Methods to identify mutations associated with human genetic diseases

Abstract

This disclosure provides a bacterial or yeast cell comprising a heterologous gene encoding an enzyme that is substituted for the endogenous bacterial or yeast counterpart gene. In one aspect, the heterologous gene encodes a human enzyme related to a human metabolic disorder, e.g., human glucose-6-phosphate isomerase (GPI), glucose 6-phosphate dehydrogenase (G6PD), or human argininosuccinate lysase (ASL) or a mutant or variant of each thereof. This disclosure addresses the limitation of current drug discovery methods that often rely on testing human enzymes in artificial settings. In one aspect, it provides a simpler, quicker, and more cost-effective way to test drugs in a living organism that mimics some human cellular conditions.

Description

Atty. Dkt. No.: 114198-3710METHODS TO IDENTIFY MUTATIONS ASSOCIATED WITH HUMAN GENETIC DISEASESCROSS-REFERENCE TO RELATED PATENT APPLICATION[00011 This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 744,114, filed January 10, 2025, the entire contents of which are incorporated herein by reference in their entireties.BACKGROUND

[0002] The human genome sequence varies between individuals. Everyone typically harbors between 250 to 300 loss-of-function sequence variations relative to the reference human genome. These sequence variations often underlie inherited disorders, making the identification of causal mutations an essential pursuit in human biology and personalized medicine. This imperative is underscored by recent strides in in vivo genome editing, which offer promising avenues for treating conditions like eye and liver disease.

[0003] Traditionally, the identification of genotypes associated with genetic disease has relied on direct patient analysis and subsequent documentation as case reports. Once genetic variations linked to specific disorders are mapped, they are studied through various methodologies. One common approach involves in vitro studies utilizing materials obtained from patients or recombinant proteins to characterize mutant proteins. However, this approach is constrained by sample availability and necessitates labor-intensive purification processes. Moreover, it often requires specialized assay systems tailored to individual proteins, alongside time-consuming analytical methods and instrumentation. Additionally, dilute in vitro conditions do not replicate crowded and dense gel-like physiological conditions, necessitating an in vivo system for an accurate biochemical assay.

[0004] Alternatively, large-scale computational surveys, such as genome-wide association studies based on population sequencing datasets5, have proven effective in identifying potential associations between thousands of genetic changes to pathological conditions. However, such associations do not establish causality, particularly given the vast number of uncharacterized genetic variations and confounding factors of zygosity and genetic linkage.-1- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710Furthermore, variations that induce severe pathogenicity from early onset (such as in newborns and infants) are rare in the human population, making it challenging to obtain samples or detect such variations in population databases. As a result, pathogenic mutations remain practically undetectable, while detected mutations are more likely to have only a minor impact on enzyme activity. This a need exists in the art for accurate and fast testing methods. This disclosure satisfies this need and provides related advantages as well.SUMMARY OF THE DISCLOSURE

[0005] Current drug discovery methods often rely on testing human enzymes in artificial settings, like in test tubes or petri dishes, which lack the complexity of a living system and can lead to inaccurate results. Alternatively, testing in human cells or animals is costly, timeconsuming, and ethically challenging, especially for initial screenings of many drug compounds. This system addresses these limitations by using E. coli bacteria with human enzymes added. It offers a simpler, quicker, and more cost-effective way to test drugs in a living organism that mimics some human cellular conditions.

[0006] In one aspect, this disclosure provides a high-throughput screening platform designed to measure human enzyme activity (Live E. coli Assay (LEICA)). It facilitates the rapid identification of human mutations associated with genetic diseases and compounds that modulate the activity of human enzymes. In this system, E. coli strains are engineered to express human enzymes instead of their endogenous enzymes, allowing the bacterial growth rate to be contingent to human enzyme activity. By substituting bacterial enzymes with their human counterparts, LEICA effectively connects the functionality of human enzymes to bacterial survival. This setup provides a rapid and cost-effective alternative to traditional in vitro assays and can be applied across enzymes relevant to human metabolic disorders. This system offers a simpler, quicker, and more cost-effective way to test drugs in a living organism that mimics some human cellular conditions.

