Adeno-associated virus vectors encoding modified G6PC and uses thereof

FIELD

This disclosure concerns gene therapy vectors encoding a modified glucose-6-phosphatase-.alpha. (G6PC) enzyme with increased activity and its use, such as for the treatment of glycogen storage disease (GSD) and complications associated with GSD.

BACKGROUND

Glycogen storage disease type Ia (GSD-Ia or von Gierke disease, MIM232200) is caused by a deficiency in glucose-6-phosphatase-.alpha. (G6Pase-.alpha. or G6PC), an enzyme that is expressed primarily in the liver, kidney, and intestine (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). G6Pase-.alpha., encoded by the G6PC gene, is a hydrophobic protein anchored in the endoplasmic reticulum (ER) by nine transmembrane helices (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). This enzyme catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the terminal step of glycogenolysis and gluconeogenesis. Patients affected by GSD-Ia are unable to maintain glucose homeostasis and present with fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic academia (Chou et al., Nat Rev Endocrinol 6:676-688, 2010).

There is currently no cure for GSD-Ia. Hypoglycemia can be managed using dietary therapies (Greene et al., N Engl J Med 294:423-425, 1976; Chen et al., N Engl J Med 310:171-175, 1984) that enable patients to attain near normal growth and pubertal development. However, the longer term clinical complications, and their underlying pathological processes, remain uncorrected. One of the most significant chronic risks is hepatocellular adenoma (HCA), that develops in 70-80% of GSD-I patients over 25 years old (Chou et al., Nat Rev Endocrinol 6:676-688, 2010; Labrune et al., J Pediatr Gastroenterol Nutr 24:276-279, 1997; Rake et al., Eur J Pediatr 161(Suppl 1):520-S34, 2002). HCAs in GSD-Ia patients are small, multiple, and nonencapsulated, with complications including local compression and intratumoral hemorrhage. In 10% of GSD-Ia patients, HCAs undergo malignant transformation to hepatocellular carcinoma (HCC) (Chou et al., Nat Rev Endocrinol 6:676-688, 2010; Rake et al., Eur J Pediatr 161(Suppl 1):S20-S34, 2002; Franco et al., J Inherit Metab Dis 28:153-162, 2005).

Although gene therapy studies using recombinant adeno-associated virus (AAV) carrying G6Pase-.alpha. have previously been performed in animal models of GSD-Ia, none have been capable of completely correcting hepatic G6Pase-.alpha. deficiency. Thus, a need exists for an improved gene therapy vector for the treatment of GSD-Ia and its associated complications.

SUMMARY

It is disclosed herein that the canine G6Pase-.alpha. enzyme is more active than the human G6Pase-.alpha. enzyme. An amino acid sequence alignment of the two proteins revealed that they differ primarily at 18 residues. To identify human G6Pase-.alpha. mutants with increased phosphohydrolase activity, mutants were generated that contain one or two corresponding amino acids from canine G6Pase-.alpha..

Provided herein are isolated nucleic acid molecules encoding a modified G6Pase-.alpha.. In some embodiments, the modified G6Pase-.alpha. comprises a serine to cysteine substitution at amino acid 298 of human G6Pase-.alpha., such as the modified G6Pase-.alpha. of SEQ ID NO: 8. In some examples, the nucleic acid molecules comprise the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

Further provided are vectors, such as adeno-associated virus (AAV) vectors, that include the disclosed nucleic acid molecules encoding modified human G6Pase-.alpha.. In some examples, the vectors further comprise a promoter, such as a human G6PC promoter/enhancer (GPE).

Also provided are recombinant AAV (rAAV) comprising the nucleic acid molecules encoding modified human G6Pase-.alpha.. Further provided are isolated host cells comprising the nucleic acid molecules or vectors disclosed herein. For example, the isolated host cells can be cells suitable for propagation of rAAV.

Compositions comprising the disclosed nucleic acid molecules, vectors and rAAV are further provided by the present disclosure.

Further provided is a method of treating a subject diagnosed with a glycogen storage disease. In some embodiments, the method includes selecting a subject with glycogen storage disease type Ia (GSD-Ia) and administering to the subject a therapeutically effective amount of the rAAV, or compositions comprising the rAAV, disclosed herein.

Also provided is a method of promoting glucose homeostasis; inhibiting hypoglycemia; inhibiting or preventing the development of HCA; inhibiting or preventing the development of HCC; inhibiting or preventing renal dysfunction or failure; or treating any other complication associated with GSD-Ia, in a subject with a deficiency in G6Pase-.alpha. by administering to the subject a therapeutically effective amount of the rAAV, or compositions comprising the rAAV, disclosed herein.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of the canine (SEQ ID NO: 1) and human (SEQ ID NO: 2) G6Pase-.alpha. protein sequences. The 18 amino acid residues that differ between the canine and human sequences are indicated by arrows.

FIG. 2 depicts human G6Pase-.alpha., which is anchored into the endoplasmic reticulum membrane by 9 helices, H1 to H9 (Pan et al J Biol Chem 273:6144-6148, 1998). The amino acid differences between human and canine G6Pase-.alpha. are listed.

FIG. 3 is a graph showing hepatic microsomal G6Pase-.alpha. activity in 4-week-old GSD-Ia (G6pc.sup.-/-) mice transduced at age 2 weeks with a recombinant AAV8-GPE vector (10.sup.13 vg/kg) expressing human G6PC (AAV-G6PC), codon-optimized (co) human G6PC (AAV-co-G6PC), human G6PC-S298C mutant (AAV-G6PC-S298C), or co-human G6PC-S298C mutant (AAV-co-G6PC-S298C). Hepatic microsomal G6Pase-.alpha. activity in 4-week-old wild type mice averaged 227.5.+-.17.6 nmol/min/mg. Data are presented as the mean.+-.SEM. **P<0.005.

FIG. 4 shows the nucleotide sequence of the G6PC cDNA (SEQ ID NO: 11), with the location of each of the 18 human G6PC mutants indicated. For site-directed mutagenesis, 20-nucleotide sense and antisense primers were designed such that the codon to be mutated was located in the middle of each primer.

FIG. 5 is a graph showing hepatic G6Pase-.alpha. activity in 24-day-old G6pc.sup.-/- mice transduced at age 10 days with 5.times.10.sup.11 vg/kg AAV-co-G6PC vector (n=3) or rAAV8-co-G6PC-S298C vector (n=3). Wild-type hepatic G6Pase activity averaged 174.+-.18.3 nmol/min/mg. Data represent the mean.+-.SEM. **P<0.005.

FIGS. 6A-6E show data demonstrating correction of hepatic G6Pase-.alpha. deficiency by rAAV-G6PC. L-G6pc.sup.-/- mice were treated 1.times.10.sup.12 vg/kg of rAAV-G6PC at age 10 weeks and analyzed at age 18 weeks. FIG. 6A is a graph showing hepatic G6Pase-.alpha. activity in control (n=7), L-G6pc.sup.-/- (n=7) and AAV mice (n=8). FIG. 6B shows images of representative H&E-stained livers of control, L-G6pc.sup.-/- and AAV mice. FIG. 6C is a graph showing fasting glucose tolerance (FGT) of control (n=13), L-G6pc.sup.-/- (n=6) and AAV mice (n=8). FIG. 6D is a graph showing liver weights of control, L-G6pc.sup.-/- and AAV mice (n=8). FIG. 6E is a graph showing hepatic contents of glycogen and triglyceride of control, L-G6pc.sup.-/- and AAV mice (n=8). Data represent the mean.+-.SEM. *P<0.05, **P<0.005.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on Jun. 21, 2017, 52.8 KB, which is incorporated by reference herein. In the accompanying Sequence Listing:

SEQ ID NO: 1 is the amino acid sequence of canine G6Pase-.alpha. protein.

SEQ ID NO: 2 is the amino acid sequence of human G6Pase-.alpha. protein.

SEQ ID NO: 3 is the nucleotide sequence of pTR-GPE-human G6PC having the following features: ITR--nucleotides 17-163 G6PC promoter/enhancer--nucleotides 182-3045 Stuffer--nucleotides 3051-3184 Intron--nucleotides 3185-3321 Stuffer--nucleotides 3322-3367 G6PC coding sequence--nucleotides 3368-4441 ITR--nucleotides 4674-4819.

SEQ ID NO: 4 is the nucleotide sequence of pTR-GPE-human G6PC-S298C having the following features: ITR--nucleotides 17-163 G6PC promoter/enhancer--nucleotides 182-3045 Stuffer--nucleotides 3051-3184 Intron--nucleotides 3185-3321 Stuffer--nucleotides 3322-3367 G6PC coding sequence--nucleotides 3368-4441 S298C mutation--nucleotides 4259-4261 (TCT to TGC) ITR--nucleotides 4674-4819.

SEQ ID NO: 5 is the nucleotide sequence of pTR-GPE-codon optimized (co) G6PC-S298C having the following features: ITR--nucleotides 17-163 G6PC promoter/enhancer--nucleotides 182-3045 Stuffer--nucleotides 3051-3184 Intron--nucleotides 3185-3321 Stuffer--nucleotides 3322-3367 Codon-optimized G6PC coding sequence--nucleotides 3368-4441 S298C mutation--nucleotides 4259-4261 (AGC to TGC) ITR--nucleotides 4674-4819.

SEQ ID NO: 6 is the nucleotide sequence of a modified human G6PC encoding S298C G6Pase-.alpha..

SEQ ID NO: 7 is the nucleotide sequence of a codon-optimized, modified human G6PC encoding S298C G6Pase-.alpha..

SEQ ID NO: 8 is the amino acid sequence of modified human S298C G6Pase-.alpha..

SEQ ID NO: 9 is the amino acid sequence of modified human S298C/A301V G6Pase-.alpha..

SEQ ID NO: 10 is an amino acid consensus sequence based on alignment of human and canine G6Pase-.alpha..

SEQ ID NO: 11 is the nucleotide sequence of the human G6PC coding region.

DETAILED DESCRIPTION

I. Abbreviations

AAV adeno-associated virus CBA chicken .beta.-actin CMV cytomegalovirus co codon optimized FGT fasting glucose tolerance G6P glucose-6-phosphate G6Pase-.alpha. glucose-6-phosphatase-.alpha. G6PC glucose-6-phosphatase, catalytic subunit G6PT glucose-6-phosphate transporter GPE G6PC promoter/enhancer GSD glycogen storage disease HCA hepatocellular adenoma HCC hepatocellular carcinoma ITR inverted terminal repeat ORF open reading frame rAAV recombinant AAV SEM standard error of the mean vg viral genomes vp viral particles WT wild type II. Terms and Methods

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for gene therapy. There are currently 11 recognized serotypes of AAV (AAV1-11).

Administration/Administer: To provide or give a subject an agent, such as a therapeutic agent (e.g. a recombinant AAV), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

Codon-optimized: A "codon-optimized" nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or in a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

Enhancer: A nucleic acid sequence that increases the rate of transcription by increasing the activity of a promoter.

G6PC: A gene located on human chromosome 17q21 that encodes glucose-6-phosphatase-.alpha. (G6Pase-.alpha.). G6Pase-.alpha. is a 357 amino acid hydrophobic protein having 9 helices that anchor it in the endoplasmic reticulum (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). G6Pase-.alpha. is depicted in FIG. 2. The G6Pase-.alpha. protein catalyzes the hydrolysis of glucose 6-phosphate to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis and is a key enzyme in glucose homeostasis. Deleterious mutations in the G6PC gene cause glycogen storage disease type Ia (GSD-Ia), which is a metabolic disorder characterized by severe fasting hypoglycemia associated with the accumulation of glycogen and fat in the liver and kidneys.

Glycogen storage disease (GSD): A group of diseases that result from defects in the processing of glycogen synthesis or breakdown within muscles, liver and other tissues. GSD can either be genetic or acquired. Genetic GSD is caused by any inborn error of metabolism involved in these processes. There are currently 11 recognized glycogen storage diseases (GSD type I, II, III, IV, V, VI, VII, IX, XI, XII and XIII). GSD-I consists of two autosomal recessive disorders, GSD-Ia and GSD-Ib (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). GSD-Ia results from a deficiency in glucose-6-phosphatase-.alpha.. Deficiencies in the glucose-6-phosphate transporter (G6PT) are responsible for GSD-Ib.

