that can be converted to a toxic t, z'.e., toxic to tumor cells. The prodrug is
converted to a toxic product by the suicide n. Representative examples of such prodrugs
include: FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9m{[3~fluoro»1“hydroxvu
2~propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'-
deoxy-2'-fluoroB-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-
arabinofuranosyluracil), FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-
((1 -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4(1H,3H)—dione),
ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled dine 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-,
ganciclovir- and penciclovir-like side chains for thymidine ; ifosfamide for
oxidoreductase; oxypurine arabinoside for VZV-TK; 5-fluorocytosine for cytosine
deaminase; doxorubicin for beta-glucuronidase; CB1954 and nitrofilrazone for nitroreductase;
and N—(Cyanoacetyl)-L-phenylalanine or hloropropionyl)-L-phenylalanine for
carboxypeptidase A.
] In some embodiments, the secondary therapeutic agent or polypeptide is chosen
from the group including, but are not limited to, cell cycle control agents, agents which inhibit
2014/029814
cyclin proteins, such as antisense cleotides to the cyclin A and/ or D genes, growth
factors such as, for example, epidermal growth factor (EGF), vascular endothelial growth factor
(VEGF), erythropoietin, G-CSF, , TGF-u, TGF-B, and fibroblast grth factor,
cytokines, including, but not limited to, Interleukins 1 through 13 and tumor necrosis factors,
anticoagulants, anti-platelet agents, anti-inflammatory agents, anti-angiogenic factors, tumor
suppressor proteins, clotting factors, including Factor VII, Factor VIII and Factor IX, protein S,
protein C, antithrombin III, von rand Factor, cystic fibrosis embrane tance
regulator (CFTR), and negative selective markers.
] In some embodiments, a secondary eutic agent or polypeptide is a cancer
suppressor, for example p53 or Rb, or a nucleic acid encoding such a protein or polypeptide.
Other examples of secondary therapeutic agents or polypeptides include pro-
apoptotic therapeutic proteins and ptides, for example, p15, p16, or le/WAF-l.
In some embodiments, a secondary therapeutic agent or polypeptide is a cytokine.
Examples of nes include: GM-CSF locyte macrophage colony stimulating factor);
TNF-alpha (Tumor is factor ; Interferons including, but not limited to, IFN-alpha
and IFN—gamma; and eukins including, but not limited to, Interleukin-l (ILl), Interleukin-
Beta (IL-beta), Interleukin-2 (IL2), Interleukin-4 (IL4), Interleukin-5 (IL5), Interleukin-6 (IL6),
Interleukin-8 (IL8), Interleukin-10 (ILlO), Interleukin-l2 (IL12), Interleukin-l3 (ILl3),
Interleukin- 1 4 (IL 1 4), Interleukin-15 (IL 1 5), Interleukin- 1 6 (IL 1 6), Interleukin-l 8 (ILl 8),
Interleukin-23 , Interleukin-24 (IL24), although other embodiments are known in the art.
In some embodiments, the secondary therapeutic agent or polypeptide is pro-
apoptotic. Examples of pro-apoptotic proteins or polypeptides include, but are not limited to:
Bax, Bad, Bik, Bak, Bim, cytochrome C, apoptosis-inducing factor (AIF), Puma, CT 10-
regulated kinase (CRK), Bok, glyceraldehydephosphate dehydrogenase, Prostate sis
se Protein-4 (Par-4), Smac, Kinase C8, Fas, inhibitory PAS domain protein (IPAS), and
Hrk.
In some embodiments, the secondary therapeutic agent or polypeptide is ed
in cell to cell communication. In some embodiments, the secondary therapeutic agent or
polypeptide is involved in gap cell junctions. In some embodiments, the secondary therapeutic
agent or polypeptide is a connexin. In some embodiments, the therapeutic protein or
polypeptide is a connexin chosen from the group connexin 43, connexin 32 and connexin 26.
In some embodiments, the secondary therapeutic agent or polypeptide is encoded
by the human receptor gene PiT-2 (SLC20A2) . The ropic Envelope gene product
included in the Reximmune-Cl and 2 retroviral vector binds to the PiT-2 receptor prior to target
cell infection.In some embodiments, the secondary therapeutic agent or polypeptide is encoded
by the human receptor gene PiT-l (SLC20A1) . The Gibbon Ape Luekemia Virus ( GALV)
Envelope gene t binds to the PiT-l receptor prior to target cell infection.
In some embodiments, the secondary therapeutic agent or polypeptide is an N-
terminal tion of a iral protein, wherein the N-terminal truncation comprises a
functional or binding domain of the envelope protein.
INCREASING INTRACELLULAR COMMUNICATION TO IMPROVE TREATMENT
Increase in Bystander Effect
Disclosed herein, in some embodiments, is a method of sing the HSV-TK
prodrug substrate bystander effect. As used herein, the “bystander effect” refers to the
phenomenon by which a HSV-TK positive exerts a kill effect on neighboring HSV-TK negative
cells following induction of expression of HSV-TK expression in the HSV-TK positive cells.
In some embodiments, is a method of sing the HSV-TK prodrug-mediated
bystander effect, for e after treatment with GCV, in conjunction with increasing gap
junction intracellular communication. In some embodiments, HSV-TK prodrug-mediate
bystander effect increases the kill rate by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by
70%, by 80%, by 90% or by 100% or more.
Gap junctions are regions of the cell membrane with clusters of gap junction
channels that directly connect the asm of one cell with the cytoplasm of another cell. A
gap junction channel is composed of two hemichannels (connexons) ed by each of two
neighboring cells. A connexon is comprised most often of six connexin proteins, which are a
large family of proteins having a basic structure sing four transmembrane domains, two
extracellular loops, and a cytoplasmic loops.
Gap junctions serve in various physiological roles, such as grth control and
homeostasis (i.e., rapid equilibration of ions, nutrients, and fluids between cells). In addition,
gap junctions serve as ical synapses in cells that are able to propagate electrical signals,
such as cardiac myocytes, smooth muscle cells, and neurons.
Once phosphorylated, GCV can travel through G] into adjoining cells that share
the junctions. GCV-P will be phosphorylated fiarther in those cells and trigger cell death as in the
HSK-TK expressing cell. The extend of the Bystander effect s on the existence of GAP
ons and therefore it will differ between cell types. But see, Dahle et al. “Gap onal
intercellular communication is not a major mediator in the bystander effect in photodynamic
treatment of MDCKII cells.” ion Res. 154: 331-341 (Sept. 2000).
The viral TK enzyme is sensitive to the prodrug ganciclovir (GCV) which
resembles the DNA base guanine.
When GCV is added to cell medium, the viral TK (but not the host non-viral TK)
phosphorylates the GCV, converting it into a drug as, now phosphorylated, it will compete with
dGTP for incorporation into DNA because of its similarity with guanine.
Incorporation will cause termination of the DNA chain synthesis. Transfer of
GCV-monoP into non-cancer cells will not be toxic to them unless they are actively dividing.
The normal cells at risk are only those in close contact to the viral TK-expressing cells when
treated with high levels of GCV drug.
Disclosed herein, in some embodiments, is a method of sing the viral
thymidine-kinase mediated killing of target cells in a t, the method comprising ring
vector les encoding HSV-TK in conjunction with gap junction intracellular communication
(GJIC) —increasing ent. In some embodiments, the target cells are neoplastic cells. In
some embodiments, the GJIC-increasing treatment comprises delivering to the cells a
polynucleotide sequence encoding at least one gap junction subunit. In some embodiments, the
at least one gap junction subunit is a wild type or mutant connexin. In some embodiments, the
gap junction t is chosen from the group consisting of wild type or mutant connexin 43,
connexin 30, and connexin 26. In other embodiments, the gap on subunit is conneXin 30.3,
in 3l, in 3l.l, in 32, connexin 33, connexin 37, connexin 40, connexin 45,
connexin 46 and connexin 50. In some embodiments, the gap junction subunit is modified to
prevent posttranslational modifications. In some embodiments, the GJIC-increasing treatment
comprises delivering to the cells a polynucleotide sequence encoding E-cadherin.
In some embodiments, a GJIC-increasing treatment comprises delivery of a
compound to a subject. In some embodiments, the GJIC-increasing treatment comprises
delivering to the subject a compound from the group comprising: gemcitabine; cAMP; a retinoic
acid; a carotenoid; a glucocorticoid, a flavanoid, apigenin, and/or lovastatin.
In some embodiments, the GJIC-increasing treatment comprises proteasome
inhibition. In some embodiments, the GJIC-increasing treatment ses proteasome
inhibition by administration ofN—Acetyl-Leu-Leu-Nle-CHO (ALLN) and/or chloroquine.
In some ments, the GJIC-increasing treatment comprises radiation
treatment.
In some embodiments, the GJIC-increasing ent comprises electrical
ent.
Methods of Detection
Disclosed herein, in some embodiments, is a method of measuring the HSV-TK-
mediated bystander , the method comprising: a) transfecting cells with a polynucleotide
sequence encoding HSV-TK and a first fluorescent protein; b) transfecting cells with a second
polynucleotide sequence encoding a second fluorescent protein that is optically discernible from
the first fluorescent protein; c) treating the cells with titrated doses of gancyclovir; and d)
measuring the relative amount of expression of the first fluorescent protein and the second
fluorescent protein.
In one embodiment, red fluorescent proteins (RFPs) are used to quantitate the
number of target tumor cells transduced with both the first cent protein fluorescent
n and the second and Hygro® can be used to select a population of tumor cells in which all
express both Hygro® and HSV-TK. RFPs are commercially ble and are contemplated for
use herein (see, for e, RFPs described in literature references l-l4 below.
In another embodiment, green fluorescent proteins (GFP) are used to tate
the number of transduced target tumor cells. GFPs are are commercially available and are
contemplated for use herein including, but not limited to, enhanced green fluorescent n
(EGFP).
PLASMIDS AND PRODUCTION OF HSV-TK
In some embodiments, disclosed herein are, nucleic acid molecules encoding
HSV-TK, or mutants and/or derivatives thereof, which are operably linked to suitable
transcriptional or translational regulatory elements. In some embodiments, suitable regulatory
elements are derived from bacterial, fungal, viral, mammalian, insect, or plant genes. Selection
of appropriate regulatory elements is dependent on the chosen host cell and, in some
embodiments, includes: a transcriptional promoter and enhancer or RNA polymerase g
sequence, and a ribosomal binding sequence, including a translation initiation signal.
bed herein are plasmids, comprising a nucleic acid sequence encoding
HSV-TK, or a mutant and/or variant thereof, as described above. In some embodiments,
disclosed herein are plasmids encoding HSV-TK fused to a second peptidic component. In
some embodiments, the second peptidic component is a eutic agent or polypeptide. In
some embodiments, the second ic component is a diagnostic polypeptide.
] In some embodiments, disclosed herein is a y of both viral and non-viral
vectors suitable for directing the expression of the nucleic acid molecules encoding HSV-TK
disclosed herein.
] In some embodiments, disclosed herein are ds for transfecting and
producing delivery vectors or therapeutic vectors for use in therapeutic and diagnostic
procedures. In l, such plasmids provide nucleic acid sequences that encode components,
viral or non-viral, of targeted vectors disclosed herein. Such plasmids e nucleic acid
sequences that encode, for example, the MoMLV pe protein. In some embodiments, the
2014/029814
MOMLV envelope protein is modified to contain a collagen binding domain. Additional
plasmids can include a nucleic acid sequence operably linked to a promoter. The ce
generally encodes a Viral gag-pol polypeptide. The d further includes a nucleic acid
sequence operably linked to a er, and the sequence encodes a polypeptide that confers
drug resistance on the producer cell. An origin of replication is also included. In some
embodiments, additional ds comprise an improved HSV-TK encoding sequence, as
disclosed herein, 5 ’ and 3 ’ long terminal repeat ces; a ‘I’ retroviral packaging ce, a
CMV enhancer upstream of the 5’ LTR promoter, a nucleic acid sequence operably linked to a
promoter, and an SV40 origin of replication.
In some embodiments, the polynucleotide encoding HSV-TK is under the control
of a le promoter. Suitable promoters include, but are not limited to, the retroviral LTR; the
SV40 promoter; the cytomegalovirus (CMV) er; the Rous Sarcoma Virus (RSV)
promoter; the histone promoter; the polIII er, the B-actin promoter; inducible promoters,
such as the MMTV promoter, the metallothionein promoter; heat shock promoters; adenovirus
promoters; the albumin promoter; the ApoAI promoter; B19 irus promoters; human
globin promoters; Viral thymidine kinase promoters, such as the Herpes Simplex Virus
thymidine kinase promoter; retroviral LTRs; human growth e ers, and the MXIFN
inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some
embodiments, a tissue specific promoters is chosen from the group including the nase
related promoters (TRP-l and TRP-2), DF3 enhancer (for breast cells), SLPI promoter
(secretory leucoprotease inhibitor--expressed in many types of carcinomas), TRS (tissue specific
regulatory sequences), u-fetoprotein promoters (specific for normal hepatocytes and transformed
hepatocytes, respectively), the carcino-embryonic antigen promoter (for use in transformed cells
of the intestinal tract, lung, breast and other tissues), the tyrosine hydroxylase promoter
(for melanocytes), choline acetyl transferase or neuron specific enolase promoters for use in
lastomas, the regulatory sequence for glial fibroblastomas, the tyrosine hydroxylase
promoter, c-erb B-2 promoter, PGK promoter, PEPCK promoter, whey acidic promoter (breast
tissue), and casein promoter t ) and the adipocyte P2 promoter. In some
embodiments, the promoter is a Viral-specific promoter (6.g. , retroviral promoters, as well as
others such as HIV promoters), hepatitis, herpes (e.g., EBV). In some embodiments, the
promoter is the native HSV-TK promoter. In some embodiments, the promoter is a bacterial,
fungal or parasitic (e.g. , malarial) -specific promoter is utilized in order to target a specific cell
or tissue which is infected with a Virus, ia, fungus or parasite.
In some embodiments, the delivery vectors or therapeutic vectors may include a
targeting moiety that targets the delivery vectors or therapeutic vectors to a desired cell or
system. In some embodiments, the targeting moiety refers to a ligand expressed by the delivery
vector or therapeutic vector that is associated with the delivery vehicle and target the e to a
cell or . In some embodiments, the ligand may include, but is not limited to, antibodies,
receptors and proteins that bind to ar components exposed in or on the ed cell or
system. In some embodiments, the exposed ar components may include collagen. In some
embodiments, the ligand binding to exposed cellular components comprises proteins that include
a collagen binding domain.