[0007] In one aspect, Applicant provides herein a bacterial or yeast cell comprising a heterologous gene that encodes an enzyme is substituted for the endogenous bacterial or yeast counterpart gene. In one aspect, the bacterial or yeast cell further comprises the deletion or inactivation of at least one or all endogenous bacterial or yeast counterpart genes capable of-2- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710catalyzing the same biochemical reaction as the counterpart heterologous enzyme or enzymes. In a further aspect, the cell further couples cell growth to the activity of a heterologous enzyme or enzymes. In one aspect, the heterologous gene is an animal gene, a mammalian gene, a human gene, a gene from a pathogen, or a gene from a parasite. In a further aspect, the heterologous gene is a human gene. In a further aspect, the bacterial or yeast cell is genetically modified to delete or inactivate the heterologous gene that encodes an enzyme is substituted for the endogenous bacterial or yeast counterpart gene. In one embodiment, least one or all endogenous bacterial or yeast genes capable of catalyzing the same biochemical reaction as the heterologous enzyme or enzymes are deleted or inactivated, thereby coupling cell growth to the activity of the heterologous enzyme or enzymes encoded by the heterologous gene or genes. In a further embodiment, the bacterial or yeast cell further lacks or inactivates additional bacterial or yeast genes in the same or intersecting metabolic pathway, thereby isolating the heterologous enzyme’s catalytic activity from alternative metabolic flux routes. Non-limiting examples of such include a human enzyme selected from the group of human glucose-6-phosphate isomerase (GPI), human glucose-6-phosphate dehydrogenase (G6PD), and human argininosuccinate lyase (ASL), and variants of each thereof. Examples of such G6PD genes include polynucleotides encoding proteins from the group of SEQ IDNOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence, and polynucleotides from the group of SEQ ID NOS: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence. Examples of such GPI genes include polynucleotides encoding proteins from the group of SEQ ID NOS: 39, 41, 43, 45, 47, 49, and 51, and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence and polynucleotides from the group of SEQ ID NOS: 38, 40, 42, 44, 46, 48, and 50, and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence. Examples of such ASL genes include polynucleotides encoding proteins from the group of SEQ ID NOS: 53, 55, 57, 59, and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence and polynucleotides from the group of SEQ ID NOS: 52, 54, 56, 58, and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence.-3- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0008] In another aspect, the bacterial or yeast cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate dehydrogenase (zwf) gene and comprises a human GPI gene inserted into the chromosome, the pgi locus, or expressed by a plasmid DNA. In an alternate embodiment, the bacterial or yeast cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate dehydrogenase (zwf) gene and comprises a human G6PD gene inserted into the chromosome or expressed by a plasmid DNA. Non-limiting examples of such are described herein.|0009] Also provided are population and cultures of these cells and use thereof, as described herein.

[0010] In one aspect, Applicant provided a bacterial cell comprising a human gene that is substituted for the endogenous bacterial counterpart gene. In one aspect, the human gene encodes a human enzyme, optionally related to a human metabolic disorder. Examples of such are provided herein and incorporated by reference into this paragraph. In one aspect, the human enzyme is human glucose-6-phosphate isomerase (GPI) or human argininosuccinate lysase (argH) or a mutant thereof. Non-limiting examples of such are described herein.

[0011] In another aspect, the bacterial cell is anE. coli cell.

[0012] In one embodiment, the endogenous bacterial gene is knocked-out or has reduced expression. Non-limiting examples of such include a cell having a double-knockout bacterial phosphoglucose isomerase (pgi) gene and bacterial glucose-6-phosphate dehydrogenase (zwf) gene. In one aspect, the cell further comprises a human GPI gene or G6PD gene. In a yet further aspect, the cell has a double-knocked out phosphoglucose isomerase (pgi) gene and a knocked out glucose-6-phosphate dehydrogenase (zwf) gene and further comprises a human GPI gene or G6PD gene. Examples of such G6PD genes include polynucleotides encoding proteins from the group of SEQ IDNOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence, and polynucleotides from the group of SEQ ID NOS: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence. Examples of such GPI genes include polynucleotides encoding proteins from the group of SEQ ID NOS: 39, 41,-4- 4923-5828-5447.1Atty. Dkt. No.: 114198-371043, 45, 47, 49, and 51, and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence and polynucleotides from the group of SEQ ID NOS: 38, 40, 42, 44, 46, 48, and 50, and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence. Examples of such ASL genes include polynucleotides encoding proteins from the group of SEQ ID NOS: 53, 55, 57, 59, and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence and polynucleotides from the group of SEQ ID NOS: 52, 54, 56, 58, and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence.