Glycogen storage disease type Ia (GSD-Ia): Also known as von Gierke disease, GSD-Ia is the most common glycogen storage disease, having an incidence of about 1 in 100,000 live births. GSD-Ia is a genetic disease resulting from deficiency of the enzyme glucose-6-phosphatase-.alpha. (G6Pase-.alpha.). Deficiency in G6Pase-.alpha. impairs the ability of the liver to produce free glucose from glycogen and from gluconeogenesis. Patients affected by GSD-Ia are unable to maintain glucose homeostasis and present with fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic academia (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). There is currently no cure for GSD-Ia.

Intron: A stretch of DNA within a gene that does not contain coding information for a protein. Introns are removed before translation of a messenger RNA.

Inverted terminal repeat (ITR): Symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating AAV integrating vectors.

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus or cell) has been substantially separated or purified away from other biological components in the cell or tissue of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

Modified: In the context of the present disclosure, a "modified" G6PC or G6Pase-.alpha. refers to a G6PC nucleic acid sequence or a G6Pase-.alpha. amino acid sequence that comprises at least one nucleic acid or amino acid substitution, deletion or insertion compared to the wild type sequence (such as compared to the human G6PC coding sequence set forth as nucleotides 3368-4441 of SEQ ID NO: 3, or relative to the human G6Pase-.alpha. amino acid sequence set forth as SEQ ID NO: 2).

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: "Preventing" a disease (such as GSD-Ia) refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease.

Promoter: A region of DNA that directs/initiates transcription of a nucleic acid (e.g. a gene). A promoter includes necessary nucleic acid sequences near the start site of transcription. Typically, promoters are located near the genes they transcribe. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is isolated in whole or in part from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.

Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.

Similarly, a recombinant virus is a virus comprising sequence (such as genomic sequence) that is non-naturally occurring or made by artificial combination of at least two sequences of different origin. The term "recombinant" also includes nucleic acids, proteins and viruses that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule, protein or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule (such as a recombinant nucleic acid molecule encoding G6Pase-.alpha.) has been packaged.

Sequence identity: The identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

Serotype: A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.

Stuffer sequence: Refers to a sequence of nucleotides contained within a larger nucleic acid molecule (such as a vector) that is typically used to create desired spacing between two nucleic acid features (such as between a promoter and a coding sequence), or to extend a nucleic acid molecule so that it is of a desired length. Stuffer sequences do not contain protein coding information and can be of unknown/synthetic origin and/or unrelated to other nucleic acid sequences within a larger nucleic acid molecule.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.

Synthetic: Produced by artificial means in a laboratory, for example a synthetic nucleic acid can be chemically synthesized in a laboratory.

Therapeutically effective amount: A quantity of a specified pharmaceutical or therapeutic agent (e.g. a recombinant AAV) sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition.

Vector: A vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. In some embodiments herein, the vector is an AAV vector.

Unless otherwise explained, 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 disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. "Comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

III. Introduction

Glycogen storage disease type Ia (GSD-Ia) is an inherited disorder of metabolism associated with life-threatening hypoglycemia, hepatic malignancy, and renal failure caused by the deficiency of glucose-6-phosphatase-.alpha. (G6Pase-.alpha. or G6PC), and primarily affecting the liver and kidney. Current therapy fails to prevent long-term complications in many patients, including growth failure, gout, pulmonary hypertension, renal dysfunction with risk for renal failure, osteoporosis, and hepatocellular adenomas (HCA) that can undergo malignant transformation to hepatocellular carcinoma (Chou et al., Curr Mol Med 2:121-143, 2002; Chou et al., Nat Rev Endocrinol 6:676-688, 2010). Therefore, the development of new therapies, such as gene therapies, is justified as a potentially curative treatment for GSD-Ia (Chou et al., Expert Opin Biol Ther 11:1011-1024, 2011).

Recombinant AAV (rAAV) vectors encoding functional G6Pase-.alpha. have been described (reviewed in Chou and Mansfield, Expert Opin Biol Ther 11:1011-1024, 2011). Previous studies using the mouse model of GSD-Ia have shown that recombinant AAV expressing G6Pase-.alpha. directed by the CBA promoter/CMV enhancer (Ghosh et al., Gene Ther 13:321-329, 2006), the canine G6PC promoter (Koeberl et al., Gene Ther 13:1281-1289, 2006), or the human G6PC promoter at nucleotides -298 to +128 of the G6PC 5' flanking region (Koeberl et al., Mol Ther 16:665-672, 2008) deliver the G6Pase-.alpha. transgene to the liver and achieve extended correction of this disorder. However, while these studies have shown promise, none have been capable of completely correcting hepatic G6Pase-.alpha. deficiency.

An example of one prior study is described by Koeberl et al. (Mol Ther 16:665-672, 2008) who developed a double-stranded, rAAV vector (rAAV-G6Pase) that was capable of treating GSD-Ia mice and GSD-Ia dogs. In addition, Yiu et al. (Mol Ther 18:1076-1084, 2010) and Lee et al. (Hepatology 56:1719-1729, 2012) describe the development of a similarly efficacious, single-stranded rAAV vector, rAAV-GPE-G6PC. The two vectors differ in several respects. First, rAAV-GPE-G6PC is a single-stranded rAAV vector while rAAV-G6Pase is a double-stranded rAAV vector. The double-stranded rAAV vector was developed to circumvent the rate-limiting step in the life cycle of AAV by converting single-stranded DNA genome into double-stranded DNA genome (Chou et al., Expert Opin Biol Ther 11:1011-1024, 2011). Second, the expression of G6Pase-.alpha. in rAAV-G6Pase is directed by the human G6PC minimal promoter/enhancer at nucleotides -382 to -1 (Koeberl et al., Mol Ther 16:665-672, 2008). The expression of G6Pase-.alpha. in rAAV-GPE-G6PC is directed by the human G6PC promoter/enhancer at nucleotides -2864 to -1 (GPE) (Yiu et al., Mol Ther 18:1076-1084, 2010; Lee et al., Hepatology 56:1719-1729, 2012). Third, the rAAV-GPE-G6PC vector contains an intron that is absent from the rAAV-G6Pase vector. Both vectors efficiently delivered the G6Pase-.alpha. transgene to the liver and corrected murine GSD-Ia (Koeberl et al., Mol Ther 16:665-672, 2008; Yiu et al., Mol Ther 18:1076-1084, 2010; Lee et al., Hepatology 56:1719-1729, 2012).

A comparative study was performed to determine which vector, rAAV-G6Pase or rAAV-GPE-G6PC, was more efficacious in treating GSD-Ia. The results showed unequivocally that the rAAV-GPE-G6PC vector was more efficient than the rAAV-G6Pase vector in correcting murine GSD-Ia. It was determined that the enhancer elements upstream of the G6PC minimal promoter were required for efficient hepatic transgene expression (Lee et al., Mol Genet Metab 10:275-280, 2013). Additional studies were carried out to determine whether the intron contained within the rAAV-GPE-G6PC vector played a role in directing hepatic G6Pase-.alpha. expression. A double-stranded rAAV vector (referred to as rAAV-miGPE-G6PC) expressing human G6Pase-.alpha. directed by the human G6PC minimal promoter at nucleotides -382 to -1, and containing the intron present in the rAAV-GPE-G6PC vector, was generated. The results showed that the rAAV-GPE-G6PC vector was more efficient than the rAAV-miGPE-G6PC vector in treating murine GSD-Ia, supporting the conclusion that the enhancer elements upstream the G6PC minimal promoter are required for efficient hepatic transgene expression (Lee et al., Mol Genet Metab 10:275-280, 2013).

Recombinant AAV vectors comprising a G6PC promoter/enhancer (GPE) element are further described in PCT Publication No. WO 2015/081101, which is herein incorporated by reference. The recombinant vectors include a GPE, a synthetic intron, and the G6PC coding region. The G6PC coding region is optionally codon-optimized for expression in human cells. The recombinant vectors further include stuffer nucleic acid sequence situated between the G6PC promoter/enhancer and the intron, as well as between the intron and the G6PC coding sequence; and 5' and 3' inverted terminal repeat (ITR) sequences.

It is disclosed in PCT Publication No. WO 2015/081101 that a G6Pase-.alpha. expressing recombinant AAV with the G6PC promoter/enhancer (rAAV-GPE-G6PC) is significantly more efficient in directing in vivo hepatic transgene expression than another G6Pase-.alpha. expressing recombinant AAV having an alternative promoter/enhancer (i.e. the chicken .beta.-actin promoter/CMV enhancer). Over a 24-week study period, G6PC-deficient mice (a model for GSD-Ia) treated with rAAV-GPE-G6PC exhibited complete normalization of hepatic G6PC deficiency as evidenced by normal levels of blood glucose, blood metabolites, hepatic glycogen and hepatic fat (see also Yiu et al., Mol Ther 18:1076-1084, 2010). Furthermore, a longer-term study of rAAV-GPE-G6PC-treated G6pc.sup.-/- mice demonstrated that gene therapy mediated by rAAV-GPE-G6PC was efficacious for at least 70-90 weeks in mice expressing more than 3% hepatic G6Pase-.alpha.. In particular, rAAV-GPE-G6PC-treated mice exhibited normal hepatic fat storage, normal blood metabolite and glucose tolerance profiles, reduced fasting blood insulin levels, and had no evidence of hepatic abnormalities, such as hepatocellular adenoma (see also Lee et al., Hepatology 56:1719-1729, 2012).

Further disclosed in PCT Publication No. WO 2015/081101 was the finding that the upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in an animal model of GSD-Ia. Specifically, it was demonstrated that treatment with rAAV-GPE-G6PC, which comprises the G6PC promoter/enhancer at nucleotides -2684 to -1 (relative to the G6PC start site) produces significantly higher levels of hepatic G6Pase-.alpha. expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in a mouse model of GSD-Ia, compared to a G6Pase-.alpha. expressing recombinant AAV containing only a 383 bp minimal G6PC promoter/enhancer (see also Lee et al., Mol Genet Metab. 110(3):275-280, 2013).

Also disclosed in PCT Publication No. WO 2015/081101 was the finding that stuffer nucleotide sequences present between the G6PC promoter/enhancer and the intron, as well as between the intron and the G6PC coding sequence, are important for liver transduction and expression of G6Pase-.alpha.. In particular, recombinant AAV produced from plasmid UF11-K29-G6PC, which lacks the stuffer sequences, exhibited G6Pase activity of 7.3 nmol/min/mg. In comparison, recombinant AAV produced from plasmid UF11-GPE-G6PC, exhibited G6Pase activity of 33.0 nmol/min/mg.

In addition, data disclosed in PCT Publication No. WO 2015/081101 demonstrated that codon-optimization of the G6PC coding sequence increased efficiency of translation approximately 1.5- to 2.5-fold, resulting in significantly greater G6Pase-.alpha. expression in the liver following administration of rAAV-co-GPE-G6PC (containing a codon-optimized G6PC nucleic acid sequence), compared with administration of rAAV-GPE-G6PC, which encodes wild-type G6PC.

Thus, recombinant AAV comprising the G6PC promoter/enhancer at nucleotides -2684 to -1, a synthetic intron, stuffer sequences flanking the intron, and the G6PC coding region (wild-type or codon-optimized) are important features for efficient hepatic transgene expression and treatment of GSD-Ia in vivo.

The present disclosure shows for the first time that the human G6PC can be modified at specific positions to enhance phosphohydrolase activity of the encoded G6Pase-.alpha. enzyme. The modified G6Pase-.alpha. coding sequences can be incorporated into rAAV gene therapy vectors to enhance efficacy in the treatment of glycogen storage disease.