The plasmids disclosed herein may be produced by genetic engineering
techniques known to those skilled in the art. In addition, the plasmids may be readily expressed
by a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast
or other fungi, viral, insect, or plant cells. s for transforming or transfecting such cells to
s foreign DNA are well known in the art (see, e.g., Itakura et al., US. Pat. No. 4,704,362;
Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., US. Pat. No. 4,801,542; l
et al., US. Pat. No. 4,935,349; Hagen et al., US. Pat. No. 4,784,950; Axel et al., US. Pat. No.
4,399,216; Goeddel et al., US. Pat. No. 4,766,075; and Sambrook et al. Molecular g. A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989; for plant cells see
Czako and Marton, Plant l. 104: 071, 1994; and Paszkowski et al., Biotech.
24:387-392, 1992).
Protocols for the transfection of mammalian cells are well known to those of
ordinary skill in the art. Representative methods include calcium and/or magnesium phosphate
mediated transfection, electroporation, lipofection, retroviral, lentiviral ,adenoviral and
protoplast fusion-mediated.
In some embodiments, HSV-TK, or a mutant thereof, is prepared by ing the
ector systems described above, in order to express the recombinant thymidine kinase
mutants. inantly produced thymidine kinase mutants may be fiarther purified according
to s well known in the art.
In some embodiments, the nucleic acid molecules described herein are introduced
into a wide variety of host cells. Representative examples of such host cells include plant cells,
eukaryotic cells, and prokaryotic cells. In some embodiments, the nucleic acid molecules are
introduced into cells from a vertebrate or warm-blooded animal, such as a human, macaque,
dog, cow, horse, pig, sheep, rat, hamster, mouse or fish cell, or any hybrid thereof
In some ments, the nucleic acid molecules described herein are introduced
into a mammalian cell. In some embodiments, the mammalian cell is chosen from the group
including COS, BHK, CHO, HeLa, 293 and NS-1 cells. In some embodiments, suitable
expression vectors for directing expression in mammalian cells include a promoter, as well as
other transcriptional and translational control sequences. Common promoters include SV40,
MMTV, metallothionein-l, adenovirus Ela, Cytomegalovirus Immediate Early Promoter, and
the Cytomegalovirus Immediate Late Promoter.
In some embodiments, the nucleic acid molecules described herein are introduced
into a yeast or fiangi cell. Yeast and fiangi host cells suitable for carrying out the present
invention include, among others Saccharomyces pombe, Saccharomyces cerevisiae, the genera
Pichz’a or Klayveromyces and various species of the genus Aspergillas. le expression
vectors for yeast and fungi include, among others, YCp 50 for yeast, and the amdS cloning
vector pV3. In some embodiments, transformation of yeast is accomplished either by
preparation of spheroplasts of yeast with DNA or by ent with alkaline salts such as LiCl.
In some embodiments, transformation of fiangi is d out using polyethylene .
In some embodiments, the nucleic acid les described herein are uced
into a bacterial cell. Bacterial host cells suitable for carrying out the present invention include E.
coli, B. sabtz’lz’s, Salmonella typhz'marz’am, and various s within the genus' Pseudomonas,
Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of
ordinary skill in the art. Representative examples of bacterial host cells include DH50L
(Stratagene, La Jolla, Calif).
In some embodiments, ial expression vectors comprise a promoter which
functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of
replication. Representative promoters include the B-lactamase (penicillinase) and lactose
promoter system, the T7 RNA polymerase promoter, the lambda promoter, the trp promoter and
the tac promoter. Representative selectable markers include various antibiotic ance markers
such as the kanamycin or ampicillin resistance genes. In some embodiments, plasmids suitable
for orming host bacterial cells include, among others, pBR322, the pUC plasmids pUC l 8,
pUCl9, pUCl 18, pUCl l9, pNH8A, pNHl6a, pNH18a, and Bluescript M13 (Stratagene, La
Jolla, Calif.).
In some embodiments, the nucleic acid molecules described herein are expressed
in non-human transgenic animals such as mice, rats, rabbits, sheep, dogs and pigs. In some
embodiments, an expression unit, including a nucleic acid molecule to be expressed together
with appropriately positioned expression control sequences, is introduced into pronuclei of
fertilized eggs, for example, by microinjection. In some embodiments, integration of the injected
DNA is ed by blot is ofDNA from tissue samples. In some embodiments, the
uced DNA is orated into the germ line of the animal so that it is passed on to the
's progeny. In some embodiments, tissue-specific expression is ed through the use
of a tissue-specific promoter, or through the use of an inducible promoter, such as the
othionein gene promoter, which allows regulated expression of the transgene.
In some embodiments, the nucleic acid molecules described herein are introduced
into host cells by a wide variety of mechanisms, including for example calcium phosphate-
mediated ection; lipofection; gene gun; electroporation; retroviral, adenoviral, protoplast
fusion-mediated transfection or DEAE-dextran mediated transfection.
VECTORS AND METHODS OF TION THEREOF
] Disclosed herein is a vector particle, comprising an improved HSV-TK encoding
sequence, as described above, which is to be expressed in a desired cell. In some embodiments,
the vector particle is a viral vector particle. In some embodiments, the viral vector particle is a
retroviral vector particle.
In some embodiments, a vector le comprising an improved HSV-TK
encoding sequence contains or expresses a wide variety of additional nucleic acid molecules in
addition to the improved HSV-TK encoding sequence. In some embodiments, the vector
additionally expresses a lymphokine, antisense sequence, toxin or “replacement” protein (e.g.,
adenosine deaminase). entative examples of lymphokines e, for example, IL-l, IL-
2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-lO, IL-l l, IL-12, IL-l3, IL-l4, IL-l5, GM-CSF,
G-CSF, M-CSF, alpha-interferon, nterferon, gamma interferon, and tumor necrosis factors
(TNFs). Representative es of antisense sequences include, but are not limited to:
antisense myc, antisense p53, antisense ms, as well as antisense sequences which block the
expression or production of viruses such as HIV, HBV and HCV. Representative examples of
toxins include, but are not limited to: ricin, abrin, diphtheria toxin, cholera toxin, gelonin,
botulinum, pokeweed antiviral protein, tritin, Shigella toxin, and monas exotoxin A.
Representative examples of suicide genes e, but are not limited to: a cytosine deaminase, a
VSV-tk, IL-2, nitroreductase (NR), ylesterase, beta-glucuronidase, rome p450,
beta-galactosidase, diphtheria toxin A-chain (DT-A), carboxypeptide G2 (CPG2), purine
nucleoside phosphorylase (PNP), and deoxycytidine kinase (dCK). In some instances, the
vector additionally expresses a yeast and/or a bacterial cytosine deaminase.
Additional eutic sequences include, but are not limited to, Yeast or
ial Cytosine Deaminase, other suicide genes and other apoptotic genes, guanylate
, p53
kinase, IL-12 and other immune stimulatory or cytokine genes, GFP, RFP, iRFP, LUC2, GLUC
and other fluorescent and bioluminescent genes, Cyclin A, D and other cell cycle regulatory
genes, Viral genes, ial genes, human genes, synthetic genes, SIRNA, RNAi, Micro RNA,
antisense of genes, inhibitory or stimulatory sequences, genes captured from library strategies,
repeat sequence, replication sequence, promoter or enhancer sequence, DNA binding ces,
any therapeutic sequence, etc.
In some embodiments, a polynucleotide sequence encoding a or to a
gamma retrovirus is included. Disclosed herein in the present application are experiments
demonstrating that that the receptor binding domain (RBD) of amphotropic viral vector
envelope gene product binds to a PiT-2 receptor on the cell membrane of target cells and allows
for enhancement of viral vector transduction. Using a topological model for PiT-2 and a murine
leukemia virus (A-MuLV) receptor-binding assay on CHO-Kl and BHK cells, Feldman et al.
(Eiden MV. J Virol. (2004) 78: 595—602) identified the extracellular domain one (ECDl) of the
human PiT-2 receptor as being important for amphotrophic virus g and infection. Studies
by Bottger and Petersen (2004) showed that the part needed for binding the virus could be
narrowed down to the 182 aa N-Term region and 170 aa C-Term region.
Accordingly, also provided herein in select embodiments are polynucleotide
sequences encoding a mutated form of thymidine kinase from human simplex virus (HSV-TK),
wherein the encoded HSV-TK includes a polynucleotide sequence to encode PiT-2, PiT-l,
MCAT and other ors used by gamma retrovirus.
GAP JUNCTION INTRACELLULAR COMMUNICATION
In some embodiments, a vector particle additionally ses a gap junction
intracellular communication (GJIC)-increasing treatment, as bed . In some
embodiments, a vector le additionally expresses one or more genes which encode proteins
that facilitate or increase the biological activity of ine kinase. In some embodiments, a
vector further comprises a sequence encoding a DNA polymerase (e.g., a Herpes DNA
polymerase) and/or guanylate kinase.
One of the most frequently used delivery systems for achieving gene therapy
involves viral s, most ly adenoviral and retroviral vectors. Exemplary viral-based
vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO
94/03622; WO 93/25698; WO 93/25234; US. Pat. No. 5,219,740; WO 30; WO
93/10218; US. Pat. No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805;
each of which is incorporated by reference with t to the disclosures regarding recombinant
retroviruses), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC
VR-67; ATCC VR-1247), Ross River virus (ATCC VR—373; ATCC VR-1246) and Venezuelan
equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)),
and adeno-associated virus (AAV) s (see, e. g., W0 94/12649, WO 93/03769; W0
W0 2014/153258
93/19191; W0 94/28938; W0 84 and W0 95/00655). Administration ofDNA linked to
killed adenovirus as described by Curiel (Hum. Gene Ther. (1992) 3:147) can also be employed.
Retroviruses generally have three common open reading frames, gag, pol, and
env, which encode the matrix, gag and nucleocapsid structural proteins, encode enzymes
including e transcriptase, integrase and protease, and encode envelope proteins and
transmembrane fusogenic proteins, tively. Generally, retroviral vector particles are
ed by packaging cell lines that provide the necessary gag, pol, and env gene products in
trans. This approach results in the production of retroviral vector les which transduce
mammalian cells, but are incapable of filrther replication after they have integrated into the
genome of the cell.
For gene delivery purposes, a viral particle can be developed from a virus that is
native to a target cell or from a virus that is non-native to a target cell. Generally, it is desirable
to use a non-native virus vector rather than a native virus vector. While native virus vectors may
possess a natural y for target cells, such viruses pose a greater hazard since they possess a
greater potential for propagation in target cells. In this regard, animal virus vectors, wherein they
are not naturally designed for propagation in human cells, can be useful for gene delivery to
human cells. In order to obtain sufficient yields of such animal virus s for use in gene
delivery, however, it is necessary to carry out tion in a native animal packaging cell.
Virus vectors produced in this way, however, normally lack any components either as part of the
envelope or as part of the capsid that can provide m for human cells. For e, current
practices for the production of non-human virus vectors, such as ecotropic mouse (murine)
retroviruses like MMLV, are produced in a mouse packaging cell line. Another component
required for human cell tropism must be provided.
In general, the ation of a viral vector (without a helper virus) proceeds in a
packaging cell in which nucleic acid sequences for packaging components are stably integrated
into the cellular genome and nucleic acid coding for viral nucleic acid is introduced in such a
cell line.
] In some embodiments, the retroviral plasmid vector includes a polynucleotide
comprising the improved HSV-TK encoding sequence, and the expression vehicle including the
polynucleotide comprising the improved HSV-TK encoding ce are transduced into a
packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (2'.e.,
unmodified) env retroviral proteins. Examples of such ing cell lines include, but are not
limited to, the PESOl, PA3 l7 (ATCC No. CRL 9078),'-2,—AM, PAlZ, Tl9-l4X, VT-l9-l7-H2,
TCRE, TCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human
Gene Therap, Vol. 1, pgs. 5-14 (1990), which is orated herein by reference in its entirety,
or the 293T cell line (U. S. Patent No. 5,952,225). The vector(s) may be transfected into the
packaging 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 generally generate infectious retroviral vector particles which include the
first, or unmodified wild-type retroviral envelope protein, a chimeric retroviral envelope protein,
and a polynucleotide encoding the therapeutic or diagnostic agent.
In some embodiments, there is provided a packaging cell which includes
polynucleotides encoding the gag and pol proteins, a polynucleotide encoding a first retroviral
envelope protein free of non-retroviral peptides (which, in some embodiments, is a ype
retroviral envelope protein), and a polynucleotide encoding a chimeric retroviral envelope
protein. In some embodiments, a producer cell for generating retroviral vector particles which
e the first and chimeric envelope proteins is ed by introducing into such packaging
cell either a iral vector particle or a retroviral d vector, in each case including a
polynucleotide encoding the eutic or diagnostic agent. In some ments, the producer
cell line thus generates infectious retroviral vector particles including the polynucleotide
comprising the ed HSV-TK encoding sequence.
In some embodiments, disclosed herein is a kit for the production of viral vectors,
the kit comprising: a) a container containing a first plasmid comprising a nucleic acid ce
encoding a iral envelope protein, wherein the c acid sequence is operably linked to a
promoter; b) a container containing a second d comprising: a nucleic acid sequence
operably linked to a promoter, wherein the sequence encodes a viral gag-pol polypeptide, a
nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a
polypeptide that confers drug resistance on the producer cell, and an SV40 origin of replication;
c) a container containing a third plasmid comprising: an improved HSV-TK ng sequence
operably linked to a er, 5 ’ and 3 ’ long terminal repeat sequences (LTRs), a ‘I’ retroviral
packaging sequence, a CMV promoter upstream of the 5’ LTR; a nucleic acid sequence operably
linked to a promoter, wherein the ce encodes a polypeptide that confers drug resistance
on the producer cell, an SV40 origin of replication, d) a container containing a producer cell that
expresses SV40 large T n; and e) instructions for transiently transfecting the er cell
of d) with the plasmids of a), b), and c) and culturing the transfected producer cell under
conditions that allow viral les to be produced.
It is recognized that the delivery vectors or therapeutic vectors disclosed herein
include viral and non-viral particles. Non-viral delivery systems, such as microparticles or
nanoparticles including, for e, cationic liposomes and polycations, provide alternative
methods for delivery systems and are encompassed by the present disclosure. Non-viral
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particles include encapsulated proteins, including wholly or partially assembled viral
particles, in lipid bilayers. Methods for encapsulating viruses into lipid bilayers are known in
the art. They include passive entrapment into lipid bilayer-enclosed vesicles (liposomes), and
incubation of virions with liposomes (US. Pat. No. 5,962,429; Fasbender, et al., J. Biol. Chem.
79-6489; Hodgson and Solaiman, Nature Biotechnology -342 (1996)). Without
being limited by a theory, we assume that acidic proteins exposed on the surface of a virion
provide an interface for complexation with the ic cationic polymer component of the
delivery vector or therapeutic vector and serve as a “scaffold” for the bilayer formation by the
neutral lipid component.
Examples of non-viral delivery systems include, for example, Wheeler et al., US.
Pat. Nos. 5,976,567 and 5,981,501. These s disclose preparation of serum-stable plasmid-
lipid particles by contacting an aqueous solution of a plasmid with an c solution
containing cationic and non-cationic lipids. Thierry et al., US. Pat. No. 6,096,335 disclose
preparation of a complex comprising a ly c biologically active substance, a cationic
constituent, and an anionic constituent. Allen and Stuart, 98/12937 (WO 98/5 8630)
disclose forming polynucleotide-cationic lipid particles in a lipid solvent suitable for
solubilization of the cationic lipid, adding neutral vesicle-forming lipid to the t containing
the particles, and evaporating the lipid solvent to form liposomes having the polynucleotide
entrapped within. Allen and Stuart, US. Pat. No. 6,120,798, disclose forming polynucleotide-
lipid microparticles by dissolving a polynucleotide in a first, e. g., aqueous, solvent, dissolving a
lipid in a , e.g., c, t immiscible with said first solvent, adding a third solvent
to effect formation of a single phase, and r adding an amount of the first and second
solvents to effect formation of two liquid phases. Bally et al. US. Pat. No. 5,705,385, and
Zhang et al. US. Pat. No. 6,110,745 disclose a method for preparing a lipid-nucleic acid particle
by contacting a nucleic acid with a solution containing a non-cationic lipid and a cationic lipid to
form a nucleic acid mixture. Maurer et al., PCT/CA00/00843 (WO 01/06574) disclose a
method for preparing fillly lipid-encapsulated therapeutic agent particles of a charged
therapeutic agent including combining preformed lipid vesicles, a charged therapeutic agent, and
a destabilizing agent to form a mixture thereof in a destabilizing solvent that destabilizes, but
does not disrupt, the vesicles, and subsequently removing the destabilizing agent.
A Particle-Forming Component (“PFC”) typically comprises a lipid, such as a
ic lipid, optionally in combination with a PFC other than a cationic lipid. A cationic lipid
is a lipid whose molecule is capable of electrolytic dissociation producing net positive ionic
charge in the range ofpH from about 3 to about 10, preferably in the physiological pH range
from about 4 to about 9. Such cationic lipids encompass, for e, cationic detergents such
as cationic amphiphiles having a single hydrocarbon chain. Patent and scientific literature
describes numerous cationic lipids having nucleic acid transfection-enhancing ties. These
transfection-enhancing cationic lipids include, for example: 1,2-dioleyloxy(N,N,N—
hylammonio)propane chloride-, DOTMA (US. Pat. No. 4,897,355); DOSPA (see
Hawley-Nelson, et al., Focus 15(3):73 (1993)); N,N-distearyl-N,N-dimethyl-ammonium
bromide, or DDAB (US. Pat. No. 5,279,833); 1,2-dioleoyloxy(N,N,N-trimethylammonio)
propane chloride-DOTAP tatos, et al., Biochemistry 27: 3917-3925 ); glycerol
based lipids (see Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990); l-PE (US.
Pat. No. 5,980,935); lysinyl-PE (Puyal, et al. J. Biochem. 228:697 (1995)), lipopolyamines (US.
Pat. No. 5,171,678) and cholesterol based lipids (WO 93/05162, US. Pat. No. 5,283,185);
CHIM (1-(3-cholesteryl)-oxycarbonyl-aminomethylimidazole); and the like. Cationic lipids for
transfection are reviewed, for example, in: Behr, Bioconjugate Chemistry, 5 :3 82-3 89 (1994).
Preferable ic lipids are DDAB, CHIM, or combinations thereof. Examples of cationic
lipids that are cationic detergents include (C12-C18)-alkyl- and (C 12-C18)-alkenyl-
trimethylammonium salts, N-(C12-C18)-alkyl- and 2-C18)-alkenyl-pyridinium salts, and
the like.
In some embodiments, the size of a delivery vector or therapeutic vector formed
is within the range of about 40 to about 1500 nm. In some ments, the delivery vector or
therapeutic vector is in the range of about 50-500 nm in size. In some embodiments, the
delivery vector or eutic vector is in the range of about 20-150 nm in size. This size
selection advantageously aids the delivery vector, when it is administered to the body, to
penetrate from the blood vessels into the diseased tissues such as malignant tumors, and transfer
a therapeutic nucleic acid therein. It is also a characteristic and advantageous property of the
ry vector that its size, as measured for example, by dynamic light ring method, does
not substantially increase in the presence of ellular biological fluids such as in Vitro cell
culture media or blood plasma.
Alternatively, in some embodiments, cells which produce retroviruses are
injected into a tumor. In some embodiments, the retrovirus-producing cells so introduced are
engineered to ly produce a delivery vector, such as a Viral vector particle, so that
continuous productions of the vector ed within the tumor mass in situ. In some
embodiments, proliferating tumor cells are transduced in vivo by proximity to retroviral vector-
producing cells.
METHODS OF USE
In some embodiments, disclosed herein is a method of providing to target cells a
cleotide encoding HSV-TK, as disclosed herein, the method comprising and then
ng the cells to an appropriate substrate which is converted to a toxic substance to kill
those cells expressing the mutant HSV-l thymidine kinase gene as well as those in the vicinity
of the mutant HSV-l thymidine kinase gene-expressing cells, z'.e. cells. The mutant
, bystander
HSV-l thymidine kinase gene can be administered directly to the targeted or d cells or
systemically in ation with a targeting means, such as through the ion of a particular
viral vector or delivery formulation. Cells can be treated in viva, within the patient to be treated,
or treated in vitro, then injected into the patient. Following introduction of the mutant HSV-l
thymidine kinase gene into cells in the patient, the prodrug is administered, systemically or
y, in an effective amount to be converted by the mutant HSV-l thymidine kinase into a
ient amount of toxic substance to kill the targeted cells. A nucleoside analog which is a
substrate for HSV-l TK to e a toxic substance which kills target cells is referred to herein
as a “prodrug”.
In some embodiments, disclosed herein is a method of killing a cell, the method
comprising: i) introducing into the cell a polynucleotide or vector as disclosed herein; ii)
allowing or directing the cell to express thymidine kinase; and iii) contacting the cell with an
agent that is converted by thymidine kinase to a cytotoxic agent.
In some embodiments of the present invention there is provided herein a method
of preventing graft-versus-host disease (GvHD) in a patient comprising: (i) administering to a
host T-cells genetically engineered to include a polynucleotide or vector of the present
invention; and (ii) administering to said host, prior to the occurrence of graft-versus-host
disease, an agent capable of being converted by thymidine kinase to a cytotoxic agent in an
amount effective to kill genetically ered T-cells capable of effecting GvHD. During an
allogeneic bone marrow transplant, alloreactive T lymphocytes can be removed from the graft in
order to prevent graft versus host disease. GvHD occurs when T-cells in the lanted stem
cell graft attack the transplant recipient's body. However, l of the s can increase the
incidence of disease relapse, graft rejection and reactivation of viral infection. To counter the
possibility of GvHD, allogeneic bone marrow transplant patients can be treated by introducing
donor T lymphocytes after a delay following the allogeneic bone marrow transplant. However,
delayed introduction of donor T lymphocytes following allogeneic bone marrow transplant is
limited by GvHD, a nt and potentially lethal complication of the treatment. By
stering to a transplant recipient T-cells genetically engineered to e a polynucleotide
encoding a “suicide gene,” the T-cells can be killed if they begin to attack the transplant
ent’s body.
In some embodiments, the iral vector particles, which include a chimeric
retroviral envelope protein and a cleotide encoding a therapeutic agent, are stered
to a host in order to express the therapeutic agent in the host. In some embodiments, the
polynucleotide encoding a therapeutic agent is a polynucleotide encoding HSV-TK, or a mutant
and/or variant thereof, as disclosed herein.
In some embodiments, cells are obtained from a patient, and retroviral vector
particles are used to introduce a therapeutic agent or polypeptide into the cells, and such
modified cells are administered to the patient. In some embodiments, retroviral vector particles
are administered to the patient in viva, whereby the retroviral vector particles transduce cells of
the patient in viva.
In some ments, disclosed herein is a method of delivering a therapeutic
agent or ptide to a site of tissue injury in a subject, comprising directly or enously
delivering to the site of tissue injury a iral particle comprising: i) a chimeric retroviral
envelope protein and ii) at least one polynucleotide encoding a therapeutic ptide, wherein
the viral particle binds to en exposed at the site of tissue injury and expresses the
therapeutic polypeptide at the site of tissue injury. In some embodiments, the tissue injury is
selected from the group consisting of tissue injury due to tumor invasion, vascular lesion,
ulcerative lesions, atory tissue injury, laser injury to eyes, surgery, arthritic joints, scars,
and keloids. In some embodiments, the tissue injury is a lesion of tissue due to growth of a
tumor in the host.
In some embodiments, therapeutic vectors, as disclosed herein, are employed in
the treatment of cancer, including malignant and nonmalignant tumors. In some ments,
the therapeutic vectors filrther comprise an extracellular matrix binding e or peptide
domain. In some embodiments, the extracellular matrix binding peptide or peptide domain is a
collagen binding domain or peptide. In some embodiments, the tumors include, but are not
limited to, all solid tumors.
In some embodiments, eutic vectors, as sed , are employed in
the treatment of cancer being selected from the group consisting of breast cancer, skin ,
bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gall
bladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon,
stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating
and papillary type, metastatic skin carcinoma, melanoma, osteosarcoma, Ewing’s a,
veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell
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tumor, primary brain tumor, acute and chronic cytic and granulocytic tumors, hairy-cell
tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neurons,
intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoid s tumor, Wilm’s
tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma,
lastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion,
mycosis fiJngoide, myosarcoma, ’s sarcoma, osteogenic and other sarcoma,
malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma
multiforma, leukemias, lymphomas, ant melanomas, and epidermoid carcinomas. In
other embodiments, the cancer being treated is pancreatic cancer, liver cancer, breast cancer,
osteosarcoma, lung cancer, soft tissue sarcoma, cancer of the larynx, melanoma, n cancer,
brain cancer, Ewing’s sarcoma or colon cancer.
In other embodiments, the cancer to be treated is chosen from the group
consisting of primary hepatocellular carcinoma, metastatic breast carcinoma to liver, metastatic
pancreatic cancer to liver, metastatic gastric cancer to liver, metastatic esophageal cancer to
liver, metastatic lung cancer to liver, metastatic melanoma to liver, metastatic ovarian oma
to liver and metastatic kidney cancer to liver.
The therapeutic vectors may be stered alone or in conjunction with other
therapeutic treatments or active agents. Examples of other active agents that may be used
include, but are not limited to, chemotherapeutic agents, nflammatory agents, protease
inhibitors, such as HIV protease inhibitors, nucleoside analogs, such as AZT. In some
embodiments, the methods 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 eutic 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 ent routes (6.g. , the retroviral vector is administered intravenously and
the one or more agents are administered orally).
The dosage of the therapeutic Viral particles lies preferably within a range of
circulating concentrations that e the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the route of administration
utilized. A eutically effective dose can be estimated initially from cell culture assays. A
dose may be formulated in animal models to achieve a ating plasma concentration range
that es the IC50 (z'.e., the concentration of the test compound which achieves a half-
maximal infection or a half-maximal inhibition) 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 therapeutically effective of the retroviral particles
disclosed herein to be administered to a t in need of treatment may be determined in a
y 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 obtained. In some embodiments,
treatment may be continuous for days, weeks, or years, or may be at intervals with intervening
rest periods. In some embodiments, the dosage is modified in accordance with other treatments
the individual may be receiving. However, the method of ent is in no way limited to a
particular concentration or range of the retroviral particle and may be varied for each individual
being treated and for each tive 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 ining dosage include,
but are not d to, tumor size, alteration in the level of tumor markers used in clinical testing
for particular malignancies. In some embodiments, the ng 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, including but not limited to the therapeutic retroviral
particles that are specifically to the cell or system of st, may be systemically or regionally
(locally) delivered to a subject in need of treatment. For e, the therapeutic vectors may
be systemically administered intravenously. Alternatively, the therapeutic vectors may also be
administered intra-arterially. The therapeutic s may also be administered topically,
intravenously, intra-arterially, intra-tumorally, intracolonically, intratracheally, intraperitoneally,
intranasally, intravascularly, intrathecally, intracranially, intramarrowly, intrapleurally,
intradermally, subcutaneously, intramuscularly, cularly, intraosseously and/or
intrasynovially or sterotactically. A combination of delivery modes may also be used, for
e, a t may e the eutic vectors both systemically and regionally (locally)
to improve tumor responses with treatment of the therapeutic vectors.
In some embodiments, multiple therapeutic s (e.g., first and second
therapeutic course) are administered to a t in need of treatment. In some embodiments,
the first and/or second eutic course is stered intravenously. In other embodiments,
the first and/or second therapeutic course is stered Via intra-arterial infusion, including
but not limited to infusion through the hepatic artery, cerebral artery, coronary artery, pulmonary
artery, iliac artery, celiac trunk, gastric artery, splenic , renal artery, gonadal artery,
subclaVian artery, vertebral artery, axilary artery, brachial artery, radial artery, ulnar artery,
carotid artery, femoral artery, inferior mesenteric artery and/or superior mesenteric artery. Intra-
arterial infusion may be accomplished using endovascular procedures, percutaneous ures
or open surgical approaches. In some embodiments, the first and second therapeutic course may
be stered sequentially. 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 administered sequentially or simultaneously with the first and second eutic
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 e, in some embodiments, a patient in need
thereofmay be systemically administered, e.g., intravenously administered, 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 I x 1022 TVP delivery vector on a cumulative basis. The first
therapeutic course may be systemically administered. Alternatively, 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 infusion with at least 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 I x 1022 TVP
delivery vector on a cumulative basis.