IV. Overview of Several Embodiments

It is disclosed herein that the canine G6Pase-.alpha. enzyme is more active than the human G6Pase-.alpha. enzyme. An amino acid alignment of the two proteins revealed that they differ in sequence at 18 residues. To identify human G6Pase-.alpha. mutants with increased phosphohydrolase activity, mutants were generated that contain at least one corresponding amino acid from canine G6Pase-.alpha..

Provided herein are recombinant nucleic acid molecules, AAV vectors and recombinant AAV that can be used in gene therapy applications for the treatment of glycogen storage disease, specifically GSD-Ia.

In some embodiments, provided is an isolated nucleic acid molecule encoding a modified G6Pase-.alpha., wherein the modified G6Pase-.alpha. comprises a serine to cysteine substitution at amino acid 298 of human G6Pase-.alpha. (the amino acid sequence of wild type human G6Pase-.alpha. is set forth herein as SEQ ID NO: 2). The modified G6Pase-.alpha. can include modifications at additional residues so long as the protein retains enzymatic activity. For example, the modified G6Pase-.alpha. can include substitutions at other residues that differ between the canine and human G6Pase-.alpha. sequences, such residues include positions 3, 54, 139, 196, 199, 242, 247, 292, 301, 318, 324, 332, 347, 349, 350 and/or 353 of the human G6Pase-.alpha. (set forth as SEQ ID NO: 2). FIG. 1 shows an alignment of the human and canine G6Pase-.alpha. protein sequences, and Table 2 provides a summary of the amino acid differences between human and canine G6Pase-.alpha.. The present disclosure contemplates nucleotide substitutions that alter the amino acid sequence at one or more of the residues listed above and in Table 2.

In some examples, the nucleic acid molecule encodes a modified G6Pase-.alpha. having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2, and having a serine to cysteine substitution at amino acid 298. In particular examples, the amino acid sequence of the modified G6Pase-.alpha. comprises or consists of SEQ ID NO: 8 (human S298C G6Pase-.alpha.). In other particular examples, the amino acid sequence of the modified G6Pase-.alpha. comprises or consists of SEQ ID NO: 9 (human S298C/A301V G6Pase-.alpha.). In non-limiting examples, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6 (modified human G6PC encoding S298C G6Pase-.alpha.) or SEQ ID NO: 7 (codon-optimized, modified human G6PC encoding S298C G6Pase-.alpha.).

Also provided herein are vectors comprising the isolated nucleic acid molecules encoding modified G6Pase-.alpha.. In some embodiments, the nucleic acid molecule encoding the modified G6Pase-.alpha. is operably linked to a promoter, such as a G6PC promoter. In some examples, the G6PC promoter is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 182-3045 of SEQ ID NO: 4 (human G6PC promoter/enhancer) or at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to 182-3045 of SEQ ID NO: 5 (human codon-optimized G6PC promoter/enhancer). In non-limiting examples, the G6PC promoter comprises nucleotides 182-3045 of SEQ ID NO: 4 or nucleotides 182-3045 of SEQ ID NO: 5.

In some examples, the vector comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 182-4441 of SEQ ID NO: 4 or nucleotides 182-4441 of SEQ ID NO: 5. In specific non-limiting examples, the vector comprises nucleotides 182-4441 of SEQ ID NO: 4 or nucleotides 182-4441 of SEQ ID NO: 5.

In some embodiments, the vector is an AAV vector. The AAV serotype can be any suitable serotype for delivery of transgenes to a subject. In some examples, the AAV vector is a serotype 8 AAV (AAV8). In other examples the AAV vector is a serotype 1, 2, 3, 4, 5, 6, 7, 9, 10, 11 or 12 vector (i.e. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11 or AAV12). In yet other examples, the AAV vector is a hybrid of two or more AAV serotypes (such as, but not limited to AAV2/1, AAV2/7, AAV2/8 or AAV2/9). The selection of AAV serotype will depend in part on the cell type(s) that are targeted for gene therapy. For treatment of GSD-Ia, the liver and kidney are the relevant target organs.

When an AAV vector is used, the vector can include inverted terminal repeats (ITRs). In some embodiments, the AAV vector comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 17-4819 of SEQ ID NO: 4 or nucleotides 17-4819 of SEQ ID NO: 5. In some examples, the AAV vector comprises nucleotides 17-4819 of SEQ ID NO: 4 or nucleotides 17-4819 of SEQ ID NO: 5.

Also provided herein are isolated host cells comprising the nucleic acid molecules or vectors disclosed herein. For example, the isolated host cell can be a cell (or cell line) appropriate for production of recombinant AAV (rAAV). In some examples, the host cell is a mammalian cell, such as a HEK-293, BHK, Vero, RD, HT-1080, A549, Cos-7, ARPE-19, or MRC-5 cell.

Further provided are rAAV comprising a nucleic acid molecule disclosed herein. In some embodiments, the rAAV is rAAV8 and/or rAAV2. However, the AAV serotype can be any other suitable AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11 or AAV12, or a hybrid of two or more AAV serotypes (such as, but not limited to AAV2/1, AAV2/7, AAV2/8 or AAV2/9). Compositions comprising a rAAV disclosed herein and a pharmaceutically acceptable carrier are also provided by the present disclosure. In some embodiments, the compositions are formulated for intravenous or intramuscular administration. Suitable pharmaceutical formulations for administration of rAAV can be found, for example, in U.S. Patent Application Publication No. 2012/0219528 (herein incorporated by reference).

Further provided are methods of treating a subject diagnosed with a glycogen storage disease, comprising selecting a subject with GSD-Ia and administering to the subject a therapeutically effective amount of a rAAV (or a composition comprising a rAAV) disclosed herein.

Methods of promoting glucose homeostasis; inhibiting hypoglycemia; inhibiting or preventing the development of hepatocellular adenoma (HCA); inhibiting or preventing the development of hepatocellular carcinoma (HCC); inhibiting or preventing renal dysfunction or failure; inhibiting or preventing growth retardation; inhibiting or preventing hepatomegaly; inhibiting or preventing nephromegaly; inhibiting or preventing hyperlipidemia; inhibiting or preventing pulmonary hypertension; or treating, preventing or inhibiting any other complication associated with GSD-Ia, in a subject with a deficiency in glucose-6-phosphatase-.alpha. (G6Pase-.alpha.) are also provided by the present disclosure. In some embodiments, the methods include administering to the subject a therapeutically effective amount of a rAAV (or a composition comprising a rAAV) disclosed herein. In some embodiments, the subject with a deficiency in G6Pase-.alpha. is a subject that has GSD-Ia. Thus, in some examples, the method includes selecting a subject with GSD-Ia.

In some embodiments of the methods disclosed herein, the rAAV is administered intravenously.

In some embodiments, the rAAV is administered at a dose of about 1.times.10.sup.10 to about 1.times.10.sup.14 viral particles (vp)/kg. In some examples, the rAAV is administered at a dose of about 1.times.10.sup.11 to about 8.times.10.sup.13 vp/kg or about 1.times.10.sup.12 to about 8.times.10.sup.13 vp/kg. In other examples, the rAAV is administered at a dose of about 1.times.10.sup.13 to about 6.times.10.sup.13 vp/kg. In specific non-limiting examples, the rAAV is administered at a dose of at least about 1.times.10.sup.10, at least about 5.times.10.sup.10, at least about 1.times.10.sup.11, at least about 5.times.10.sup.11, at least about 1.times.10.sup.12, at least about 5.times.10.sup.12, at least about 1.times.10.sup.13, at least about 5.times.10.sup.13, or at least about 1.times.10.sup.14 vp/kg. In other non-limiting examples, the rAAV is administered at a dose of no more than about 1.times.10.sup.10, no more than about 5.times.10.sup.10, no more than about 1.times.10.sup.11, no more than about 5.times.10.sup.11, no more than about 1.times.10.sup.12, no more than about 5.times.10.sup.12, no more than about 1.times.10.sup.13, no more than about 5.times.10.sup.13, or no more than about 1.times.10.sup.14 vp/kg. In one non-limiting example, the rAAV is administered at a dose of about 1.times.10.sup.12 vp/kg. In another non-limiting example, the rAAV is administered at a dose of about 1.times.10.sup.11 vp/kg. The rAAV can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

V. Modified Human G6PC/G6Pase-.alpha. Sequences

It is disclosed herein that the canine G6Pase-.alpha. enzyme is more active than the human G6Pase-.alpha. enzyme. As shown in FIG. 1 and Table 2, the two proteins differ in sequence at 18 residues. Using site-directed mutagenesis, human G6Pase-.alpha. mutants with increased phosphohydrolase activity were identified. The human G6Pase-.alpha. mutants that were generated contained one or more corresponding amino acids from canine G6Pase-.alpha. at position(s) 3, 54, 139, 196, 199, 242, 247, 292, 298, 301, 318, 324, 332, 347, 349, 350 and/or 353 of SEQ ID NO: 2. The human G6Pase-.alpha. sequence and a human/canine G6Pase-.alpha. consensus sequence are set forth below.

TABLE-US-00001 Human G6Pase-.alpha. (SEQ ID NO: 2): MEEGMHVLHD FGTQSTHYLQ VHYQDSQDWF TLVSVTADLR HAFYVLEPTW FHLQEAVGIK 60 LLWVAVTGDW LHLVFKWILF GQRPYWWVLD TDYYSHTSVP LIKQFPVTCE TGPGSPSGHA 120 MGTAGVYYVM VTSTLSTFQG KTKPTYRFRC LHVILWLGEW AVQLHVCLSR TYLAAHEPHQ 180 VVAGVLSGTA VAFTFSHTHS TYHASLKKYF LITFFLFSFA TGFYLLLKGL GVDLLWTLEK 240 AQRWCEQPEW VHIDTTPFAS LLKHLGTLFG LGLALHSSMY RESCKGKLSK WLPFRLSSTV 300 ASLVLLHVFD SLKPPSQVEL VFYVLSFCKS AVVPLASVSV TPYCLAQVLG QPHKKSL 357 Human/Canine G6Pase-.alpha. consensus sequence (SEQ ID NO: 10): MEXGMHVLHD FGTQSTHYLQ VHYQDSQDWF TLVSVTADLR HAFYVLEPTW FHLKEAVGIK 60 LLWVAVTGDW LHLVFKWILF GQRPYWWVLD TDYYSHTSVP LIKQFPVTCE TGPGSPSGHA 120 MGTAGVYYVM VTSTLSTFXG KXKPTYRFRC LHVILWLGEW AVQLHVCLSR TYLAAHEPHQ 180 VVAGVLSGTA VAETEXHIXS TYHASLKKYF LITFFLFSFA TGFYLLLKGL GVDLLWTLEK 240 AXRWCEXPEW VHIDTTPFAS LLKHLGTLFG LGLALHSSMY RESCKGKLSK WXPERLSXTV 300 XSLVLLHVFD SLKPPSQXEL VFYXLSFCKS AXVPLASVSV TPYCLAXVXX QPXKKSL 357

In some embodiments, provided herein is a modified G6Pase-.alpha. comprising SEQ ID NO: 10, wherein X at amino acid residue 3=K or E; X at amino acid residue 54=R or Q; X at amino acid residue 139=R or Q; X at amino acid residue 142=K or I; X at amino acid residue 196=R or S; X at amino acid residue 199=Q or H; X at amino acid residue 242=R or Q; X at amino acid residue 247=R or Q; X at amino acid residue 292=F or L; X at amino acid residue 298=C or S; X at amino acid residue 301=V or A; X at amino acid residue 318=T or V; X at amino acid residue 324=T or V; X at amino acid residue 332=A or V; X at amino acid residue 347=R or Q; X at amino acid residue 349=F or L; X at amino acid residue 350=D or G; or X at amino acid residue 353=D or H, or any combination thereof.

In particular examples, the modified G6Pase-.alpha. sequence comprises a S298C mutation, or comprises S298C and A310V mutations, as set forth below. In non-limiting examples, the modified G6Pase-.alpha. sequence comprises or consists of SEQ ID NO: 8 or SEQ ID NO: 9.