In yet other embodiments, a t in need thereofmay receive a ation,
either sequentially or concurrently, of systemic and intra-arterial infusions administration of
high doses of delivery vector. For example, a patient in need thereofmay be first systemically
administered with at least 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, 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 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis, followed by an
additional 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 fmay receive a combination of intra-arterial
infusion and systemic administration of delivery vector in high doses. For example, a patient in
need thereofmay be first 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, ed 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 cumulative
basis. The therapeutic courses may also be administered simultaneously, i.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 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP ry
vector on a cumulative basis, together with a therapeutic course of intra-arterial infilsion, e.g.,
hepatic arterial on, 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.
In still other embodiments, a t in need thereofmay onally e,
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 I x 1019 TVP, at least I x 1020 TVP, at least I x 1021 TVP or at least I x 1022 TVP
delivery vector on a tive basis.
In some embodiments, the subject in need of treatment is administered
systemically (e.g., intravenously) a dose of at least I x 1011 TVP, followed by the administration
via intra-arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1011 TVP. In
other ments, the patient in need of treatment may be administered ically (e.g.,
intravenously) a cumulative dose of at least I x 1012 TVP, followed by the administration via
intra-arterial infilsion (e.g., hepatic-arterial infusion) of a dose of at least I x 1012 TVP. In one
embodiment, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least I x 1013 TVP, followed by the administration via intra-arterial
infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1013 TVP. In yet other
embodiments, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least I x 1014 TVP, concurrently with the stration via intra-
arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1014 TVP. In still other
embodiments, the t in need of ent may be administered systemically (e.g.,
intravenously) a dose of at least I x 1015 TVP, together with the administration via intra-arterial
on (e.g., hepatic-arterial infusion) of a dose of at least I 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 I x 1016 TVP, concurrently with the administration via intra-
arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1016 TVP. In still other
ments, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least I x 10137TVP, er with the administration via intra-arterial
infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1017 TVP.
A subject in need of treatment may also be administered, either systemically or
localized (for example intra-arterial infusion, such as hepatic arterial infilsion) a eutic
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 , at least nine months, at least ten
months, at least eleven months, 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 manner, z'.e.,
for an undefined or indefinite period of time.
stration of the therapeutic vector may also occur in a periodic manner,
e.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 ent 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 ry vector takes place over a r period of time.
In one embodiment, the subject is allowed to rest 1 to 2 days between the first
therapeutic course and second therapeutic . In some embodiments, the subject is allowed
to rest 2 to 4 days between the first therapeutic course and second therapeutic course. In other
embodiments, the subject is allowed to rest at least 2 days n 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 ments, 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 d to rest at least 2 weeks
between the first and second therapeutic course. In one embodiment, the subject is allowed to
rest at least one month n the first and second therapeutic course. 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.
In some embodiments, the therapeutic vector is administered to increase local
concentration of the peptide or vector. In some embodiments, the therapeutic vector is
administered via intra-arterial infilsion, which increases local tration of the therapeutic
vector to a specific organ . In yet other embodiments, the therapeutic vector is
administered intra-tumorally. Dependent upon the on of the target lesions, in some
embodiments, catheterization of the hepatic artery is followed by on into the
pancreaticoduodenal, right hepatic, and middle hepatic artery, respectively, in order to locally
target hepatic s. In some embodiments, localized distribution to other organ systems,
ing the lung, gastrointestinal, brain, reproductive, splenic or other defined organ ,
of the peptide or delivery vector is accomplished via catheterization or other localized delivery
. In some embodiments, intra-arterial infusions are accomplished via any other available
arterial source, including but not limited to infusion 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
artery, ulnar artery, carotid artery, femoral artery, inferior eric artery and/or superior
mesenteric . In some embodiments, arterial infusion is accomplished using
scular procedures, percutaneous procedures or open surgical approaches.
Formulations
Pharmaceutical compositions comprising a therapeutic vector can be formulated
in any conventional manner by mixing a selected amount of the therapeutic vector with one or
more physiologically acceptable carriers or excipients. For example, the therapeutic vector may
be ded in a carrier such as PBS (phosphate buffered saline). The active compounds can
be administered by any appropriate route, for example, orally, parenterally, intravenously,
ermally, 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, , 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, dichlorotetra-fluoroethane, carbon
dioxide or other suitable gas. In some embodiments, a pressurized aerosol dosage unit or a valve
to deliver a metered . In some embodiments, capsules and cartridges (e.g., of gelatin) for
use in an r or insufflator are formulated containing a powder mix of a therapeutic
compound and a le powder base such as lactose or starch.
In some embodiments, the pharmaceutical itions are ated for oral
administration as tablets or capsules prepared by conventional means with pharmaceutically
acceptable excipients such as g agents (e.g., pregelatinized maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g, magnesium stearate, talc or );
disintegrants (e.g, potato starch or sodium starch ate); 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 formulated as a dry product for
constitution with water or other suitable vehicle before use. In some embodiments, such liquid
preparations are prepared by conventional means with pharmaceutically acceptable additives
such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily
, ethyl alcohol or fractionated vegetable oils); and vatives (e.g., methyl or propyl-p-
hydroxybenzoates or sorbic acid). In some embodiments, the preparations also contain buffer
salts, flavoring, ng 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 ated for
buccal in the form of tablets or lozenges formulated in conventional manner.
In some embodiments, the therapeutic vector is formulated for parenteral
administration by injection, e.g., by bolus ion, or continuous infusion. In some
embodiments, formulations for injection are in unit dosage form, e.g., in ampoules or in multi-
dose ners, with an added preservative. In some embodiments, the compositions are
formulated as suspensions, solutions or emulsions in oily or aqueous vehicles. In some
embodiments, the ations comprise formulatory agents such as ding, stabilizing
and/or dispersing . Alternatively, 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 ations are administered by
tation (for example, subcutaneously or intramuscularly) or by uscular injection.
Thus, for example, in some embodiments, the therapeutic compounds are formulated with
suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil)
or ion exchange resins, or as sparingly e derivatives, for example, as a gly soluble
salt.
In some ments, 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
pinal application. In some embodiments, such solutions, particularly those intended for
ophthalmic use, are formulated as 0.0l%-10% 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
absorption, 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 presented 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 blister pack. In some
embodiments, the pack or dispenser device is accompanied by instructions for administration.
In some embodiments, the active agents are packaged as es of manufacture
containing packaging material, an agent provided herein, and a label that tes the disorder
for which the agent is provided.
Animal Models
In some embodiments, 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 retroviral vector particles are administered
in varying doses to different animals of the same species. The s 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 stered to a human patient.
Also provided are kits or drug delivery systems comprising the compositions for
use in the methods described herein. All the essential materials and reagents required for
stration of the retroviral particles disclosed herein may be assembled in a kit (6.g.
packaging cell uct or cell line, cytokine sion vector). The components of the kit
may be provided in a variety of formulations as bed above. The one or more therapeutic
retroviral particles may be ated 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 provided in
dried or lyophilized forms. When reagents or components are provided as a dried form,
reconstitution 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
placed.
The kits disclosed herein may also comprise ctions regarding the dosage
and/or administration information for the retroviral particle. Instructions can include ctions
for practicing any of the methods described herein including treatment methods. Instructions can
additionally include indications of a actory clinical endpoint or any e symptoms that
may occur, or additional information required by regulatory agencies such as the Food and Drug
stration for use on a human subject.
WO 53258 2014/029814
The instructions 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 component of the kit. Instructions may additionally be included on a er
readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-
ROM/RAM, magnetic tape, electrical e media such as RAM and ROM, IC tip and hybrids
of these such as magnetic/optical e media.
In some ments, the kits or drug delivery systems include a means for
ning the vials in close ment for commercial sale such as, e.g., injection or blow-
molded plastic containers into which the desired vials are retained. Irrespective of the number or
type of containers, the kits may also se, or be packaged with, an instrument for assisting
with the inj /administration or placement of the ultimate complex composition within the
body of a subject. Such an instrument may be an applicator, inhalant, e, pipette, forceps,
measured spoon, eye dropper or any such medically approved delivery vehicle.
Packages and kits can further include a label specifying, for example, a product
description, mode of administration and/or indication of ent. Packages provided herein
can include any of the compositions as described 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 appropriate written instructions.
Kits, therefore, can additionally include labels or instructions for using the kit components in
any method described herein. A kit can e 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
methods described herein, the following examples are provided for illustration purposes.
Example 1: Cell Line Generation
First, iral supernatant is generated by transfection of a 3 or 4 d
system with calcium phosphate t into 293 T cells. Supernatant is filtered through a 0.45
um filter. Filtered supernatant can be used fresh, stored up to 48 hours at 40 C, or stored at -80°
Cell lines are generated by seeding l x 104 cells/well in a 6 well tissue culture
dish. The next day retroviral supernatant is added with 8 ug/mL polybrene for 16-24 hours and
WO 53258
selected with the appropriate dose of selection drug (G418 ,hygromycin or puromycin). The
dose of the selection drug is the minimum amount to cause 100% kill on V-TK cells at
least 4 days post on of drug, in order to avoid excessive toxicity to cells.
Example 2: GCV Sensitivity Assay
Cells expressing HSV-TK, or a mutant and/or variant thereof, are seeded at l X
105 in 6 well dishes. The next day, 5 serial 10 fold dilution of GCV are added with a final
concentration ranging from 1 mM to 01 um. Three (3) days after GCV treatment, methylene
blue is added to stain live cells.
Example 3: Bystander Assay
Cells are seeded at l-4 x 104 cells/well in a 96 well plate, in triplicate, with
mixtures of TK cells ranging from 0-100%. The next day GCV is added at doses ranging from
um to 1 mM. Cells plates at confluency are split 1:30 into 3 , 20-24 hours after GCV
on. 5 days later, cells are analyzed by Presto Blue for live cell metabolism and read on a
microplate reader. Cell plates at sub-confluency are analyzed 3 days after GCV treatment by
Presto Blue.
In one assay, the inventors used HSV-TK clonal cell lines were generated; using
Neomycin-HSV-TK, Hygromycin-HSV-TK, Red Fluorescent protein (RFP)-HSV-TK cell lines
and several mutants of HSV-TK gene were compared.
] RexC2 carries an improved version of the Herpes simplex virus (HSV)
Thymidine Kinase gene (TK). A cellular host that has been efficiently infected duced)
with RxC2 will integrate the viral TK in its genome and express this enzyme. HSV-TK
phosphorylates the DNA base thymidine for its incorporation into newly synthesized DNA in
dividing cells.
A typical 96-well plate plan for cell g and GCV treatment is shown in the
table below (HK=HSV-TK):
NO NO NO NO
media 20 uM 20 uM 20 uM 10 uM 10 uM 10 uM GCV GCV GCV GCV
III-“mu
nmedia media media media media media media media media media media
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
media TK TK TK TK TK TK TK TK TK TK
IIIIIIIIIIIICmedia TK TK TK TK TK TK TK TK TK TK
uIIIIIIIIIIImedia TK TK TK TK TK TK TK TK TK TK
nIIIIIIIIIIImedia TK TK TK TK TK TK TK TK TK TK
% 25% 25% 25% 25% 25% 25% 25% 25% 25%
I: media
100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
media TK TK TK TK TK TK TK TK TK TK
nmedia media media media media media media media media media media
Graphic results of a bystander assay experiment are shown in Figures 19 and 20.
] More than 40 bystander assays were performed using different mutants HSV-TK
and clonal populations.
The data was compiled with GCV sensitivity and enzyme kinetics measurements
and viral titer of tion for the mutants with potential.
The careful examination of all these parameters allowed the selection of the
mutant HSV-TK168dmNES to be the TK gene in Reximmune C-2.
Exam le 4: uantitation of S liced Form of TK RNA b Real Time PCR
The unspliced and truncated form of HSV-TK are subcloned into a pCR2.l
TOPO vector (Invitrogen). Two quantitative real time PCRs are set-up with two different sets of
primers and probes able to selectively amplify and detect the unspliced and spliced form of
HSV-TK, using the TaqMan®/ABI PRISM 7700 sequence detection system. For the HSV-TK
unspliced form, primers and probe are designed in the spliced region of the HSV-tk gene
Real Time PCR for the unspliced form is med in a 25 ul reaction e
containing 100-500 ng of genomic DNA or 10 ul of cDNA, 1X TaqMan® Universal PCR
Master Mix, 300 nM of each of the two primers TKwtfor (5'-CGG CGG TGG TAA TGA CAA
G-3') and Tkwtrev (5'-GCG TCG GTC ACG GCA TA-3') and 200 nM of TKwt MGB probe (5'-
FAM CCA GAT AAC AAT GGG C-3').
A ® probe assing the splice on is designed to selectively
detect the HSV-TK spliced form. Quantitative Real time PCR specific for the TK spliced
(truncated) form was performed in a 25 ul reaction mixture containing 100-500 ng of genomic
DNA or 10 ul of cDNA, 1X Master Mix (PE Applied Biosystems) 300 nM of each of the two
primers. l cycling conditions are as follows: l activation ofUNG at 50 0C for 2 min,
followed by activation of Taq Gold and inactivation ofUNG at 95 0C for 15 min. Subsequently,
40 cycles of amplification are performed at 95 0C for 15 s and 60 0C for l min. Both PCRs are
performed in parallel in MicroAmp® optical 96-well reaction plates (Applied Biosystems) using
the ABI Prism 7700 ce Detection Systems (Applied Biosystems). Mean baseline
fluorescence was calculated from PCR cycles 3 to 15, and Ct was defined as the PCR cycle in
which the normalized fluorescence intensity of the er dye d 0.05. Two standard
curves with known copy numbers (from 10<6 >to 4 copies/reaction) are generated in each
TaqMan® assay by plotting the Ct values against the logarithm of the initial input ofDNA
amount. Standard dilutions and cDNA samples are analyzed in duplicate and triplicate,
respectively.
Example 5: Clinical Trial
A dose escalation trial was conducted to evaluate the safety, pharmacokinetics,
and pharmacodynamics of Reximmune-C2 dine Kinase and GM-CSF Genes) in
refractory subjects with primary hepatocellular carcinoma or tumors metastatic to the liver.
Background and Rationale
Reximmune-C2 is sed of a genetic delivery platform containing an
internal d that encodes for eutic proteins of interest. The c delivery platform
has been dosed in over 280 subjects worldwide; approximately 270 subjects were treated with
the vector containing dnGl as a payload -G) and 16 subjects with thymidine kinase (vTK)
and the immune ator Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) as a
payload (Reximmune-C). The genetic ry platform is a highly engineered combinant
Mouse Moloney Viral vector (MoMLV). Previously, a Phase 1 dose tion trial was
performed investigating the combination of Rexin-G and Reximmune-C in subjects with
refractory primary or metastatic solid tumors (Genevieve Trial). This proposed Phase I al
trial (entitled eve 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 subject were recruited over 3 dose levels
with the mean exposure in the t 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 ined safe and effective (optimal) dose of Rexin-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. Unrelated 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 e
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 uation of this Phase 1 trial is that: (l) thymidine kinase
itself could prove to be an effective anticancer agent particularly in subjects whose tumors
demonstrate a bystander effect; (2) stration of the genetic delivery platform 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 bute to an immunological effect and enhanced tumor cell kill through
tumor associated antigens through recruitment of the appropriate immune cells.
The tribution of the viral particles is highest to the liver, followed by
, then lung — this is the rationale for focusing initially on hepatocellular tumors where the
dose intensity should be the highest. 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
nce to a "protein" is a reference to one or more ns, and includes equivalents f
known to those skilled in the art and so forth.
Unless defined ise, 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 belongs. Specific methods, s, 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 t 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 phrases 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
clearer understanding of certain aspects of the t invention.
Example 6: Clinical Trial for gene y applications.
This clinical trial is divided into two phases: Phase IA in which Reximmune-C2
was administered as a single intravenous dose on three out of five days . Valganciclovir (the oral
form of lovir) dosing is ted on day 8 for 5 days irrespective of the PET scan results.
An approximately one week drug holiday follows. Each cycle will be of three weeks duration.
There will be three patients in the first and subsequent s until a t
experiences Dose Limiting ty (DLT) or two instances ofNCI-CTC Grade 2 ties
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 ts and the dose level will not be exceeded if 2 or more patients exhibit
DLTs.
Once the Maximum Administered Dose (MAD) is reached, a d Fibonacci
schedule will be followed starting with the cohort dose which had no DLTs and continuing until
dose-limiting toxicities are observed in two ts at a dose level. Once the Recommended
Phase 2 Dose (RP2D) is defined, 6-12 ts will be recruited.
Phase IB is designed to explore the activity of Reximmune-C2 in ts of a
defined tumor type and stage based on the Phase IA data and who are [18F]FHBG scan ve
day three to six after one dose (RP2D) of Reximmune-C2. If the scan is positive, the t is
accepted into the Phase IB treatment 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 Reximmune-C2 and occurring during the first cycle (3 weeks) of drug
administration:
Grade 4 neutropenia (i.e., absolute 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 thrombocytopenia (< 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 nonhematological
toxicity (except Grade 3 ion 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 appropriate use of
premedications (by Common Toxicity Criteria defined as omatic bronchospasm,
requiring parenteral medications(s), with or without urticaria; allergy-related edema-
angioedema”).
Reximmune-C2 is infused intravenously over 15-60 minutes (depending on the
dose) Via an infusion pump. une-C2 is provided in 30 ml vials stored at -80 °Ci 10 0C.
WO 53258
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
activity and clinical responses to Reximmune-C2 treatment will be described.
The starting dose in this trial is based on: human clinical safety experience with
the related vector platform drug products Rexin-G and Reximmune-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 m tolerated dose
(MTD), dose limiting toxicity (DLT), safety, and a ended Phase 2 dose (RP2D) of
Reximmune-C2 administered over a three week cycle consisting of a series of three doses given
intraveneously within five days in week 1, followed 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 pharmacokinetics of
une-C2; (ii) assessment of the surrogate of HSV-TK-m2 n expression from
Reximmune-C2 via serial [18F]FHBG PET and/or SPECT g; (iii) description and
assessment of any preliminary evidence of anti-tumor activity of Reximmune-C2; and (iv) to
provide clinical research testing for dies to ector gp70 env, replication-competent
retrovirus in peripheral blood cytes (PBLs); vector integration into genomic DNA of
PBLs, and circulating hGM-CSF protein.
Methods
Study Design: Parallel group, open label dose escalation, three-center clinical
trial.
Stratification: None.
Therapy: Reximmune-C2 will be administered as an intravenous infusion 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, additional patients will be
recruited. In Phase IB ts 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 ons occur.
Statistical Methods: ptive statistics will be used for tical 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
treated 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 portion.
Enrollment ia
Subjects must meet all of the following inclusion criteria to be eligible for
randomization into the study:
1. Diagnosis of histologically documented, advanced 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, chemotherapy, or surgical
procedures 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 on.
6. Patients may be tis 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 anti-seizure medicines but must
not be on steroids.
8. ky performance status must be 2 70.
9. Life ancy of at least 3 months.
10. Patients must be able to travel to St. Luke’s Medical Center for the PET
scans.
ll. Required ne laboratory data include:
Absolute neutrophil count
3 _ 9
2 mm [SI units 10 /L]
(ANC)
obin 2 8.0 gm/dL [SI units mmol/L]
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 starting 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 procedures to be followed, the
experimental nature of the therapy, alternatives, potential benefits, side effects, risks, and
discomforts.
13. Willing and able to comply with scheduled visits, treatment plan, and
laboratory tests.
The ce of any of the following will exclude a subject from study
enrollment
] 1. Concurrent therapy with any anticancer therapy including any other
igational 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
ive contraception, must be surgically sterile, or must be postmenopausal. Male subjects
must agree to use effective contraception or be surgically sterile. The definition of effective
contraception will be based on the nt of the Investigator or a designated associate. All at-
risk female subjects must have a negative ncy test within 7 days prior to the start of study
treatment.
4. Clinically significant cardiac e (New York Heart Association, Class III
or IV).
5. ia or altered mental status that would prohibit informed consent.
6. Other severe, acute, or chronic l or psychiatric condition or laboratory
ality that may increase the risk associated with study participation or study drug
stration or may interfere with the interpretation of study results and, in the judgment of
the pal 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. Patient must not be taking steroids at the time of screening.
Rationalefor the Starting Dose and Schedule
Reximmune-C has been dosed in 16 patients over a range of 1.0, 2.0 or 3.0 x1010
cfu (Dose Levels 1, II, 111 respectively on day 3 of the cycle). 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 nonserious adverse events occurred in 2 patients and both
were Grade 2. Four patients experienced serious adverse events, all of which were deemed not
related to the study drug. The trial was closed prior to determining the optimal 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 HSV-TK-ml study. Future dosing will proceed using total
viral particles (TVP)/ml which is a more accurate measure of titer than cfu per mL.
The le is based on the rationale that Reximmune-C2 re will not
uce all of the tumor cells. Therefore, patients will be dosed three times in a cycle over a
period of 5 days.
The time n 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 ial toxicity of
valganciclovir and the published observations that 5 days of lovir should be sufficient to
kill the majority of cells containing HSV-TK-m2, 5 days of therapy was chosen. Due to the
ial 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 ated antigens
(TAAs) that may appear during tumor cell sis.
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 distribution is primarily to the liver, toxicities will be carefully monitored
there and because of the implications, the bone marrow.
This clinical protocol calls for the administration of Reximmune-C2 via
intravenous on to patients with advanced malignancies, either primary hepatocellular or
tumors metastatic to the liver. There will be two parts: Phase IA (dose escalation 3 doses/week
every three weeks) and Phase IB (pre-screening after one dose of Reximmune-C2 and an
[18F]FHBG scan). If the PET scan is positive, the patient will ue 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 escalation will follow an accelerated titration design, incorporating
three ts per dose level until either one instance of DLT or two instances ofNCI-CTC
Grade 2 toxicities attributed to the study drug t nausea/vomiting, fatigue, anorexia,
alopecia or anemia) are observed. Thereafter, dosing in the clinical protocol will follow a
d Fibonacci schedule until dose-limiting toxicities are achieved.
Trial Design
This is a Phase 1, abel, 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 infilsion over 15-60 minutes. It is
pated that 33-70 patients will be d during the course of the study.
For Phase IA, the dose of Reximmune-C2 will be escalated from 11 TVP.
In the accelerated dose escalation phase, cohorts of three patients will be enrolled at each dose
level. The dose tion increment will be 100% until a DLT or two CTC Grade 2 or greater
toxicities are observed. When the accelerated dose escalation ends, the dose escalation for a
new patient in the standard dose escalation will follow a modified Fibonacci scheme (i.e., dose
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 une-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 fiarther 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 cokinetic 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, ng, and
safe disposal of biotherapeutic agents in an appropriate environment.
Reximmune C2 is a Moloney Murine replication incompetent retrovector particle
containing the genes encoding for a HSV-TK-m2 and hGM-CSF. The drug product contains
DMEM (low glucose), RD-Retrovector Particles, L-glutamine, Sodium pyruvate, human serum
albumin, n-butyric acid, Pulmozyme®, magnesium and other ents.
Drug product is available in one vial size: 30 mL type 1 clear glass vials with a
mm finish (containing 25 mL of 31 0 TVP). The vials are closed with 20 mm Teflon
coated serum stoppers and 20 mm flip-off lacquered flip tops.
Reximmune-C2 will be administered intravenously by lI‘lfiISlOIl pump over 15
minutes 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. s 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 red carefiJlly. Dosage adjustment is
required based on nine nce 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 ing
formula:
] 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.
Table I. Valganciclovir Dosing for Renally ed 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 e of the Phase 1 study is to establish the MTD, DLT, safety and a
RP2D of the investigational agent. T0Xic 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 response 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 Evaluable for response - All patients who have received at least a single cycle
of treatment and had tumor re-assessment will be considered ble for
response. In addition, those patients who develop early progressive 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 ive tumor
assessments made according to the Immune-Related Response Criteria (irRC) system of
evaluation and treatment decisions 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 se
Criteria vs RECIST 1.1 are as follows: (1) the ance of measurable umor ty may
take longer for immune therapies than for xic 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 cient” PD (e. g. small new s 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 Response
Criteria are listed below:
Table 11. Comparison ofWHO RECIST and Immune-Related Response 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 bute to defining Contribute to defining irCR
BOR of CR, PR, SD, and PD (complete earance
required)
CR Disappearance 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% se in SPD of all 2 50% decrease in tumor burden
index lesions compared with compared with baseline in two
baseline in two observations 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 non-index lesions
PD At least 25% se 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 baseline imaging-based tumor ments are to be performed within 14
days prior to the start of treatment. For the purposes of this study, all patients’ tumor
assessments should be re-evaluated starting 9 weeks after initiation of ent 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 ssion must have
progression confirmed no less than 6 weeks after the first documentation of progression.
The same method of assessment and the same technique should be used to
characterize each identified and reported lesion at baseline and during -up. Imaging-
based evaluation is preferred to evaluation by clinical examination when both methods have
been used to assess the antitumor effect of treatment. All ements 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 contiguous reconstruction algorithm. This applies to the
chest, abdomen, and pelvis.
Chest CT will used for assessment of pulmonary lesions.
Clinical s will only be considered measurable when they are superficial
(e. g., skin nodules, palpable lymph nodes). In the case of skin lesions, documentation 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
une-C2 in Phase IB. In Phase IA additional [18F]FHBG PET-CT scans can be obtained
in subsequent cycles at the discretion 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 se evaluation, e. g., visceral lesions. It is a possible
alternative to clinical ements of cial palpable nodes, SC lesions, and thyroid
nodules. Ultrasound might also be useful to confirm the complete disappearance of superficial
lesions usually assessed by clinical examination.
] Endoscopy, laparoscopy, and radionuclide scan should not be used for response
assessment.
All patients’ files and radiological images must be available for source
verification and may be submitted for extramural review for final assessment of mor
activity.
Measurability of Tumor s
At baseline, tumor lesions will be rized by the Investigator as measurable
or non-measurable by the criteria as bed 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 lesions will only be
considered measurable when they are superficial (e. g., skin nodules, palpable
lymph nodes).
0 asurable: All other lesions, 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 confirmed
and followed by imaging ques, cystic lesions, usly irradiated
lesions, and disease nted 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
] Recording Tumor Measurements
All measurable lesions up to a maximum of 10 lesions, representative of all
involved , 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 recorded as the baseline. The sum
of the longest diameters is to be used as reference to fithher 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 identified as non-target lesions
and should also be recorded at baseline. Measurements are not required and these lesions should
be ed as “present” or “absent.”
Definitions of Tumor Response
WO 53258
Immune-Related Response Criteria criteria will be followed for assessment of
tumor response.
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 Partial se (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 relative to nadir um ed tumor burden) confirmed
by a repeat, consecutive assessment no less than 6 weeks fiom the
date first documented lesions recorded since the treatment started, or
the appearance of one or more new lesions.
0 Stable Disease (irSD) is defined as not meeting the criteria for irCR or
irPR, in absence of irPD.
Non-Target Lesions for Solid Tumors
The cytological confirmation of the neoplastic origin of any effilsion that appears
or worsens during ent when the able tumor has met criteria for response or irSD is
mandatory to differentiate between response or irSD and irPD.
Confirmation of Tumor se
To be assigned a status of irPR or irCR, changes in tumor measurements in
patients with responding 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 interval of 6 weeks.
When both target and non-target lesions are present, individual assessments will be recorded
separately. The l ment of response will involve all parameters as depicted in Table
111.
The best overall response is the best response recorded from the start of the
treatment until disease progression/recurrence g 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 measurement and confirmation criteria.
Patients will be defined as being not evaluable (NE) for response if there is no
post-randomization gic assessment. These patients will be d as failures in the
is of tumor response data.
al Efficacy Assessment: Performance Status.
Patients will be graded according to the Kamofsky performance status scale.
Tumor Marker Response
] Method of Assessment
While not a fillly validated measure of y in many malignancies, serial
determinations of tumor markers may allow tion 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 decrease or se will not be assessed as an objective 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 censored at the last date of
follow-up.