TABLE-US-00002 Human G6Pase-.alpha. S298C (SEQ ID NO: 8): MEEGMHVLHD FGTQSTHYLQ VHYQDSQDWF TLVSVTADLR HAFYVLEPTW FHLQEAVGIK 60 LLWVAVTGDW LHLVFKWILF GQRPYWWVLD TDYYSHTSVP LIKQFPVTCE TGPGSPSGHA 120 MGTAGVYYVM VTSTLSTFQG KTKPTYRFRC LHVILWLGEW AVQLHVCLSR TYLAAHEPHQ 180 VVAGVLSGTA VAFTFSHTHS TYHASLKKYF LITFFLFSFA TGFYLLLKGL GVDLLWTLEK 240 AQRWCEQPEW VHIDTTPFAS LLKHLGTLFG LGLALHSSMY RESCKGKLSK WLPFRLSCTV 300 ASLVLLHVFD SLKPPSQVEL VFYVLSFCKS AVVPLASVSV TPYCLAQVLG QPHKKSL 357 Human G6Pase-.alpha. 5298C/A310V (SEQ ID NO: 9): MEEGMHVLHD FGTQSTHYLQ VHYQDSQDWF TLVSVTADLR HAFYVLEPTW FHLQEAVGIK 60 LLWVAVTGDW LHLVFKWILF GQRPYWWVLD TDYYSHTSVP LIKQFPVTCE TGPGSPSGHA 120 MGTAGVYYVM VTSTLSTFQG KTKPTYRFRC LHVILWLGEW AVQLHVCLSR TYLAAHEPHQ 180 VVAGVLSGTA VAFTFSHTHS TYHASLKKYF LITFFLFSFA TGFYLLLKGL GVDLLWTLEK 240 AQRWCEQPEW VHIDTTPFAS LLKHLGTLFG LGLALHSSMY RESCKGKLSK WLPFRLSCTV 300 VSLVLLHVFD SLKPPSQVEL VFYVLSFCKS AVVPLASVSV TPYCLAQVLG QPHKKSL 357

In some embodiments, the modified G6Pase-.alpha. comprising an S298C mutation is encoded by a nucleic acid sequence set forth below.

TABLE-US-00003 Human G6PC S298C (SEQ ID NO: 6): ATG GAGGAAGGAA TGAATGTTCT CCATGACTTT GGGATCCAGT CAACACATTA CCTCCAGGTG AATTACCAAG ACTCCCAGGA CTGGTTCATC TTGGTGTCCG TGATCGCAGA CCTCAGGAAT GCCTTCTACG TCCTCTTCCC CATCTGGTTC CATCTTCAGG AAGCTGTGGG CATTAAACTC CTTTGGGTAG CTGTGATTGG AGACTGGCTC AACCTCGTCT TTAAGTGGAT TCTCTTTGGA CAGCGTCCAT ACTGGTGGGT TTTGGATACT GACTACTACA GCAACACTTC CGTGCCCCTG ATAAAGCAGT TCCCTGTAAC CTGTGAGACT GGACCAGGGA GCCCCTCTGG CCATGCCATG GGCACAGCAG GTGTATACTA CGTGATGGTC ACATCTACTC TTTCCATCTT TCAGGGAAAG ATAAAGCCGA CCTACAGATT TCGGTGCTTG AATGTCATTT TGTGGTTGGG ATTCTGGGCT GTGCAGCTGA ATGTCTGTCT GTCACGAATC TACCTTGCTG CTCATTTTCC TCATCAAGTT GTTGCTGGAG TCCTGTCAGG CATTGCTGTT GCAGAAACTT TCAGCCACAT CCACAGCATC TATAATGCCA GCCTCAAGAA ATATTTTCTC ATTACCTTCT TCCTGTTCAG CTTCGCCATC GGATTTTATC TGCTGCTCAA GGGACTGGGT GTAGACCTCC TGTGGACTCT GGAGAAAGCC CAGAGGTGGT GCGAGCAGCC AGAATGGGTC CACATTGACA CCACACCCTT TGCCAGCCTC CTCAAGAACC TGGGCACGCT CTTTGGCCTG GGGCTGGCTC TCAACTCCAG CATGTACAGG GAGAGCTGCA AGGGGAAACT CAGCAAGTGG CTCCCATTCC GCCTCAGCTG CATTGTAGCC TCCCTCGTCC TCCTGCACGT CTTTGACTCC TTGAAACCCC CATCCCAAGT CGAGCTGGTC TTCTACGTCT TGTCCTTCTG CAAGAGTGCG GTAGTGCCCC TGGCATCCGT CAGTGTCATC CCCTACTGCC TCGCCCAGGT CCTGGGCCAG CCGCACAAGA AGTCGTTGTA A Human codon-optimized G6PC S298C (SEQ ID NO: 7): ATG GAAGAGGGCA TGAACGTGCT GCACGACTTC GGCATCCAGA GCACCCACTA TCTGCAGGTC AACTACCAGG ACAGCCAGGA CTGGTTCATC CTGGTGTCCG TGATCGCCGA CCTGCGGAAC GCCTTCTACG TGCTGTTCCC CATCTGGTTC CATCTGCAAG AAGCCGTCGG CATCAAGCTG CTGTGGGTGG CCGTGATCGG CGATTGGCTG AACCTGGTGT TCAAGTGGAT CCTGTTCGGC CAGCGGCCCT ATTGGTGGGT GCTGGACACC GACTACTACA GCAACACCAG CGTGCCCCTG ATCAAGCAGT TCCCCGTGAC CTGCGAGACA GGCCCTGGCT CTCCTTCTGG CCACGCCATG GGAACAGCCG GCGTGTACTA CGTGATGGTC ACCAGCACCC TGAGCATCTT CCAGGGCAAG ATCAAGCCCA CCTACCGGTT CCGGTGCCTG AACGTGATCC TGTGGCTGGG CTTCTGGGCC GTGCAGCTGA ACGTGTGCCT GAGCCGGATC TACCTGGCCG CCCACTTCCC ACATCAAGTG GTGGCCGGCG TGCTGAGCGG AATCGCCGTG GCCGAGACAT TCAGCCACAT CCACAGCATC TACAACGCCA GCCTGAAGAA GTACTTCCTG ATCACATTCT TTCTGTTCAG CTTCGCCATC GGCTTCTACC TGCTGCTGAA GGGCCTGGGC GTGGACCTGC TGTGGACCCT GGAAAAGGCC CAGCGGTGGT GCGAGCAGCC CGAGTGGGTG CACATCGACA CCACCCCCTT CGCCAGCCTG CTGAAGAACC TGGGCACCCT GTTTGGACTG GGCCTGGCCC TGAACAGCAG CATGTACAGA GAGAGCTGCA AGGGCAAGCT GAGCAAGTGG CTGCCCTTCC GGCTGAGCTG CATCGTGGCC AGCCTGGTGC TGCTGCACGT GTTCGACAGC CTGAAGCCCC CCAGCCAGGT GGAACTGGTG TTTTACGTGC TGAGCTTCTG CAAGAGCGCC GTGGTGCCCC TGGCCTCCGT GTCTGTGATC CCCTACTGCC TGGCTCAGGT GCTGGGCCAG CCCCACAAGA AGTCCCTCTG A

VI. Recombinant AAV for Gene Therapy Applications

AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome. Both sense and antisense strands of AAV DNA are packaged into AAV capsids with equal frequency.

The AAV genome is characterized by two inverted terminal repeats (ITRs) that flank two open reading frames (ORFs). In the AAV2 genome, for example, the first 125 nucleotides of the ITR are a palindrome, which folds upon itself to maximize base pairing and forms a T-shaped hairpin structure. The other 20 bases of the ITR, called the D sequence, remain unpaired. The ITRs are cis-acting sequences important for AAV DNA replication; the ITR is the origin of replication and serves as a primer for second-strand synthesis by DNA polymerase. The double-stranded DNA formed during this synthesis, which is called replicating-form monomer, is used for a second round of self-priming replication and forms a replicating-form dimer. These double-stranded intermediates are processed via a strand displacement mechanism, resulting in single-stranded DNA used for packaging and double-stranded DNA used for transcription. Located within the ITR are the Rep binding elements and a terminal resolution site (TRS). These features are used by the viral regulatory protein Rep during AAV replication to process the double-stranded intermediates. In addition to their role in AAV replication, the ITR is also essential for AAV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

The left ORF of AAV contains the Rep gene, which encodes four proteins--Rep78, Rep 68, Rep52 and Rep40. The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2 and VP3). The AAV capsid contains 60 viral capsid proteins arranged into an icosahedral symmetry. VP1, VP2 and VP3 are present in a 1:1:10 molar ratio (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

AAV is currently one of the most frequently used viruses for gene therapy. Although AAV infects humans and some other primate species, it is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. Because of the advantageous features of AAV, the present disclosure contemplates the use of AAV for the recombinant nucleic acid molecules and methods disclosed herein.

AAV possesses several desirable features for a gene therapy vector, including the ability to bind and enter target cells, enter the nucleus, the ability to be expressed in the nucleus for a prolonged period of time, and low toxicity. However, the small size of the AAV genome limits the size of heterologous DNA that can be incorporated. To minimize this problem, AAV vectors have been constructed that do not encode Rep and the integration efficiency element (IEE). The ITRs are retained as they are cis signals required for packaging (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

Methods for producing rAAV suitable for gene therapy are well known in the art (see, for example, U.S. Patent Application Nos. 2012/0100606; 2012/0135515; 2011/0229971; and 2013/0072548; and Ghosh et al., Gene Ther 13(4):321-329, 2006), and can be utilized with the recombinant nucleic acid molecules, vectors and methods disclosed herein.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Construction and Characterization of Human G6PC (G6Pase-.alpha.) Mutants for Use in AAV-Mediated Gene Therapy

This example describes the generation of 18 human G6PC mutants and the identification of specific G6Pase-.alpha. mutants with increased phosphohydrolase activity.

Construction of G6PC Mutants

To construct human G6PC mutants, the pSVL vector, comprising nucleotides 1 to 1074 of human G6PC cDNA (the entire coding region, with the initiation codon ATG at nucleotides 1-3; SEQ ID NO: 11), was used as a template. For PCR-directed mutagenesis, the template was amplified using two outside PCR primers matching nucleotides 1 to 20 (sense) and 1055 to 1074 (antisense) that flanked the 20 nucleotide long sense and antisense mutant primers with the codon to be mutated in the middle (see FIG. 4 and Table 1 below). The template for the hG6PC-S298C/A301V double mutant was the pSVL-hG6PC-S298C mutant. The mutated sequences were cloned in pSVL and verified by DNA sequencing.

TABLE-US-00004 TABLE 1 Nucleotide changes in human G6PC mutants Mutation Nucleotide/codon changes Sequences in human G6PC R3K GAA (R) .fwdarw. AAA (K) nucleotides 7-9 Q54R CAG (Q) .fwdarw. CGT (R) nucleotides 160-162 Q139R CAG (Q) .fwdarw. CGG (R) nucleotides 415-417 I142K ATA (I) .fwdarw. AAA (K) nucleotides 424-426 S196R AGC (S) .fwdarw. CGC (R) Nucleotides 586-588 H199Q CAC (H) .fwdarw. CAG (Q) nucleotides 595-597 Q242R CAG (Q) .fwdarw. AGO (R) nucleotides 724-726 Q247R CAG (Q) .fwdarw. CGG (R) nucleotides 739-741 L292F CTC (L) .fwdarw. TTC (F) nucleotides 874-876 S298C TCT (S).fwdarw. TGC (C) nucleotides 892-894 A301V GCC (A) .fwdarw. GTG (V) nucleotides 901-903 V318T GTC (V) .fwdarw. ACT (T) nucleotides 952-954 V324T GTC (V) .fwdarw. ACC (T) nucleotides 970-972 V332A GTA (V) .fwdarw. GCA (A) nucleotides 994-996 Q347R CAG (Q) .fwdarw. CGG (R) nucleotides 1039-1041 L349F CTG (L) .fwdarw. TTC (F) nucleotides 1045-1047 G350D GGC (G) .fwdarw. GAC (D) nucleotides 1048-1050 H353D CAC (H) .fwdarw. GAC (D) nucleotides 1057-1059

Expression in COS-1 Cells and Phosphohydrolase Assays

COS-1 cells were grown at 37.degree. C. in HEPES-buffered Dulbecco modified minimal essential medium supplemented with 4% fetal bovine serum. The G6PC constructs were transfected into COS-1 cells by the DEAE-dextran/chloroquine method. After incubation at 37.degree. C. for 2 days, the transfected cultures were harvested for phosphohydrolase activity. Briefly, reaction mixtures (50 .mu.l) containing 50 mM cacodylate buffer, pH 6.5, 10 mM G6P and appropriate amounts of cell homogenates were incubated at 37.degree. C. for 10 minutes as previously described (Lei et al., Science 262:580-583, 1993).

Statistical Analysis

The unpaired t test was performed using the GraphPad Prism Program, version 4 (GraphPad Software, San Diego, Calif.). Values were considered statistically significant at p<0.05.

Results

The phosphohydrolase activities of human and canine G6Pase-.alpha. were compared by in vitro expression assays. The results demonstrated that the canine enzyme was about 5-fold more active than the human enzyme. A sequence alignment of the canine and human sequences showed that the two enzymes differ by 18 amino acid residues (FIG. 1 and Table 2).

TABLE-US-00005 TABLE 2 Amino acid differences between human and canine G6Pase-.alpha. Amino Amino Amino Amino Amino acid acid acid acid acid Canine K3 R54 R139 K142 R196 Human E3 Q54 Q139 I142 S196 Canine Q199 R242 R247 F292 C298 Human H199 Q242 Q247 L292 S298 Canine V301 T318 T324 A332 R347 Human A301 V318 V324 V332 Q347 Canine F349 D350 D353 Human L349 G350 H353

To determine which amino acid substitutions result in increased enzymatic activity of canine G6Pase-.alpha., 18 human G6PC mutants were constructed via site-directed mutagenesis. Each mutant carried one of the corresponding amino acids from canine G6Pase-.alpha.. Phosphohydrolase activities of the 18 human G6Pase-.alpha. mutants were examined by transient expression assays. The results showed that the human G6PC-S298C construct was the most active, with 2.14-fold higher activity than the activity of the G6PC-WT construct (Table 3). The G6PC-A301V mutant was also more active, being 1.35-fold more active than the G6PC-WT. Next, a double S298C/A301V mutant (hG6PC-S298C/A301V) was constructed. This double mutant was equally as active as the single hG6PC-S298C mutant (Table 3).

TABLE-US-00006 TABLE 3 Phosphohydrolase activity of human G6PC mutants Phosphohydrolase activity G6PC construct Location (nmol/min/mg) pSVL-hG6PC-WT 164.5 .+-. 9.5 (100%) pSVL-hG6PC-E3K N-terminal 156.0 .+-. 6.3 pSVL-hG6PC-Q54R C1 169.6 .+-. 7.3 pSVL-hG6PC-Q139R C2 113.1 .+-. 6.7 pSVL-hG6PC-I142K C2 141.0 .+-. 4.4 pSVL-hG6PC-S196R H5 90.5 .+-. 2.5 pSVL-hG6PC-H199Q C3 154.0 .+-. 7.4 pSVL-hG6PC-Q242R L3 174.5 .+-. 4.sup. pSVL-hG6PC-Q247R L3 143.8 .+-. 16.4 pSVL-hG6PC-L292F H8 140.3 .+-. 20.3 pSVL-hG6PC-S298C H8 351.8 .+-. 16.4 (214%) pSVL-hG6PC-A301V H8 221.7 .+-. 12.5 (135%) pSVL-hG6PC-S298C/A301V H8 353.7 .+-. 18.8 (215%) pSVL-hG6PC-V318T L4 156.7 .+-. 16.3 pSVL-hG6PC-V324T H9 117.6 .+-. 8.2 pSVL-hG6PC-V332A H9 160.5 .+-. 13.1 pSVL-hG6PC-Q347R C-terminal 162.5 .+-. 7.9 pSVL-hG6PC-L349F C-terminal 186.4 .+-. 5.9 (113%) pSVL-hG6PC-G350D C-terminal 146.6 .+-. 16.7 pSVL-hG6PC-H353D C-terminal 164.7 .+-. 2.8

Phosphohydrolase activity of COS-1 cells transfected with a human (h) G6PC wild-type (WT) or a hG6PC mutant construct in a pSVL vector. The data represent the mean.+-.SEM. H, L, and C denote the locations of the mutations in helices 1 to 9, luminal loops 1 to 4 or cytoplasmic loops 1 to 4, respectively (FIG. 2). Numbers in parentheses are % of hG6PC-WT activity. Phosphohydrolase assays were performed in duplicate.

It has been previously shown that the structural integrity of transmembrane helices is vital to the stability and enzymatic activity of G6PC, and non-helical mutants play no essential role in the stability of G6PC (Shieh et al., J Biol Chem 277:5047-5053, 2002). In accordance with these previous observations, mutations of amino acid residues in helix 8 of G6PC markedly altered enzymatic activity.

To further confirm the efficacy of the hG6PC-S298C construct, phosphohydrolase activities of two human G6PC constructs, pSVL-G6PC-WT and pSVL-G6PC-S298C, were compared. Transient expression assays showed that the pSVL-G6PC-S298C construct was 1.7-fold more efficacious than the pSVL-G6PC construct (Table 4).

Studies have shown that codon optimization strategies can increase translation efficiency (Huston et al., Mol Ther 9:1867-1877, 2011). Codon-optimized (co) human G6PC is 1.46-fold more active than the G6PC-WT construct (Table 4). To examine the impact of codon optimization, pSVL-co-G6PC-S298C, which expresses a codon optimized human G6PC-S298C, was constructed. Transient expression assays showed that the pSVL-co-G6PC-S298C construct was 2.9-fold more efficacious than the pSVL-G6PC-WT construct (Table 4).

TABLE-US-00007 TABLE 4 Phosphohydrolase activity of hG6PC-WT, hG6PC-S298C, co-hG6PC, co-hG6PC-S298C, and canine G6PC Phosphohydrolase activity hG6PC constructs (nmol/min/mg) pSVL-hG6PC-WT 131.1 .+-. 3.8 (100%) pSVL-hG6PC-S298C 223.1 .+-. 7.1 (170%) pSVL-co- hG6PC 191.5 .+-. 6.0 (146%) pSVL-co- hG6PC-S298C 382.8 .+-. 23.5 (292%) pSVL-canine G6PC 725.7 .+-. 66.5 (554%)

Phosphohydrolase activity of COS-1 cells transfected with hG6PC-WT, hG6PC-S298C, co-hG6PC, co-hG6PC-S298C, or canine G6PC construct in a pSVL vector. The data represent the mean.+-.SEM of three independent experiments using three separate batches of each construct. Phosphohydrolase assays were performed in duplicate. Numbers in parentheses are % of hG6PC-WT activity.

Recombinant AAV8-GPE-G6PC, AAV8-GPE-co-G6PC, AAV8-GPE-G6PC-S298C and AAV8-GPE-co-G6PC-S298C vectors were constructed. Hepatic G6Pase activity was examined in GSD-Ia (G6pc-/-) mice infused with AAV8-GPE-G6PC, AAV8-GPE-G6PC-S298C, AAV8-GPE-co-G6PC, or AAV8-GPE-co-G6PC-S298C vectors. In agreement with in vitro expression studies, in vivo studies showed that the AAV8-GPE-G6PC-S298C and AAV8-co-G6PC-S298C vectors directed hepatic G6PC expression that was 3.4-fold over that of the AAV8-GPE-G6PC vector (FIG. 3).

Example 2: Evaluation of Minimal Vector Dose Required to Correct Hepatic G6Pase-.alpha. Deficiency

This example describes studies to determine the minimal dose needed to restore G6Pase-.alpha. activity to a level that prevents HCA/HCC development and maintains glucose homeostasis.

GSD-Ia is characterized by impaired glucose homeostasis and the long-term complication of hepatocellular adenoma (HCA) (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). The inventor has previously shown that rAAV8-G6PC-treated G6pc-/- mice expressing .gtoreq.3% of normal hepatic G6Pase-.alpha. activity (which is equivalent to .gtoreq.5 units of G6Pase-.alpha. activity; 1 nmol/min/mg is defined as one unit of G6Pase-.alpha. activity) maintain glucose homeostasis to age P70-P90 weeks and do not develop HCA (Lee et al., Hepatology 56:1719-1729, 2012; PCT Publication No. WO 2015/081101, which is herein incorporated by reference).

The present study was conducted using purified, accurately tittered rAAV8 vectors (supplied by Dimension Therapeutics, Cambridge, Mass.) to determine the minimal dosage of rAAV vector required to correct hepatic G6Pase-.alpha. deficiency using G6pc-/- mice. Ten-day-old G6pc-/- mice were infused with 5.times.10.sup.11 vg/kg of rAAV8-co-G6PC or rAAV8-co-G6PC-S298C. At age 24-days (2 weeks post-infusion), hepatic G6Pase-.alpha. activity in rAAV-co-G6PC-S298C- and rAAV8-co-G6PC-treated G6pc-/- mice was 18.6.+-.1.1 and 7.7.+-.1.1 units, respectively (FIG. 5).

It has been shown that the efficiency and persistence of AAV-mediated hepatic gene transfer are lower during early development because the fast rate of hepatocellular proliferation associated with liver growth can dilute out the number of cells effectively infected with AAV (Yiu et al., Mol Ther 18:1076-1084, 2010). In two-week-old G6pc-/- mice infused with 1.5.times.10.sup.13 vg/kg of rAAV-G6PC, hepatic G6Pase-.alpha. activity was 174.0.+-.22.4 units at age 24 weeks. In four-week-old G6pc-/- mice infused with 1.times.10.sup.13 vg/kg of rAAV-G6PC, hepatic G6Pase-.alpha. activity was 335.6.+-.40.2 units at age 24 weeks, which is 2.9-fold higher than the G6Pase-.alpha. activity in mice infused at age 2 weeks with the same vector dosage.

Therefore, it is expected that if the same dosage of rAAV-co-G6PC-S298C is infused into 4-week-old G6pc-/- mice, hepatic G6Pase-.alpha. activity will be restored to 53.94 (18.6.times.2.9) units at age 24 weeks, well above the minimal hepatic G6Pase-c activity (5 units) required to maintain glucose homeostasis and prevent HCA/HCC formation. This also meets the requirement of rAAV-mediated human clinical gene therapy trial for the treatment of GSD-Ia, which requires administration of .ltoreq.1.times.10.sup.12 vg/kg AAV.

To compare the relative efficacy of the four different vectors, 10-day-old G6pc-/- mice are infused with 5.times.10.sup.11 vg/kg of accurately tittered rAAV8-G6PC or rAAV8-G6PC-S298C vector and hepatic G6Pase-.alpha. activity of the treated G6pc-/- mice is examined at age 24 days. In addition, 10-day-old G6pc-/- mice are infused with 5.times.10.sup.12 vg/kg of accurately tittered rAAV8-G6PC, rAAV8-G6PC-S298C, rAAV8-co-G6PC or rAAV8-co-G6PC-S298C and hepatic G6Pase-.alpha. activity of the treated G6pc-/- mice is examined at age 12 weeks. The results of this study will demonstrate the stability of transgene expression from age 24 days to age 12 weeks.

Untreated G6pc-/- mice have a short lifespan. To more accurately determine the minimal dosage of rAAV vector required to correct hepatic G6Pase-.alpha. deficiency in adult mice, L-G6pc-/- mice, which have a liver-specific G6Pase knockout and survive to adulthood, were used as described below.

G6pc.sup.fx/fx mice contain exon 3 of the G6pc gene flanked with loxP sites (Peng et al., Genesis 47:590-594, 2009). The G6pc.sup.fx/fx mice were crossed with SA.sup.creERT2/w mice (Schuler et al., Genesis 39:167-172, 2004), which express a tamoxifen-dependent Cre-recombinase under the control of the serum albumin promoter to produce G6pc.sup.fx/fx.SA.sup.creERT2/w mice. The liver-specific G6pc knockout (L-G6pc-/-) were generated by tamoxifen-mediated excision of the G6pc exon 3 in three-week-old G6pc.sup.fx/fx.SA.sup.creERT2/w mice. It is expected that 100% of L-G6pc-/- mice will develop HCA/HCC at age 54 weeks (51 week post G6pc gene excision).