Tumor response 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 confirmed documentation of
tumor progression or to death due to any cause. For patients who do not have objective
evidence of tumor progression and who are either removed from study treatment or are given
antitumor treatment other than the study treatment, TTP will be censored. A tumor marker
increase g criteria for tumor marker progression does not constitute adequate objective
evidence of tumor progression. However, such a tumor marker increase should prompt a repeat
radiographic evaluation to nt whether or not objective tumor progression has ed.
TTF is defined as the time from treatment to first ed documentation of
tumor progression, or to off-treatment date, or to death due to any cause, ver 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 awal 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 ts who are given antitumor treatment, other than the study treatment, before the
first of objective tumor progression, off-study date, or death. A tumor marker increase meeting
ia for tumor marker progression does not constitute adequate objective evidence of
treatment failure. However, such a tumor marker increase should prompt a repeat radiographic
2014/029814
tion 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 baseline or to death, due to any
cause, in the absence of previous documentation of definitive confimed performance status
worsening. For patients who do not have definitive performance status worsening and who are
either removed from study or are given antitumor 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 se from baseline was < 5% or to death due to any cause in the
absence of previous documentation of ive weight loss. For patients who do not have
ive 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 treatment, time to first
occurrence of new 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.
Treatment stration Assessment
For both Phase IA and 1B: dose intensity is defined as the total dose/cycle times
the number of weeks between 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.
Example 7: RxCZ-GCV Kill Assay
Kill assays were ted as follows. The percentage of cell kill by GCV after
treatment with RXC2 depends on the infectability (transducibility) of the cancer cells tested.
Cells for each cell line were plated in a 6 well dish. The following day, the cells were
uced with ector containg the EGFP (Enhanced Green Fluorescent Protein Gene)
diluted 1:5. After 48 hours cells were collected. The fluorescent and non-fluorescent cells were
d using an automated fluorescent cells counter to determine the percent uced. The
efficiency of transduction was examined using a virus ng the gene for Green fluorescent
protein where ransduction ncy is shown in decreasing order.
—«EGFP + cells <%>
BREAST Hs578T 66 --/- 5
BREAST HCC-38 59 +/- 2.3
SKIN A375 57.7 --/- 7.1
LUNG NCI-H460 25.9 +/— 1.7
LIVER SkHepl 21.4 --/— 4
The same viral preparation was used for all cell lines shown here (titer 2.72E+lO
TVP)
Example 8: is of Reximmune—C2 mediated GCV kill of cell lines expressing PiT-2
Cell lines expressing PiT-2 were established by transduction of target cells with a
E-Rex expression retroviral vector containing the PiT-2 and Neomycin Resistance genes. Stable
cell lines were then drug selected (G418) to establish a pure population of PiT-2 expressing
cells. The cell lines were verified by amphotropic retrovial vector transduction of the LUC-2
gene into PiT2 expressing cells followed by bioluminescent analysis. For Reximmune-C2 cell
kill is, PiT2 expressing cell lines were then plated in 48 well plates. The following day
cells were transduced with the Reximmune-C2 retrovector. After transduction, cells were
exposed to a daily dose of 20-40 uM GCV. After four days of GCV treatment the cells were
analyzed for cell ity using the PrestoBlue reagent. This reagent is a resazurin-based
solution that in the presence of the reducing nment of viable cells converts the t into
fluorescence that is quantitated using absorbance measurements.
Human colon cancer lines HCT-lS demonstrated poor HSV-TK-GCV kill and
RKO cell line demonstrated no cell kill following Reximmune-C2 transduction and GCV
exposure. PiT-2 expressing HCT-lS and RKO lines were ted and their transduction
efficiency examined; resutls are ed in the following table.
Cell Line EGFP+ cells (%)
PiT—2-CHO-K1 34 +/- 2.9
PiT—2-MIA-PaCa-2 78.6 +/- 2.2
PiT—2-HA-HCT-15 14.9 +/- 1.2
PiT—2-RKO 43.1 +/- 1.6
A considerable increase of LNCE-RVE transduction efficiency was observed in
all PiT-2 expressing cell lines demonstrating that the kill activity is increased when target cells
express PiT2.
Using cells lines expressing PiT-2, the data shows that the requirement of PiT-2
receptor presence for LNCE-RVE transduction is reflected by the level of EGFP expression in
cells ed by cent microscopy (data not shown). The requirement of PiT-2 presence
for Reximmune-C2 infectivity has also been shown by a GCV cell kill assay. Therefore, PiT-2
represents a good biomarker for Reximmune-C2.
It was determined that Pit2 expression correlated to Reximmune-C2 mediated
GCV cell kill. HSV-TK-GCV kill of CHO-Kl parent line versus PIT2 expressing CHO-Kl
lines.
Figures 25 and 26 e graph results -GCV kill after single or triple
transduction in various cell lines following single or triple transduction in the absence of PiT-2
(panel A of each figure) or presence of PiT-2 (panel B of each figure).
] Figure 27 es the results of TK-GCV kill after triple transduction with
Reximmune-C2 in a MIA-PaCa-2 human atic carconima cell line. GCV kill was ive
at the higher concentrations of TVP.
Figure 28 provides the results of HSV-TK-GCV kill after triple transduction of
PiTMIA-PaCa-2 cells with Reximmune-C2. GCV kill of RxC2-triple transduced PiTMIA-
PaCa2 human atic carconima cell line. The presence of PiT-2 dramatically increased the
amount of cell killing at lower concentrations of TVP.
Figure 32 illustrates a graph of RxC2-tranduced CHO-Kl cell lines after four
days in GCV.
Figure 33 rates a graph of RxC2-tranduced PiTHA-CHO-Kl cell lines
after four days in GCV.
It is very apparent that, even at the lowest concentration of GCV, the presence of
PiT-2 allows for significantly greater transduction and cell killing.
Example 9: Transduction ncy versus GCV Kill After Reximmune C2 triple
transduction
To demonstrate transduction efficiency and GCV kill, cells were plated into 48
well plates. The next day cells are transduced with Reximmune-C2 diluted in the range of l :40
to 1:5120. Following the last of three transductions, the cells were exposed a daily doses of
GCV (20-40 uM) for four days. One day following the last dose of GCV the cells were
analyzed using the Prestoblue reagent for cell viability. This reagent is a resazurin-based
on that in the presence of the reducing environment of viable cells converts the reagent into
fluorescence that is quantitated using absorbance measurements. The results are ed as
percent kill based on the non-transduced cell viability.
A549 16.3 --/- 1.5
Figure 31 is a graph depicting the percentage of GCV kill after Reximmune-C2
triple transduction of various cancer cell lines. The graph demonstrates the variation in GCV kill
amongst the different cell lines. The cell lines are comparable across each dilution converted to
the total virus les/mL against the percent cell kill. The table gives the transduction
efficiencies for the cell lines ented in the graph. The percent ncy does not seem to
have a direct correlation with the cell kill, but a trend is evident in which higher efficiency leads
to higher cell kill.
Exam le 10: Immunohistochemistr IHC of mutant HSV-TK cellular rotein ex ression
Either Reximmune Cl or C2 ds were transiently trasfected into 293T cells
and incubated under standard condition on tissue culture slides aparatus, a couple days later cells
were fixed with about 2% formalin, washed with PBS and bilized with 0.1% triton x 100
or equivalent detergent. Primary anti HSV-TK antibody (Santa Cruz Biotechnology) at effective
dilution is incubated with these cells 4 degrees C overnight. Cells are washed and incubated for
1-2 hours with secondary anti y antibody conjugated with horse radish peroxidase (
HRPO) at ambient room temperature. Cells are again washed and HRPO detection stain reagent
is applied for 5-30 minutes at room temperature. IHC images are acquired with a light
microscope fitted with a CCD l camera, pictures are ed with image analysis
re. Note: IHC in this example can also be described as ImmunoCyto Chemistry (ICC).
Wild type vector was found to localize to the nucleus ( As determined with
fluorescent genes fused to wild type HSV-TK), Data not shown..
RexCl distributes between nucleus and cytoplasm in fluorescent fusion (data not
shown), but mostly r in Immunohistochemistry (IHC; see, Figure 37, left panel).
ReXC2 is almost entirely cytoplasmic in fluorescent fusion (data not shown), with
some shift to the cytoplasm seen in IHC (see, Figure 37, right panel).
PCT/U82014/029814
Example 11: Improved s
The effect of various mutations were compared to previously disclosed constructs
such as those described by Margaret Black. Rescue of BL21 DE3 tk(-) Cells by HSV-TK
Variant pET Constructs is shown in the ing table:
ConcentrationTh midine m /mL
{onsti11m
W“ -----
(imNES
dmNES
The following table depicts GCV Kill after Rescue of BL21 DE3 tk(-) Cells by
HSV-TK Variant pET ucts.
Growth afie: 24 hr 3nwbaiion at 3?“;
m: pTK# = pET30a-based bacterial protein expression vector encoding an HSV-TK gene or
variant; pTKl = wild-type ; pTK2 = HSV-TK NESdmNLS Al67Y(SR39); pTK3 =
HSV-TK(SR39) (As in Reximmune-Cl); pTK4 = HSV-TK-NESdmNLS Al67F; pTKS = HSV-
TK-NESdmNLS Al68H ( As in Reximmune-CZ); pET24a = empty expression vector as
negative control; GCV = ganciclovir (at the indicated concentrations), IPTG = isopropyl b-D-l-
thiogalactopyranoside (as lac operon inducer for HSV-TK protein expression); 2xYT = 2x
yeast/tryptone bacterial media in agar plates, where the trials in the column so labeled lack both
IPTG and GCV. All of these HSV-TK’s are codon optimized for expression in prokaryotes and
expressed in the IPTG inducible pET30a plasmid. Note; HSV-TK Mutants which do not have
Thymidine enzymatic ty will not support the growth of these TK minus bacterial cells.
e 12: In vitro Bystander Assays
] Experiments were conducted at our tory to demonstrate the bystander
effect in vitro on mixtures of cancer cells expressing various TK mutants with non-expressing
cells. A375 human melanoma and C6 rat glioma stable pure tion cell lines were
established containing the A168H mutated HSV-TK-m2 gene. The bystander assays were
conducted by plating the cancer cells with mixtures of the parental non-HSV-TK-m2 cells with
the corresponding HSV-TK-m2 cell line ranging from 0-lOO% HSV-TK-m2. The mixtures of
cancer cells were subsequently exposed to 5-20 uM GCV and cell kill is plotted in the figures
below. The results clearly show significant increases in the mixed populations over what would
be considered tical, t a bystander effect.
More than 40 bystander assays were performed using different mutant TK and
clonal populations. The data was compiled with GCV sensitivity and enzyme kinetic
measurements as well as viral titer of production for the mutants with potential.
Figure 29 provides graphic results from one bystander in vitro assay for various
mutants. The data support that mutated HSV-TK Al68H gene has a higher cell kill and
bystander effect than the HSV-TK 167 or Margaret Black mutants.
Figure 30 provides a c from a bystander in vitro assay where C6-Hygro-TK
clones were treated with 20 mM GCV. The data further support that HSV-TK Margaret Black
mutants had the lowest cell kill of the other mutants tested.
Analysis of all of the mutants identified mutant TKl68dmNES to be a lead
candidate for the TK gene in une C-2.
Example 13: Seguences of modified TK molecules
HSV-TK Splice Sites Removal; optimized TKl [salice sites ted]
ATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAG
CCGCGGCCACAGCAACGGCAGCACCGCCCTGCGCCCCCGCCGCCAGCAGGAGGCCA
CCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGC
CCCCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCG
CGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCA
GCGAGACCATCGCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATC
AGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATCACCATGGGCATGCC
CTACGCCGTGACCGACGCCGTGCTGGCCCCCCACATCGGCGGCGAGGCCGGCAGCA
GCCACGCCCCCCCCCCCGCCCTGACCATCTTCCTGGACCGCCACCCCATCGCCTTCA
TGCTGTGCTACCCCGCCGCCCGCTACCTGATGGGCAGCATGACaCCaCAaGCCGTGCT
GGCCTTCGTGGCCCTGATCCCCCCCACCCTGCCCGGCACCAACATCGTGCTGGGCGC
CCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGC
GCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAAC
CGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAG
CGGCACCGCCGTGCCCCCCCAGGGCGCCGAGCCCCAGAGCAACGCCGGCCCCCGCC
CCCACATCGGCGACACCCTGTTCACCCTGTTCCGCGCCCCCGAGCTGCTGGCCCCCA
ACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTG
CGCAGCATGCACGTGTTCATCCTGGACTACGACCAGAGCCCCGCCGGCTGCCGCGA
CGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACCACCCCCGGCA
GCATCCCCACCATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCC
AACTAA
] Codon-optimized, all putative splice acceptor sites ablateda TK1 with
RE’s,+Kozaka 2XTK A168H gLIF. . .AHL!
gKaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGC
CAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCM:
TIKKQCCaCGgCGCCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCC
ACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACC
CAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCAT
GACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCC
AGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACC
AGCGCCCAGAJtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCa
CACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCMXhCCMXhCTGACCCTGATC
TTCGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGG
GAfihCCBCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCG
GCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCC
AAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCG
CGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGC
GCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCthCAGGGCGCCGAGCCa
CAGAGCAACGCCGGaCCaCGaCCMQACATCGGCGACACCCTGTTCACCCTGTTCCGgG
CaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGG
TGGCCAAGCGCCTGCGCumATGCACGTGTTCATCCTGGACTACGACCAGga
CCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCA
CGTGACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGC
GAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTga:
] HSV-TK Splice Sites Removal Improves Codon Optimization
gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGC
CAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaC
TGCGgCCaCGgCGCCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCC
ACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACC
CAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCAT
GACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCC
AGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACC
AGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaC
ACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTT
QGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCt
ccATGACaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCA
CCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAG
CGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGT
GTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCG
AGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAG
AGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCC
aGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGT
GCTGGCCAAGCGCCTGCGthcATGCACGTGTTCATCCTGGACTACGACCAGtcaCCgG
@GGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTG
ACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGA
TGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTgtca
HSV-TK NLS Removal and tute in NES
gtcaGCGGCCGCACCGGTACGCGTCCACCflGCCAGCTACCCCGGCCA
CCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCA
GCACCGCaCTGCGgCCaCGgCGCCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGA
AGATGCCCACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCA
CCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCC
GAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTA
CACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGG
TGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGC
TGGCaCCaCACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGAC
CTTCGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACC
TGATGGGthcATGACaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaC
TGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGC
CTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCAT
CGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCA
GCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCC
GAGCCaCAGAGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGT
TCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGC
CCTGGACGTGCTGGCCAAGCGCCTGCGthcATGCACGTGTTCATCCTGGACTACGAC
CAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAG
ACCCACGTGACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCG
CCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTgtca
HSV-TK NLS Removal
gtcaGCGGCCGCACCGGTACGCGTCCACCflGCCAGCTACCCCGGCCACCAGCACGC
CAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaC
TGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCC
ACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACC
CAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCAT
GACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCC
AGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACC
AGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaC
ACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTT
QGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCt
ccATGACaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCA
CCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAG
CGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGT
GTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCG
AGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAG
AGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCC
aGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGT
GCTGGCCAAGCGCCTGCGthcATGCACGTGTTCATCCTGGACTACGACCAGtcaCCgG
EGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTG
CCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGA
TGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTgtca
HSV-TK Custom Codon Optimization
gtcaGCGGCCGCACCGGTACGCGTCCACCflGCCCTGCAGAAAAAGCTGGAAGAGC
TGGAACTGGATGGCTCTTATCCT
GGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGACATTCTAAT
GGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG
AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGA
ACCACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTA
TGTGCCTGAACCTATGACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTA
ATATCTATACAACACAGCATAGACTGGATCAAGGAGAAATTTCTGCCGGAGATGCT
GCCGTGGTGATGACATCTGCTCAGATTACAATGGGAATGCCTTATGCTGTGACAGAT
GCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGCTCTCATGCACCACCACC
AGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGTGTTATCCGGC
AGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGCTC
TGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGAT
AGACATATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGC
CATGCTGGCTGCCATTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCT
GCAGTGTGGAGGCTCTTGGAGAGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGC
CACCACAGGGAGCCGAACCACAGAGCAATGCTGGACCACGACCACATATCGGAGA
CACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGGCACCAAATGGAGACCTGT
ACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAGATCTATGCATG
TGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTGCAGC
CTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATC
TGTGATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATAGGGATC
CCTCGAGAAGCTTgwa
HSV-TK NLS Removal NES and Addition
gKmGCGGCCGCACCGGTACGCGTCCACCAIQGCCCTGCAGAAAAAGCTGGAAGAGC
TGGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCC
GCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCflHKKXhfiKhGGATCTCAGCAG
GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACAT
CGACGGflXhCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGG
GCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTG
GGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCCTGGACCAaGG
CGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATUKhATGGG
CATGCCCTACGCCGTGACCGACGCCGTGCTGGCflXhCACATCGGCGGCGAGGCCGG
CAGCAGCCALIKhCCkaKKhGCaCTGACCCTGATCTTCGACCGgCACCCaATCGCaC
ACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCKmATGACaCCaCAaGCCGTGC
TGGCCTTCGTGGCCCTGATCCCflXhAfihCTGCCCGGCACCAACATCGTGCTGGGCGC
CCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGC
GCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAAC
CGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAG
CGGCACCGCCGTGCCflXhCAGGGCGCCGA£KKhCAGAGCAACGCCGGflXhCGflXh
CACATCGGCGACACCCTGTTCACCCTGTHXXQGCflXhGAGCTGCTGGCflXhAACG
GCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCGCt
WATGCACGTGTTCATCCTGGACTACGACCAGwflXgGCCGGCTGCCGCGACGCCCTG
CTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACMUhCCCGGCAGCATCCCa
ACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATA
GGGATCCCTCGAGAAGCTTgwa
HSV-TK Custom Codon zation
gKmGCGGCCGCACCGGTACGCGTCCACCAIQGCCCTGCAGAAAAAGCTGGAAGAGC
TGGAACTGGATGGCTCTTATCCT
GGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGACATTCTAAT
ACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG
AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGA
AAAACAACCACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTA
TGTGCCTGAACCTATGACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTA
ATATCTATACAACACAGCATAGACTGGATCAAGGAGAAATTTCTGCCGGAGATGCT
GCCGTGGTGATGACATCTGCTCAGATTACAATGGGAATGCCTTATGCTGTGACAGAT
GCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGCTCTCATGCACCACCACC
AGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGTGTTATCCGGC
AGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGCTC
TGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGAT
AGACATATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGC
CATGCTGGCTGCCATTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCT
GCAGTGTGGAGGCTCTTGGAGAGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGC
CACCACAGGGAGCCGAACCACAGAGCAATGCTGGACCACGACCACATATCGGAGA
CACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGGCACCAAATGGAGACCTGT
ACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAGATCTATGCATG
TGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTGCAGC
TGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATC
TGTGATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATAGGGATC
CCTCGAGAAGCTTgwa
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Example 14: HAT Assay
Retroviral Vectors of RexRed Super TK Al68H and RexRed TK 167F were
produced in 293T cells and used to transduce 3T3 (TK-) cells. These transduced cells were HAT
selected for 7-14 days. Untransduced 3T3 (TK-) cells will die post HAT selection. These same
cells transduced with RexRed Super TK Al68H did survive HAT selection, however 3T3(TK-)
cells transduced with RexRed TK 167F did not survive HAT selction. This is a plus/ minus cell
survival assay, surviving cells are fixed and stained with 1% methylene blue in methanol.
Previous transduction based HAT cell kill assays reveal a GCV specificity over
thymidine for the Al67F HSV-TK mutants in retroviral vectors ning the RFP marker.
That specificity is found in NIH 3T3 cells in a 72 hour and 7 day assay at lx HAT dose.
Current transduction based HAT cell kill assays reveal a GCV specificity over
thymidine for the Al67F HSV-TK s in retroviral s containing the RFP marker.
That specificity is found in NIH 3T3 cells in a 7 day assay at 2x HAT dose.
Transduction based HAT cell kill assays reveal a GCV specificity over thymidine
for the Al67F HSV-TK mutants in retroviral vectors containing the RFP marker. That
specificity is found in NIH 3T3 cells in a 72 hour assay and 7 day assay at lx HAT dose.
] Transduction based HAT cell kill assays reveal a GCV specificity over thymidine
for the Al67F HSV-TK mutants in retroviral vectors containing the HygroR marker. That
specificity is found in NIH 3T3 cells in a 72 hour and 7 day assay at lx HAT dose.
Example 15: GCV Kill Assay
Cells were seeded in a 24 well dish. Cells were transduced the next day with 6
ons of the retroviral vectors (1:4-4096). The next day 0-200 uM GCV was added to the
cells. After seven days of GCV treatment the cells were fixed and the live cells stain with 1%
methylene blue in methanol. The higher the potency of the viral mutants leads to more cell kill.
Previous transduction based HSV-TK/GCV cell kill assays reveal a y order
for Al68F, Al67F and Al68H HSV-TK mutants in retroviral s containing the RFP
. That order is Al68H > Al68F = Al67F when tested in RgA375 cells in a 72 hour and 7
day assay at high GCV dose ( 1 mM — 125 mM).
Current transduction based HSV-TK/GCV cell kill assays reveal a potency order
for Al67F and Al68H HSV-TK mutants in iral vectors containing the RFP marker. That
order is Al68H > Al67F when tested in RgA375 cells in a 7 day assay at high GCV dose
(0.2mM — 0.05mM). The addition of dm NLS or NES does not appear to change this order. The
use of JCO does appear to lower titer and aggregate HSV-TK cell kill activity.
Transduction based HSV-TK/GCV cell kill assays reveal a potency order for
Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the RFP marker.
That order is Al68H > Al68F = Al67F when tested in A375 and RgA375 cells in a 72 hour
assay at high GCV dose ( 1 mM — 125 mM).
Transduction based /GCV cell kill assays reveal a potency order for
Al68F, Al67F and Al68H HSV-TK mutants in retroviral s containing the RFP marker.
That order is Al68H > Al68F = Al67F when tested in NIH 3T3 cells in a 72 hour assay at high
GCV dose ( 1 mM — 500 mM).
Transduction based HSV-TK/GCV cell kill assays reveal a potency order for
Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the RFP marker.
That order is Al68H > Al68F = Al67F when tested in RgA375 cells in a 72 hour and 7 day
assay at high GCV dose ( 1 mM — 125 mM).
Transduction based HSV-TK/GCV cell kill assays reveal a potency order for
Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the RFP or
HygroR marker. That order is Al68H > Al68F = Al67F when tested in A375, RgA375 or NIH
3T3 cells in a 72 hour assay at high GCV dose ( 1 mM — 125 mM).
Transduction based HSV-TK/GCV cell kill assays reveal a potency order for
Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the HygroR
marker. That order is Al68H > Al68F = Al67F when tested in A375 and RgA375 cells in a 72
hour assay at high GCV dose ( 1 mM — 125 mM).
] Transduction based HSV-TK/GCV cell kill assays reveal a potency order for
Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the HygroR
marker. That order is Al68H > Al68F = Al67F when tested in NIH 3T3 cells in a 72 hour assay
at high GCV dose ( 1 mM — 500 mM).
Example 16: Hygro Resistance
[0051 1] Cell lines transduced with retrovector Hygro-HSV-TK mutants were selected in
the ce of ycin to e a pure population of cells containg the Hygro-HSV-TK
mutants and expressing the hygromycin resistence gene.
A375 une-C2 like Cell lines: A375 Hygro selected HSV-TK
dmNESAl68H cell lines have been converted to Luc(+). The above cell line has same GCV kill
as parental line. A A375 Luc(+) only cell line has same Luc activity as above cell line.
C6 Reximmune-C2 like Cell lines: C6 Hygro selected HSV-TK dmNESAl68H
cell lines have been converted to Luc(+). The above cell line has same GCV kill as parental line.
A C6 Luc(+) only cell line has same Luc activity as above cell line.
2014/029814
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 d 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 s and structures within the scope of these claims and their equivalent be covered
thereby.
2014/029814
ABBREVIATIONS
ALT Alanine aminotransferase
ANC Absolute neutrophil count
AST ate aminotransferase
AUC Area under the plasma concentration-time curve
BSA Body surface area (mg/m2)
CL Systemic plasma clearance
Cmax Peak plasma concentration
CR Complete response
CRF Case report form
CT Computerized tomography
CTC Common Toxicity Criteria
DLT Dose Limiting Toxicities
EOI End of infusion
FDA Food and Drug Administration
G-CSF Granulocyte-colony stimulating factor (filgrastim, Neupogen®)
GCP Good al practice
GM-CSF ocyte-macrophage colony-stimulating factor (sargramostim,
Leukine®)
HIV Human Immunodeficiency Virus
HR Hazard ratio
IEC Independent Ethics Committee
i.p. Intraperitoneal
IRB Institutional Review Board
IV Intravenous, intravenously
LD10 or Dose that is lethal to 10% or 50% of animals
LDso
LDH Lactate dehydrogenase
MAD Maximum Administered Dose
MRI Magnetic resonance imaging
MTD Maximum tolerated dose
NCI National Cancer ute
NE Not evaluable for tumor response
NOAEL No Observed Adverse Effect Level
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Non-CR Non-complete response
Non-PD Non-progressive disease
PBMC Peripheral Blood Mononuclear Cells
PCE Propylene : Cremophor® EL: Ethanol
PD Progressive disease
PR Partial response
SAER-S Serious Adverse Event Report-Study
aneous, subcutaneously
Stable disease
Dose that is severely toxic to 10% of animals
Time to Progression
Time to Failure
Half-life
Time ofmaximum plasma concentration
Steady state volume of distribution
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-lO2-