Ten-week-old L-G6pc-/- mice were treated with 10.sup.12 vg/kg of rAAV-G6PC. The results showed that at age 18 weeks, hepatic G6Pase-.alpha. activity was restored to 68.9.+-.12.8 units (FIG. 6A). The AAV-treated mice (AAV mice) exhibited no hepatic histological abnormalities except mild glycogen storage (FIG. 6B), and displayed a normal profile of fasting glucose tolerance (FGT) (FIG. 6C). Moreover, the AAV mice exhibited normalized liver weight (FIG. 6D) and normalized hepatic levels of glycogen and triglyceride (FIG. 6E). These data indicate that it is possible to scale down to a more optimal dosage for a human clinical gene therapy trial for the treatment of GSD-Ia.

To examine the minimal dosage of rAAV8-G6PC, rAAV8-co-G6PC, rAAV8-G6PC-S298C and rAAV8-co-G6PC-S298C required to correct hepatic G6Pase-.alpha. deficiency and prevent HCA/HCC development, various doses (e.g., 1.times.10.sup.10, 3.times.10.sup.10, 1.times.10.sup.11, 3.times.10.sup.11, 1.times.10.sup.12, 3.times.10.sup.12 and 1.times.10.sup.13) of each vector are infused into 10-12-week-old L-G6pc-/- mice. At age 54 weeks, G6Pase-.alpha. activity is measured. Hepatic histology, FGT, liver weight and hepatic levels of glycogen and triglyceride are also measured and/or evaluated.

Example 3: Treatment of Human GSD-Ia Using AAV-Based Gene Therapy

This example describes an exemplary method for the clinical use of AAV vectors encoding modified G6PC for the treatment of GSD-Ia.

A patient diagnosed with GSD-Ia is selected for treatment. Typically the patient is at least 18 years old and may or may not have had pre-exposure to immunomodulation. The patient is administered a therapeutically effective amount of a recombinant AAV expressing modified G6PC, such as a rAAV comprising SEQ ID NO: 4 or SEQ ID NO: 5, as disclosed herein. The recombinant AAV can be administered intravenously. An appropriate therapeutic dose can be selected by a medical practitioner. In some cases, the therapeutically effective dose is in the range of 1.times.10.sup.10 to 1.times.10.sup.14 viral particles (vp)/kg, such as about 1.times.10.sup.11 or 1.times.10.sup.12 vp/kg. In most instances, the patient is administered a single dose. In the absence of immunomodulation, the patient is likely to tolerate only a single infusion of rAAV. If the subject has had pre-exposure immunomodulation, two or more doses may be administered. The health of the subject can be monitored over time to determine the effectiveness of the treatment.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

SEQUENCE LISTINGS

1

111357PRTCanis lupus 1Met Glu Lys Gly Met Asp Val Leu His Asp Phe Gly Ile Gln Ser Thr1 5 10 15His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45Ile Trp Phe His Leu Arg Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe65 70 75 80Gly Gln Arg Pro Tyr Trp Trp Val Met Asp Thr Asp Tyr Tyr Ser Asn 85 90 95Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125Val Met Val Thr Ser Thr Leu Ser Ile Phe Arg Gly Arg Lys Arg Pro 130 135 140Thr Tyr Arg Phe Arg Cys Leu Asn Ile Leu Leu Trp Leu Gly Phe Trp145 150 155 160Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190Glu Thr Phe Arg His Ile Gln Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys225 230 235 240Ala Arg Arg Trp Cys Glu Arg Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255Pro Phe Ala Ser Leu Leu Lys Asn Val Gly Thr Leu Phe Gly Leu Gly 260 265 270Val Thr Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285Ser Lys Trp Phe Pro Phe Arg Leu Ser Cys Ile Val Val Ser Leu Ile 290 295 300Leu Leu His Leu Phe Asp Ser Leu Lys Pro Pro Ser Gln Thr Glu Leu305 310 315 320Ile Phe Tyr Thr Leu Ser Phe Cys Lys Ser Ala Ala Val Pro Leu Ala 325 330 335Ser Val Ser Leu Ile Pro Tyr Cys Leu Ala Arg Val Phe Asp Gln Pro 340 345 350Asp Lys Lys Ser Leu 3552357PRTHomo sapiens 2Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr1 5 10 15His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe65 70 75 80Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp145 150 155 160Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys225 230 235 240Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu305 310 315 320Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350His Lys Lys Ser Leu 35537671DNAArtificial SequenceSynthetic construct (pTR-GPE-human G6PC)misc_feature(17)..(163)Inverted terminal repeatmisc_feature(182)..(3045)GPEmisc_feature(3051)..(3184)Stuffer sequencemisc_feature(3185)..(3321)Intronmisc_feature(3322)..(3367)Stuffer sequencemisc_feature(3368)..(4441)human G6PC coding sequencemisc_feature(4674)..(4819)Inverted terminal repeat 3gggggggggg ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 60gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga 120gcgcgcagag agggagtggc caactccatc actaggggtt cctagatctg aattcggtac 180ccctttgaga atccacggtg tctcgatgca gtcagctttc taacaagctg gggcctcacc 240tgttttccca cggataaaaa cgtgctggag gaagcagaaa ggggctggca ggtggaaaga 300tgaggaccag ctcatcgtct catgactatg aggttgctct gatccagagg gtccccctgc 360ctggtggccc accgccagga agactcccac tgtccctgga tgcccagagt gggatgtcaa 420ctccatcact tatcaactcc ttatccatag gggtattctt cctgaggcgt ctcagaaaac 480agggccctcc ccatatgctg accacataat agaacccctc ccaactcaga gaccctggct 540gctagctgcc ctggcatgac ccagacagtg gcctttgtat atgtttttag actcaccttg 600actcacctct gaccatagaa actctcatcc cagaggtcac tgcaatagtt actccacaac 660agaggcttat ctgggtagag ggaggctccc tacctatggc ccagcagccc tgacagtgca 720gatcacatat accccacgcc ccagcactgc ctgccacgca tgggcttact ttacacccac 780ccacagtcac caacacatta cctgctctcc aaggttaggc gtggcaggag aagtttgctt 840ggaccagcag aaaccatgca gtcaaggaca actggagtca gcatgggctg ggtgcgagcc 900cttggtgggg tggggaggag actccaggtc atacctcctg gaggatgttt taatcatttc 960cagcatggaa tgctgtcaac ttttgccaca gattcattag ctctgagttt cttttttctg 1020tccccagcta ccccttacat gtcaatatgg acttaatgat gggaaattca ggcaagtttt 1080taaacatttt attccccctg gctcttatcc tcaaaaaatg catgaatttg gaggcagtgg 1140ctcatgcctg taatcccaat gctttgctag gttgaggcgg gaggatcact tgaagccagg 1200aatttgagac cagcctgggc cgcatagtga gaccccgttt ctacaaaaat aaataaataa 1260ataataaata atagtgatat gaagcatgat taaatagccc tattttttaa aatgcatgag 1320ttcgttacct gattcattcc ctggttcctt tcacagtcct ccgtgaccca agtgttaggg 1380ttttggtctc tctactattt gtaggctgat atatagtata cacacacaca cacacacaca 1440tatacacaca cacagtgtat cttgagcttt cttttgtata tctacacaca tatgtataag 1500aaagctcaag atatagaagc cctttttcaa aaataactga aagtttcaaa ctctttaagt 1560ctccagttac cattttgctg gtattcttat ttggaaccat acattcatca tattgttgca 1620cagtaagact atacattcat tattttgctt aaacgtatga gttaaaacac ttggccaggc 1680atggtggttc acacctgtaa tcccagagct ttgggaagcc aagactggca gatctcttga 1740gctcaggaat tcaagaccag cctgggcaac atggaaaaac cccatctcta caaaagatag 1800aaaaattagc caggcatggt ggcgtgtgcc tgtggtccca gctactcagg aggctgaggt 1860gggaggatca cattagccca ggaggttgag gctgcagtga gccgtgatta tgccactgca 1920ctccagcctg ggagacagag tgagaccctg tttcaaaaaa aagagagaga aaatttaaaa 1980aagaaaacaa caccaagggc tgtaacttta aggtcattaa atgaattaat cactgcattc 2040aaaaacgatt actttctggc cctaagagac atgaggccaa taccaggaag ggggttgatc 2100tcccaaacca gaggcagacc ctagactcta atacagttaa ggaaagacca gcaagatgat 2160agtccccaat acaatagaag ttactatatt ttatttgttg tttttctttt gttttgtttt 2220gttttgtttt gttttgtttt agagactggg gtcttgctcg attgcccagg ctgtagtgca 2280gcggtgggac aatagctcac tgcagactcc aactcctggg ctcaagcaat cctcctgcct 2340cagcctcctg aatagctggg actacaaggg tacaccatca cacacaccaa aacaattttt 2400taaatttttg tgtagaaacg agggtcttgc tttgttgccc aggctggtct ccaactcctg 2460gcttcaaggg atcctcccac ctcagcctcc caaattgctg ggattacagg tgtgagccac 2520cacaaccagc cagaacttta ctaattttaa aattaagaac ttaaaacttg aatagctaga 2580gcaccaagat ttttctttgt ccccaaataa gtgcagttgc aggcatagaa aatctgacat 2640ctttgcaaga atcatcgtgg atgtagactc tgtcctgtgt ctctggcctg gtttcgggga 2700ccaggagggc agacccttgc actgccaaga agcatgccaa agttaatcat tggccctgct 2760gagtacatgg ccgatcaggc tgtttttgtg tgcctgtttt tctattttac gtaaatcacc 2820ctgaacatgt ttgcatcaac ctactggtga tgcacctttg atcaatacat tttagacaaa 2880cgtggttttt gagtccaaag atcagggctg ggttgacctg aatactggat acagggcata 2940taaaacaggg gcaaggcaca gactcatagc agagcaatca ccaccaagcc tggaataact 3000gcaagggctc tgctgacatc ttcctgaggt gccaaggaaa tgaggtctag agaagcttta 3060ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct 3120cgaacttaag ctgcagtgac tctcttaagg tagccttgca gaagttggtc gtgaggcact 3180gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg 3240tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac 3300tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggccctgca 3360ggccaccatg gaggaaggaa tgaatgttct ccatgacttt gggatccagt caacacatta 3420cctccaggtg aattaccaag actcccagga ctggttcatc ttggtgtccg tgatcgcaga 3480cctcaggaat gccttctacg tcctcttccc catctggttc catcttcagg aagctgtggg 3540cattaaactc ctttgggtag ctgtgattgg agactggctc aacctcgtct ttaagtggat 3600tctctttgga cagcgtccat actggtgggt tttggatact gactactaca gcaacacttc 3660cgtgcccctg ataaagcagt tccctgtaac ctgtgagact ggaccaggga gcccctctgg 3720ccatgccatg ggcacagcag gtgtatacta cgtgatggtc acatctactc tttccatctt 3780tcagggaaag ataaagccga cctacagatt tcggtgcttg aatgtcattt tgtggttggg 3840attctgggct gtgcagctga atgtctgtct gtcacgaatc taccttgctg ctcattttcc 3900tcatcaagtt gttgctggag tcctgtcagg cattgctgtt gcagaaactt tcagccacat 3960ccacagcatc tataatgcca gcctcaagaa atattttctc attaccttct tcctgttcag 4020cttcgccatc ggattttatc tgctgctcaa gggactgggt gtagacctcc tgtggactct 4080ggagaaagcc cagaggtggt gcgagcagcc agaatgggtc cacattgaca ccacaccctt 4140tgccagcctc ctcaagaacc tgggcacgct ctttggcctg gggctggctc tcaactccag 4200catgtacagg gagagctgca aggggaaact cagcaagtgg ctcccattcc gcctcagctc 4260tattgtagcc tccctcgtcc tcctgcacgt ctttgactcc ttgaaacccc catcccaagt 4320cgagctggtc ttctacgtct tgtccttctg caagagtgcg gtagtgcccc tggcatccgt 4380cagtgtcatc ccctactgcc tcgcccaggt cctgggccag ccgcacaaga agtcgttgta 4440agcggccgcg gggatccaga catgataaga tacattgatg agtttggaca aaccacaact 4500agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 4560accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 4620gttcaggggg aggtgtggga ggttttttag tcgaccatgc tggggagaga tctaggaacc 4680cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg 4740ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 4800cagagaggga gtggccaacc cccccccccc cccccctgca gccctgcatt aatgaatcgg 4860ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4920ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4980acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5040aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5100tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5160aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5220gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 5280acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5340accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5400ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5460gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 5520gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 5580ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 5640gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 5700cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5760cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5820gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 5880tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 5940gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6000agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6060tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6120agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6180gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6240catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6300ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 6360atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 6420tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 6480cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 6540cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 6600atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 6660aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 6720ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 6780aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 6840aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 6900cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 6960agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 7020tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 7080ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 7140gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 7200aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 7260ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 7320aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 7380agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 7440tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 7500ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 7560gccgcgctta atgcgccgct acagggcgcg tcgcgccatt cgccattcag gctacgcaac 7620tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccaggctgc a 767147671DNAArtificial SequenceSynthetic construct (pTR-GPE-human G6PC-S298C)misc_feature(17)..(163)Inverted terminal repeatmisc_feature(182)..(3045)GPEmisc_feature(3051)..(3184)Stuffer sequencemisc_feature(3185)..(3321)Intronmisc_feature(3322)..(3367)Stuffer sequencemisc_feature(3368)..(4441)human G6PC S298C coding sequencemisc_feature(4259)..(4261)Codon changemisc_feature(4674)..(4819)Inverted terminal repeat 4gggggggggg ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 60gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga 120gcgcgcagag agggagtggc caactccatc actaggggtt cctagatctg aattcggtac 180ccctttgaga atccacggtg tctcgatgca gtcagctttc taacaagctg gggcctcacc 240tgttttccca cggataaaaa cgtgctggag gaagcagaaa ggggctggca ggtggaaaga 300tgaggaccag ctcatcgtct catgactatg aggttgctct gatccagagg gtccccctgc 360ctggtggccc accgccagga agactcccac tgtccctgga tgcccagagt gggatgtcaa 420ctccatcact tatcaactcc ttatccatag gggtattctt cctgaggcgt ctcagaaaac 480agggccctcc ccatatgctg accacataat agaacccctc ccaactcaga gaccctggct 540gctagctgcc ctggcatgac ccagacagtg gcctttgtat atgtttttag actcaccttg 600actcacctct gaccatagaa actctcatcc cagaggtcac tgcaatagtt actccacaac 660agaggcttat ctgggtagag ggaggctccc tacctatggc ccagcagccc tgacagtgca 720gatcacatat accccacgcc ccagcactgc ctgccacgca tgggcttact ttacacccac 780ccacagtcac caacacatta cctgctctcc aaggttaggc gtggcaggag aagtttgctt 840ggaccagcag aaaccatgca gtcaaggaca actggagtca gcatgggctg ggtgcgagcc 900cttggtgggg tggggaggag actccaggtc atacctcctg gaggatgttt taatcatttc 960cagcatggaa tgctgtcaac ttttgccaca gattcattag ctctgagttt cttttttctg 1020tccccagcta ccccttacat gtcaatatgg acttaatgat gggaaattca ggcaagtttt 1080taaacatttt attccccctg gctcttatcc tcaaaaaatg catgaatttg gaggcagtgg 1140ctcatgcctg taatcccaat gctttgctag gttgaggcgg gaggatcact tgaagccagg 1200aatttgagac cagcctgggc cgcatagtga gaccccgttt ctacaaaaat aaataaataa 1260ataataaata atagtgatat gaagcatgat taaatagccc tattttttaa aatgcatgag 1320ttcgttacct gattcattcc ctggttcctt tcacagtcct ccgtgaccca agtgttaggg 1380ttttggtctc tctactattt gtaggctgat atatagtata cacacacaca cacacacaca 1440tatacacaca cacagtgtat cttgagcttt cttttgtata tctacacaca tatgtataag 1500aaagctcaag atatagaagc cctttttcaa aaataactga aagtttcaaa ctctttaagt 1560ctccagttac cattttgctg gtattcttat ttggaaccat acattcatca tattgttgca 1620cagtaagact atacattcat tattttgctt aaacgtatga gttaaaacac ttggccaggc 1680atggtggttc acacctgtaa tcccagagct ttgggaagcc aagactggca gatctcttga 1740gctcaggaat tcaagaccag cctgggcaac atggaaaaac cccatctcta caaaagatag 1800aaaaattagc caggcatggt ggcgtgtgcc tgtggtccca gctactcagg aggctgaggt 1860gggaggatca cattagccca ggaggttgag gctgcagtga gccgtgatta tgccactgca 1920ctccagcctg ggagacagag tgagaccctg tttcaaaaaa aagagagaga aaatttaaaa 1980aagaaaacaa caccaagggc tgtaacttta aggtcattaa atgaattaat cactgcattc 2040aaaaacgatt actttctggc cctaagagac atgaggccaa taccaggaag ggggttgatc 2100tcccaaacca gaggcagacc ctagactcta atacagttaa ggaaagacca gcaagatgat 2160agtccccaat acaatagaag ttactatatt ttatttgttg tttttctttt gttttgtttt 2220gttttgtttt

gttttgtttt agagactggg gtcttgctcg attgcccagg ctgtagtgca 2280gcggtgggac aatagctcac tgcagactcc aactcctggg ctcaagcaat cctcctgcct 2340cagcctcctg aatagctggg actacaaggg tacaccatca cacacaccaa aacaattttt 2400taaatttttg tgtagaaacg agggtcttgc tttgttgccc aggctggtct ccaactcctg 2460gcttcaaggg atcctcccac ctcagcctcc caaattgctg ggattacagg tgtgagccac 2520cacaaccagc cagaacttta ctaattttaa aattaagaac ttaaaacttg aatagctaga 2580gcaccaagat ttttctttgt ccccaaataa gtgcagttgc aggcatagaa aatctgacat 2640ctttgcaaga atcatcgtgg atgtagactc tgtcctgtgt ctctggcctg gtttcgggga 2700ccaggagggc agacccttgc actgccaaga agcatgccaa agttaatcat tggccctgct 2760gagtacatgg ccgatcaggc tgtttttgtg tgcctgtttt tctattttac gtaaatcacc 2820ctgaacatgt ttgcatcaac ctactggtga tgcacctttg atcaatacat tttagacaaa 2880cgtggttttt gagtccaaag atcagggctg ggttgacctg aatactggat acagggcata 2940taaaacaggg gcaaggcaca gactcatagc agagcaatca ccaccaagcc tggaataact 3000gcaagggctc tgctgacatc ttcctgaggt gccaaggaaa tgaggtctag agaagcttta 3060ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct 3120cgaacttaag ctgcagtgac tctcttaagg tagccttgca gaagttggtc gtgaggcact 3180gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg 3240tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac 3300tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggccctgca 3360ggccaccatg gaggaaggaa tgaatgttct ccatgacttt gggatccagt caacacatta 3420cctccaggtg aattaccaag actcccagga ctggttcatc ttggtgtccg tgatcgcaga 3480cctcaggaat gccttctacg tcctcttccc catctggttc catcttcagg aagctgtggg 3540cattaaactc ctttgggtag ctgtgattgg agactggctc aacctcgtct ttaagtggat 3600tctctttgga cagcgtccat actggtgggt tttggatact gactactaca gcaacacttc 3660cgtgcccctg ataaagcagt tccctgtaac ctgtgagact ggaccaggga gcccctctgg 3720ccatgccatg ggcacagcag gtgtatacta cgtgatggtc acatctactc tttccatctt 3780tcagggaaag ataaagccga cctacagatt tcggtgcttg aatgtcattt tgtggttggg 3840attctgggct gtgcagctga atgtctgtct gtcacgaatc taccttgctg ctcattttcc 3900tcatcaagtt gttgctggag tcctgtcagg cattgctgtt gcagaaactt tcagccacat 3960ccacagcatc tataatgcca gcctcaagaa atattttctc attaccttct tcctgttcag 4020cttcgccatc ggattttatc tgctgctcaa gggactgggt gtagacctcc tgtggactct 4080ggagaaagcc cagaggtggt gcgagcagcc agaatgggtc cacattgaca ccacaccctt 4140tgccagcctc ctcaagaacc tgggcacgct ctttggcctg gggctggctc tcaactccag 4200catgtacagg gagagctgca aggggaaact cagcaagtgg ctcccattcc gcctcagctg 4260cattgtagcc tccctcgtcc tcctgcacgt ctttgactcc ttgaaacccc catcccaagt 4320cgagctggtc ttctacgtct tgtccttctg caagagtgcg gtagtgcccc tggcatccgt 4380cagtgtcatc ccctactgcc tcgcccaggt cctgggccag ccgcacaaga agtcgttgta 4440agcggccgcg gggatccaga catgataaga tacattgatg agtttggaca aaccacaact 4500agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 4560accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 4620gttcaggggg aggtgtggga ggttttttag tcgaccatgc tggggagaga tctaggaacc 4680cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg 4740ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 4800cagagaggga gtggccaacc cccccccccc cccccctgca gccctgcatt aatgaatcgg 4860ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4920ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4980acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5040aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5100tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5160aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5220gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 5280acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5340accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5400ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5460gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 5520gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 5580ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 5640gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 5700cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5760cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5820gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 5880tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 5940gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6000agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6060tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6120agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6180gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6240catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6300ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 6360atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 6420tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 6480cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 6540cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 6600atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 6660aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 6720ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 6780aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 6840aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 6900cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 6960agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 7020tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 7080ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 7140gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 7200aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 7260ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 7320aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 7380agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 7440tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 7500ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 7560gccgcgctta atgcgccgct acagggcgcg tcgcgccatt cgccattcag gctacgcaac 7620tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccaggctgc a 767157671DNAArtificial SequenceSynthetic construct (pTR-GPE-co-G6PC-S298C)misc_feature(17)..(163)Inverted terminal repeatmisc_feature(182)..(3045)GPEmisc_feature(3051)..(3184)Stuffer sequencemisc_feature(3185)..(3321)Intronmisc_feature(3322)..(3367)Stuffer sequencemisc_feature(3368)..(4441)human codon-optimized G6PC coding sequencemisc_feature(4259)..(4261)Codon changemisc_feature(4674)..(4819)Inverted terminal repeat 5gggggggggg ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 60gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga 120gcgcgcagag agggagtggc caactccatc actaggggtt cctagatctg aattcggtac 180ccctttgaga atccacggtg tctcgatgca gtcagctttc taacaagctg gggcctcacc 240tgttttccca cggataaaaa cgtgctggag gaagcagaaa ggggctggca ggtggaaaga 300tgaggaccag ctcatcgtct catgactatg aggttgctct gatccagagg gtccccctgc 360ctggtggccc accgccagga agactcccac tgtccctgga tgcccagagt gggatgtcaa 420ctccatcact tatcaactcc ttatccatag gggtattctt cctgaggcgt ctcagaaaac 480agggccctcc ccatatgctg accacataat agaacccctc ccaactcaga gaccctggct 540gctagctgcc ctggcatgac ccagacagtg gcctttgtat atgtttttag actcaccttg 600actcacctct gaccatagaa actctcatcc cagaggtcac tgcaatagtt actccacaac 660agaggcttat ctgggtagag ggaggctccc tacctatggc ccagcagccc tgacagtgca 720gatcacatat accccacgcc ccagcactgc ctgccacgca tgggcttact ttacacccac 780ccacagtcac caacacatta cctgctctcc aaggttaggc gtggcaggag aagtttgctt 840ggaccagcag aaaccatgca gtcaaggaca actggagtca gcatgggctg ggtgcgagcc 900cttggtgggg tggggaggag actccaggtc atacctcctg gaggatgttt taatcatttc 960cagcatggaa tgctgtcaac ttttgccaca gattcattag ctctgagttt cttttttctg 1020tccccagcta ccccttacat gtcaatatgg acttaatgat gggaaattca ggcaagtttt 1080taaacatttt attccccctg gctcttatcc tcaaaaaatg catgaatttg gaggcagtgg 1140ctcatgcctg taatcccaat gctttgctag gttgaggcgg gaggatcact tgaagccagg 1200aatttgagac cagcctgggc cgcatagtga gaccccgttt ctacaaaaat aaataaataa 1260ataataaata atagtgatat gaagcatgat taaatagccc tattttttaa aatgcatgag 1320ttcgttacct gattcattcc ctggttcctt tcacagtcct ccgtgaccca agtgttaggg 1380ttttggtctc tctactattt gtaggctgat atatagtata cacacacaca cacacacaca 1440tatacacaca cacagtgtat cttgagcttt cttttgtata tctacacaca tatgtataag 1500aaagctcaag atatagaagc cctttttcaa aaataactga aagtttcaaa ctctttaagt 1560ctccagttac cattttgctg gtattcttat ttggaaccat acattcatca tattgttgca 1620cagtaagact atacattcat tattttgctt aaacgtatga gttaaaacac ttggccaggc 1680atggtggttc acacctgtaa tcccagagct ttgggaagcc aagactggca gatctcttga 1740gctcaggaat tcaagaccag cctgggcaac atggaaaaac cccatctcta caaaagatag 1800aaaaattagc caggcatggt ggcgtgtgcc tgtggtccca gctactcagg aggctgaggt 1860gggaggatca cattagccca ggaggttgag gctgcagtga gccgtgatta tgccactgca 1920ctccagcctg ggagacagag tgagaccctg tttcaaaaaa aagagagaga aaatttaaaa 1980aagaaaacaa caccaagggc tgtaacttta aggtcattaa atgaattaat cactgcattc 2040aaaaacgatt actttctggc cctaagagac atgaggccaa taccaggaag ggggttgatc 2100tcccaaacca gaggcagacc ctagactcta atacagttaa ggaaagacca gcaagatgat 2160agtccccaat acaatagaag ttactatatt ttatttgttg tttttctttt gttttgtttt 2220gttttgtttt gttttgtttt agagactggg gtcttgctcg attgcccagg ctgtagtgca 2280gcggtgggac aatagctcac tgcagactcc aactcctggg ctcaagcaat cctcctgcct 2340cagcctcctg aatagctggg actacaaggg tacaccatca cacacaccaa aacaattttt 2400taaatttttg tgtagaaacg agggtcttgc tttgttgccc aggctggtct ccaactcctg 2460gcttcaaggg atcctcccac ctcagcctcc caaattgctg ggattacagg tgtgagccac 2520cacaaccagc cagaacttta ctaattttaa aattaagaac ttaaaacttg aatagctaga 2580gcaccaagat ttttctttgt ccccaaataa gtgcagttgc aggcatagaa aatctgacat 2640ctttgcaaga atcatcgtgg atgtagactc tgtcctgtgt ctctggcctg gtttcgggga 2700ccaggagggc agacccttgc actgccaaga agcatgccaa agttaatcat tggccctgct 2760gagtacatgg ccgatcaggc tgtttttgtg tgcctgtttt tctattttac gtaaatcacc 2820ctgaacatgt ttgcatcaac ctactggtga tgcacctttg atcaatacat tttagacaaa 2880cgtggttttt gagtccaaag atcagggctg ggttgacctg aatactggat acagggcata 2940taaaacaggg gcaaggcaca gactcatagc agagcaatca ccaccaagcc tggaataact 3000gcaagggctc tgctgacatc ttcctgaggt gccaaggaaa tgaggtctag agaagcttta 3060ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct 3120cgaacttaag ctgcagtgac tctcttaagg tagccttgca gaagttggtc gtgaggcact 3180gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg 3240tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac 3300tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggccctgca 3360ggccaccatg gaagagggca tgaacgtgct gcacgacttc ggcatccaga gcacccacta 3420tctgcaggtc aactaccagg acagccagga ctggttcatc ctggtgtccg tgatcgccga 3480cctgcggaac gccttctacg tgctgttccc catctggttc catctgcaag aagccgtcgg 3540catcaagctg ctgtgggtgg ccgtgatcgg cgattggctg aacctggtgt tcaagtggat 3600cctgttcggc cagcggccct attggtgggt gctggacacc gactactaca gcaacaccag 3660cgtgcccctg atcaagcagt tccccgtgac ctgcgagaca ggccctggct ctccttctgg 3720ccacgccatg ggaacagccg gcgtgtacta cgtgatggtc accagcaccc tgagcatctt 3780ccagggcaag atcaagccca cctaccggtt ccggtgcctg aacgtgatcc tgtggctggg 3840cttctgggcc gtgcagctga acgtgtgcct gagccggatc tacctggccg cccacttccc 3900acatcaagtg gtggccggcg tgctgagcgg aatcgccgtg gccgagacat tcagccacat 3960ccacagcatc tacaacgcca gcctgaagaa gtacttcctg atcacattct ttctgttcag 4020cttcgccatc ggcttctacc tgctgctgaa gggcctgggc gtggacctgc tgtggaccct 4080ggaaaaggcc cagcggtggt gcgagcagcc cgagtgggtg cacatcgaca ccaccccctt 4140cgccagcctg ctgaagaacc tgggcaccct gtttggactg ggcctggccc tgaacagcag 4200catgtacaga gagagctgca agggcaagct gagcaagtgg ctgcccttcc ggctgagctg 4260catcgtggcc agcctggtgc tgctgcacgt gttcgacagc ctgaagcccc ccagccaggt 4320ggaactggtg ttttacgtgc tgagcttctg caagagcgcc gtggtgcccc tggcctccgt 4380gtctgtgatc ccctactgcc tggctcaggt gctgggccag ccccacaaga agtccctctg 4440agcggccgcg gggatccaga catgataaga tacattgatg agtttggaca aaccacaact 4500agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 4560accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 4620gttcaggggg aggtgtggga ggttttttag tcgaccatgc tggggagaga tctaggaacc 4680cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg 4740ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 4800cagagaggga gtggccaacc cccccccccc cccccctgca gccctgcatt aatgaatcgg 4860ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4920ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4980acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5040aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5100tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5160aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5220gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 5280acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5340accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5400ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5460gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 5520gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 5580ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 5640gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 5700cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5760cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5820gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 5880tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 5940gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6000agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6060tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6120agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6180gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6240catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6300ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 6360atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 6420tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 6480cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 6540cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 6600atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 6660aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 6720ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 6780aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 6840aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 6900cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 6960agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 7020tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 7080ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 7140gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 7200aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 7260ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 7320aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 7380agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 7440tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 7500ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 7560gccgcgctta atgcgccgct acagggcgcg tcgcgccatt cgccattcag gctacgcaac 7620tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccaggctgc a 767161074DNAArtificial SequenceSynthetic polynucleotide 6atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctgcattgta 900gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 107471074DNAArtificial SequenceSynthetic polynucleotide 7atggaagagg gcatgaacgt gctgcacgac ttcggcatcc agagcaccca ctatctgcag 60gtcaactacc aggacagcca ggactggttc atcctggtgt ccgtgatcgc cgacctgcgg 120aacgccttct acgtgctgtt ccccatctgg ttccatctgc aagaagccgt cggcatcaag 180ctgctgtggg tggccgtgat cggcgattgg ctgaacctgg tgttcaagtg gatcctgttc 240ggccagcggc cctattggtg ggtgctggac accgactact acagcaacac cagcgtgccc 300ctgatcaagc