SE UENCES
SEQ ID NO: 1: wild type HSVl-TK nucleotide sequence
atggcttcgtaccccggccatcaacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagcaaccgacg
tacggcgttgcgccctcgccggcagcaagaagccacggaagtccgcccggagcagaaaatgcccacgctactgcggg
tttatatagacggtccccacgggatggggaaaaccaccaccacgcaactgctggtggccctgggttcgcgcgacgat
atcgtctacgtacccgagccgatgacttactggcgggtgctgggggcttccgagacaatcgcgaacatctacaccac
acaacaccgcctcgaccagggtgagatatcggccggggacgcggcggtggtaatgacaagcgcccagataacaatgg
gcatgccttatgccgtgaccgacgccgttctggctcctcatatcgggggggaggctgggagctcacatgccccgccc
ccggccctcaccctcatcttcgaccgccatcccatcgccgccctcctgtgctacccggccgcgcggtaccttatggg
cagcatgaccccccaggccgtgctggcgttcgtggccctcatcccgccgaccttgcccggcaccaacatcgtgcttg
gggcccttccggaggacagacacatcgaccgcctggccaaacgccagcgccccggcgagcggctggacctggctatg
ctggctgcgattcgccgcgtttacgggctacttgccaatacggtgcggtatctgcagtgcggcgggtcgtggcggga
ggactggggacagctttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggcccacgacccc
atatcggggacacgttatttaccctgtttcgggcccccgagttgctggcccccaacggcgacctgtataacgtgttt
gcctgggccttggacgtcttggccaaacgcctccgttccatgcacgtctttatcctggattacgaccaatcgcccgc
cggctgccgggacgccctgctgcaacttacctccgggatggtccagacccacgtcaccacccccggctccataccga
cgatatgcgacctggcgcgcacgtttgcccgggagatgggggaggctaactga
SEQ ID NO: 2: wild type HSVl-TK amino acid ce
MASYPGHQ{ASAFDQAARSRGHSNRRTALR?RRQQnATnVRPnQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDD
< ?EPMTYWRVLGASETIANIYTTQHR.DQGEISAGDAAVVMTSAQITMG ?YAVTDAVLAPHIGGEAGSSHAPPPAL
H SI
W DRHPIAASLCYPAARYLMGSMTPQAVLAFVASIP?TLPGTNIVLGA.P _*J DRHIDRDAKRQRPG .RLDLAM.AAIRR*J
<._<G
bSANTVRYSQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDT.FT.FRAP?.LAPNGDLYVVFAWASDVLAK
SMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTEARnMGnAV
SEQ ID NO: 3: HSV-TK in Reximmune-C HSV-TK; SR 39 mutant and 26S
Mutation of NLS
atggcctcgtaccccggcca:caacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagca
acggatccacggcg,,gcgccc:cgccggcagcaagaagccacggaag:ccgcccggagcagaaaatgcc
cacgc,ac,gcggg,,La,a,agacgg:ccccacgggatggggaaaaccaccaccacgcaacLgc,gg,g
gcchggg,,cgcgcgacga,a,chc,achacccgagccga,gacL,achgcgggtgctgggggce,
ccgagacaatcgcgaaca,c,acaccacacaacaccgcctcgaccagggtgagatatcggccggggacgc
ggcggegg,aa,gacaagcgcccagataacaatgggca,gcc,,a,gccgLgaccgacgccgttctggc:
cceca,a,cgggggggaggc:gggagctcaca:gccccgcccccggccc:caccatcttcctcgaccgcc
chcc,LcaLchg,chacccggccgcgcgg,acc,,a,gggcagca:gaccccccaggccg:
gceggcg,ch,ggccctca:cccgccgacce,gcccggcaccaacach,ch,ggggccc:tccggag
gacagacacatcgaccgcctggccaaacgccagcgccccggcgagcggctggacctggc:atgctggctg
cgattcgccgcgtttacgggc,ac,,gccaaeacgngcggLaLchcag,gcggcggg,cg,ggcggga
ggac:ggggacagc:ttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggccca
cgaccccatatcggggacacg,La,,Laccceg,Lchggcccccgag,,gceggcccccaacggcgacc
LgLa,aachgLL,gccnggccttggacgtcttggccaaacgcctccgttccatgcacgLcEL,aLch
ggat:acgaccaatcgcccgccggc:gccgggacgcccLgcLgcaacL,acceccgggatggtccagacc
cacg:caccacccccggctccataccgacgatatgcgacctggcgcgcacgtttgcccgggagatggggg
actga
SEQ ID NO: 4 (amino acid sequence encoded by SEQ ID NO: 3)
MASYPGHQHASAFDQAARSRGHSNGSTALRPRRQQnATnVRPnQKMPTLLRVY:DGPHGMGKTTTTQL;V
D VYVPnPMTYWRVLGASnT AN YTTQHRLDQGn SAGDAAVVMTSAQITMGMPYAVTDAVLA
VV()2014/153258 PCT/L S2014/029814
PH GG flAGSSHAPPPA' J hLDRiP AhMLCYPAARYLMGSMTPQAV' .AFVAL" PPT' .PGTN"VLGALP:‘J
DRH: DRLAKRQRPGTRH DLA VYGLIJANTVRYLQCGGSWRE.|. DWGQLSGTAVPPQGAEPQSNAGP
RPH G DTLhTLh?AP*CF .AP GIJYNVFAWA' 'Q" .RSMHVF'". DYDQSPAGCR DALLQLTSGMVQT
{VTTPGS PT CDLARTEARLMGLAN
SEQ ID NO: 5: HSV-TK Sites to mutate are in bold, underlining K nuclear
localization sequence, RR, and Substrate Binding Domain, LIF and AAL
atggcctcgtaccccggccatcaacacgcgtc:gcgttcgaccaggctgcgcgttctcgc 60
M A S Y P G H Q i A S A F D Q A A R S R
ggccatagcaaccgacgtacggcg, ,gcgccthgccggcagcaagaagccacggaag:c L20
G H S NBBT A .4 A P 5 5 Q Q :3 A T :3 v
cgcccggagcagaaaatgcccacgc ,acegcggg,LLa,aLagacggtccccacgggatg L80
R P '-T‘
.L Q K M P T H H R V Y i D G P H G M
accaccaccacgcaacLgc ,gg,ggccc,ggg,ecgcgcgacgatatcgtctac 240
G K T T T T Q "
. H V A L G S R D D V Y
gtacccgagccgatgacttac:ggcggg :gctgggggc,,ccgagacaatcgcgaacatc 300
V P r
L P M T Y W R V L G A S E T A N
tacaccacacaacaccgcctcgaccagggtgagatatcggccggggacgcggcggegg,a 360
Y T T Q H R L D Q G A S A G D A A V V
atgacaagcgcccagataacaatgggcatgcc:tatgccgtgaccgacgccgttc:ggct
M T S A Q T M G M P Y A V T D A V L A
cctcatatcgggggggaggctgggagctcaca:gccccgcccccggccctcaccctcatc
P i " G G E A G S S H A P P P A L T E E
EEggaccgccatcccatcgccgccctcc:gtgctacccggccgcgcgg,acc,Langgc 540
E 3 R H P A A E :4 C Y P A A R Y :4 M G
agcatgaccccccaggcchgc,ggcg,,chggccctcatcccgccgacc,egcccggc 600
S T P Q A V L A F V A L P P T L P G
accaacatcgtgcttggggccCttccggaggacagacaca:cgaccgcctggccaaacgc 660
T " V L G A L P E D R H D R L A < R
cagcgccccggcgagcggctggacc,ggcLaLchgchgcgaLchccgcgtttacggg 720
Q R P G T. R '. D L A M L A A I R R V Y G
cLac,egccaaLacgngcggeaLc,gcagLgcggcgggtcgtggcgggaggaCtgggga 780
L L A N T V R Y L Q C G G S W R E D W G
cagc,,Lcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggccca 840
Q L S G T A V P P Q G A E P Q S A G P
cgaccccatatcggggacacge,aLLLacchg,eLcgggcccccgagttgctggccccc 900
R P i G D T L F T L F R A P H H H A P
gacc,g,a,aacg,ge,,gchgggcc,eggacgtCttggccaaacgcctccgt 960
G D L Y N V F A W A L D V L A < R L R
:ccatgcacgec,L,aLcc,gga,Lacgaccaa:cgcccgccggctgccgggacgccctg 1020
S M i V F L D Y D Q S P A G C R D A L
c,gcaacLLacc,ccggga,gg,ccagacccacg:caccacccccggc:ccataccgacg 1080
L Q L T S G M V Q T i V T T P G S P T
atatgcgacctggcgcgcacgt:tgcccgggaga:gggggaggctaac:ga
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-
—104—
2014/029814
-EEC§T§CTGTGCTACCCGGCCGCGCGGTACE (SfiQ 3 NO: 6)
—A_AGEAEGACACGATGGGCCGGCGCGCCATGG (S *1Q 3 NO: 7)
Kpn "
IIIIIIIIIII - 3’
IIIII "III
- 3’
GAGCTC IIIIIIIIIII
IIIIIIIIIII
SEQ ID NOS: 8 and 9: Sac I-Kpn I (SR39) mutant region (cut)
CACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCCEECATE
GTACGGGGCGGGGGCCGGGAGTGGEAGQAGEAGCTGGCGGTAGGGTAGCGGAE
Sac I (cut)
CTGTGCTACCCGGCCGCGCGGTAC (SfiQ 3 NO: 8)
GEAEGACACGATGGGCCGGC (SfiQ 3 NO: 9)
Kpn I(Cut)
GGTACC IIIIIIIIIII GTAC - 3’
IIIIIIIIIII
SEQ ID NOS: 10 and 11: Primers
SR39sackpn Fl
'CACATGCCCCGCCCCCGGCCCTCACCETCETCETCGACCGCCATCCCATCGCCTTCATGCTGTGCTAC
CCGGCCGCGCGGTAC 3’ (SfiQ 3 NO: 10)
SR39sackpn R1
’CGCGCGGCCGGGTAGCACAGCATGAAGGCGATGGGATGGCGGTCGAEGAAGAEGGTGAGGGCCGGGGG
ATGTGAGCT 3' (SfiQ 3 NO: 11)
SEQ ID NO: 12 Gene #3 mHSV-TK CO A168H(LIF...AHL): Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
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): :1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
GACCGGCACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
AAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
GACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
GGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 14 Gene #5 mHSV-TK CO dual mutant A167F-A168H (LIF...FHL):
Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
GACCGGCACCCAATCTTCCACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
TGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
TTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
GGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 15 Gene #6 mHSV-TK CO MB-IFL A168H(IFL...AHL): Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
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
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
CACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
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
GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
CACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
GACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
TTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
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
CTCCATGCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG
CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG
ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT
TGTCA
2014/029814
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
CGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA
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
GGGAATGCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC
TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGT
GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC
TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT
ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA
TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG
AGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT
GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG
CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG
GCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG
CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG
ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT
TGTCA
SEQ ID NO: 21 Gene #6 HSV-TK A167F NESdmNLS JCO SC: Length:1221
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT
GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA
AATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG
AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC
CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG
ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC
AAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT
GGGAATGCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC
TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTTTCGCACTGCTGT
GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC
TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT
ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA
TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG
AGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT
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
GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
aCCaCACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTC
CACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtccATGA
CaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
aCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTC
ACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCtccATGCACGTGTTCATCCTGGACTACGACCAGtc
aCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACa
ACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTgtca
SEQ ID NO: 23 — MAP Kinase Kinase Nuclear Export Polynucleotide Sequence
-H0-
2014/029814
C T GCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC
SEQ ID NO: 24 MAP Kinase Kinase Nuclear Export Polypeptide Sequence
T.QKKT. HT. *iT .DG
Claims (28)
1. A polynucleotide encoding a mutated form of thymidine kinase from a human herpes simplex virus type 1 (HSV1-TK) and at least one pro-apoptotic agent, wherein the encoded HSV1-TK comprises at least two mutated amino acids in a nuclear localization sequence (NLS) at positions corresponding to amino acid residue 25, 26, 32, or 33 of SEQ ID NO:2, and wherein the mutated HSV1-TK increases cell kill activity relative to a wild-type HSV1-TK.
2. The polynucleotide according to claim 1, wherein the at least two mutated amino acids in the NLS comprises amino acid es at positions 32 and 33 of SEQ ID NO:2, wherein each amino acid residue is independently mutated to an amino acid chosen from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid.
3. The polynucleotide according to claim 1, wherein the at least two mutated amino acids in the NLS comprises amino acid residues at positions 25 and 26 of SEQ ID NO:2, wherein each amino residue is independently mutated to an amino acid chosen from the group consisting of: glycine, serine, cysteine, glutamic acid, and aspartic acid.
4. The polynucleotide according to claim 1, wherein the encoded mutated HSV1-TK further comprises a mutation at amino acid residue positions 167 or 168 of SEQ ID NO:2, or a combination thereof, to a polar, non-polar, acidic, or basic amino acid.
5. The polynucleotide according to claim 1, wherein the encoded d HSV1-TK further ses a mutation at amino acid residue on 167 of SEQ ID NO:2, to a polar, nonpolar , basic, or acidic amino acid.
6. The polynucleotide according to claim 1, wherein the encoded mutated K further ses a on at amino acid residue position 167 of SEQ ID NO: 2, to an amino acid ed from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.
7. The cleotide according to claim 1, wherein the encoded mutated HSV1-TK r comprises a mutation at amino acid residue position 168 of SEQ ID NO:2, to a polar, nonpolar , basic, or acidic amino acid.
8. The polynucleotide according to claim 1, wherein the encoded mutated HSV1-TK further comprises a mutation at amino acid residue position 168 of SEQ ID NO:2, to an amino acid ed from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.
9. The polynucleotide according to claim 1, wherein the encoded mutated K further comprises a nuclear export signal sequence.
10. The polynucleotide according to claim 9, n the nuclear export signal sequence is LQKKLEELELDG (SEQ ID NO: 24).
11. The polynucleotide according to claim 1, n the at least one optotic agent is a cancer suppressor.
12. The polynucleotide according to claim 11, wherein the cancer suppressor is p53 or Rb.
13. The polynucleotide according to claim 1, wherein the at least one pro-apoptotic agent is selected from the group consisting of p15, p16, and p21/WAF-1.
14. The polynucleotide according to claim 1, wherein the at least one optotic agent is selected from the group consisting of Bax, Bad, Bik, Bak, Bim, cytochrome C, apoptosisinducing factor (AIF), Puma, CT 10-regulated kinase (CRK), Bok, glyceraldehydephosphate dehydrogenase, Prostate Apoptosis Response Protein-4 (Par-4), Smac, Kinase C6, Fas, inhibitory PAS domain protein (IPAS), and Hrk.
15. A retroviral vector comprising the polynucleotide of claim 1 encoding the mutated modified HSV1-TK and the at least one pro-apoptotic agent.
16. The retroviral vector of claim 15, further sing a polynucleotide encoding for a PiT-2 or PiT-1 polypeptide.
17. The retroviral vector of claim 15, further sing a polynucleotide encoding for a targeting ptide.
18. The retroviral vector of claim 17, wherein the targeting polypeptide binds to an extracellular protein.
19. Use of a retroviral vector of claim 15 in the manufacture of a medicament for treating cancer in a patient in need thereof, wherein the retroviral vector is formulated to be administered to the t prior to the patient being administered a nucleoside analogue or a prodrug thereof.
20. The use of claim 19, wherein the iral vector is formulated to be administered intravenously, uscularly, subcutaneously, intra-arterially, intra-hepatic arterially, intrathecally , intra-peritoneally and/or intra-tumorally.
21. The use of claim 19, n a cell of interest is uced with the retroviral vector in vitro, and is formulated to be administered to the patient in need thereof.
22. The use of claim 19, wherein at least 1 x 109 TVP of the retroviral vector is formulated to be administered cumulatively to the patient in need thereof.
23. The use of claim 19, wherein the prodrug is administered between about 1-2 days after administration of the retroviral vector.
24. The use of claim 19, wherein the nucleoside analogue is ganciclovir.
25. Use of retroviral vector of claim 15 in the manufacture of a medicament for inducing cell kill activity in a cancer cell of interest, wherein: a. the cancer cell is ted with the medicament; b. the cancer cell is transduced with the polynucleotide encoding the modified HSV1-TK; c. the cancer cell is contacted with a nucleoside ue or a prodrug thereof, thereby inducing cell kill activity in vivo.
26. The use of claim 25, wherein the nucleoside analogue is ganciclovir.
27. The use of claim 25, wherein the cancer cell of interest is transduced in vivo in a t in need thereof.
28. The use of claim 25, n the cancer cell in step (b) is transduced in vitro, and subsequently administered to a patient in need thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361784901P | 2013-03-14 | 2013-03-14 | |
US61/784,901 | 2013-03-14 | ||
NZ712210A NZ712210B2 (en) | 2013-03-14 | 2014-03-14 | Improved thymidine kinase gene |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ751656A NZ751656A (en) | 2021-07-30 |
NZ751656B2 true NZ751656B2 (en) | 2021-11-02 |
Family
ID=
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