agttccccgt gacctgcgag acaggccctg gctctccttc tggccacgcc 360atgggaacag ccggcgtgta ctacgtgatg gtcaccagca ccctgagcat cttccagggc 420aagatcaagc ccacctaccg gttccggtgc ctgaacgtga tcctgtggct gggcttctgg 480gccgtgcagc tgaacgtgtg cctgagccgg atctacctgg ccgcccactt cccacatcaa 540gtggtggccg gcgtgctgag cggaatcgcc gtggccgaga cattcagcca catccacagc 600atctacaacg ccagcctgaa gaagtacttc ctgatcacat tctttctgtt cagcttcgcc 660atcggcttct acctgctgct gaagggcctg ggcgtggacc tgctgtggac cctggaaaag 720gcccagcggt ggtgcgagca gcccgagtgg gtgcacatcg acaccacccc cttcgccagc 780ctgctgaaga acctgggcac cctgtttgga ctgggcctgg ccctgaacag cagcatgtac 840agagagagct gcaagggcaa gctgagcaag tggctgccct tccggctgag ctgcatcgtg 900gccagcctgg tgctgctgca cgtgttcgac agcctgaagc cccccagcca ggtggaactg 960gtgttttacg tgctgagctt ctgcaagagc gccgtggtgc ccctggcctc cgtgtctgtg 1020atcccctact gcctggctca ggtgctgggc cagccccaca agaagtccct ctga 10748357PRTArtificial SequenceSynthetic polypeptide 8Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr1 5 10 15His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe65 70 75 80Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp145 150 155 160Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys225 230 235 240Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285Ser Lys Trp Leu Pro Phe Arg Leu Ser Cys Ile Val Ala Ser Leu Val 290 295 300Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu305 310 315 320Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350His Lys Lys Ser Leu 3559357PRTArtificial SequenceSynthetic polypeptide 9Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr1 5 10 15His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe65 70 75 80Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp145 150 155 160Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys225 230 235 240Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285Ser Lys Trp Leu Pro Phe Arg Leu Ser Cys Ile Val Val Ser Leu Val 290 295 300Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu305 310 315 320Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350His Lys Lys Ser Leu 35510357PRTArtificial SequenceSynthetic polypeptideMISC_FEATURE(3)..(3)Xaa = Lys or GluMISC_FEATURE(54)..(54)Xaa = Arg or GlnMISC_FEATURE(139)..(139)Xaa = Arg or GlnMISC_FEATURE(142)..(142)Xaa = Lys or IleMISC_FEATURE(196)..(196)Xaa = Arg or SerMISC_FEATURE(199)..(199)Xaa = Gln or HisMISC_FEATURE(242)..(242)Xaa = Arg or GlnMISC_FEATURE(247)..(247)Xaa = Arg or GlnMISC_FEATURE(292)..(292)Xaa = Phe or LeuMISC_FEATURE(298)..(298)Xaa = Cys or SerMISC_FEATURE(301)..(301)Xaa = Val or AlaMISC_FEATURE(318)..(318)Xaa = Thr or ValMISC_FEATURE(324)..(324)Xaa = Thr or ValMISC_FEATURE(332)..(332)Xaa = Ala or ValMISC_FEATURE(347)..(347)Xaa = Arg or GlnMISC_FEATURE(349)..(349)Xaa = Phe or LeuMISC_FEATURE(350)..(350)Xaa = Asp or GlyMISC_FEATURE(353)..(353)Xaa = Asp or His 10Met Glu Xaa Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr1 5 10 15His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45Ile Trp Phe His Leu Xaa Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe65 70 75 80Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125Val Met Val Thr Ser Thr Leu Ser Ile Phe Xaa Gly Lys Xaa Lys Pro 130 135 140Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp145 150 155 160Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190Glu Thr Phe Xaa His Ile Xaa Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys225 230 235 240Ala Xaa Arg Trp Cys Glu Xaa Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285Ser Lys Trp Xaa Pro Phe Arg Leu Ser Xaa Ile Val Xaa Ser Leu Val 290 295 300Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Xaa Glu Leu305 310 315 320Val Phe Tyr Xaa Leu Ser Phe Cys Lys Ser Ala Xaa Val Pro Leu Ala 325 330 335Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Xaa Val Xaa Xaa Gln Pro 340 345 350Xaa Lys Lys Ser Leu 355111074DNAHomo sapiens 11atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074