[0013] In a further embodiment, the cell has a knocked out bacterial argininosuccinate lysase (argH) gene or has reduced expression of bacterial argH. In a further aspect, the cell further comprises the human argininosuccinate lysase (ASL) gene or a mutant thereof. In a yet further embodiment, the cell has a knocked out bacterial argininosuccinate lysase (argH) gene or has reduced expression of the bacterial argH gene and a human argininosuccinate lysase (ASL) gene or a mutant thereof.

[0014] In additional embodiments, the disclosed bacterial cell platform is not limited to human genes. The heterologous gene may be a non-human target gene encoding an enzyme whose activity is associated with disease pathology, pathogenicity, or therapeutic intervention. Such non-human target genes may be derived from animals, pathogens, or parasites.

[0015] In one non-limiting example, the target gene encodes a glucose-6-phosphate dehydrogenase enzyme derived from a malaria parasite. A bacterial or yeast host cell is engineered to delete or inactivate endogenous bacterial or yeast genes involved in glucose-6-phosphate metabolism, including phosphoglucose isomerase (pgi) and glucose-6-phosphate dehydrogenase (zwf), thereby rendering bacterial growth dependent on the catalytic activity of the parasite-derived enzyme.

[0016] Populations of these cells are further provided as are in vitro cultures of these cells. Further comprising are compositions comprising the cells, wherein the compositions-5- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710optionally comprise components necessary for culturing the cells, further optionally the proposed metabolic modulator.

[0017] The cells can be used to identify biological agents such as antibodies, antibody fragments or small molecules that modulate the human genes. Thus, provided herein is a method to screen for a metabolic modulator, the method comprising contacting a first population of the cells identified herein, the potential metabolic modulator and determining the growth rate of the first population. In a yet further aspect, the method further comprising determining the growth rate of a second population of these cells but grown in the absence of the potential metabolic modulator and optionally comparing the growth rate of the first population and the second population. In a yet further aspect, the determination of a higher or lower growth rate of the first population.[0018| As provided herein, Applicant noted that A. coli lacking key native enzymes for glucose metabolism could utilize glucose with the introduction of corresponding human enzymes without alterations to their coding sequence.

[0019] Applicant thus has developed an alternative approach to investigate the activities of human mutant enzymes by developing a live Escherichia coli assay (LEICA). Applicant focused on glucose-6-phosphate isomerase (GPI) and glucose-6-phosphate dehydrogenase (G6PD), which are associated with the most common human hereditary enzymopathies.

[0020] LEICA revealed other potential uses. Through experimentation with small molecules using LEICA, Applicant found that chemical compounds targeting human G6PD could either enhance or inhibit growth when administered directly to cells in culture. Applicant confirmed inhibitory effects of known G6PD inhibitors, highlighting the drug screening capability of LEICA on human drug targets. Screening a library of 160 human metabolism modulators revealed seven lead compounds, including the rediscovery of three with known G6PD inhibitory effects or antimalarial activity. Lastly, Applicant observed enhanced growth of E. coli carrying the G6PD Canton mutant when treated with the recently discovered G6PD agonist AG1. LEICA thus serves not only as a drug screening platform but also holds promise for use in developing personalized medicine. In addition, the platform enables identification of compounds that modulate enzyme activity for therapeutic or-6- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710prophylactic intervention in disease contexts, including metabolic disorders and infectious diseases. In embodiments where the substituted gene is derived from a non-human organism, such as a parasite, the disclosed system provides a means to identify candidate compounds that inhibit pathogen-associated enzymes while minimizing host toxicity. Accordingly, LEICA serves as a versatile platform for drug discovery and therapeutic development across both human and non-human enzyme targets.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGS. 1A- 1G: (FIG. 1A) bacterial system represents activities of human enzymes by growth rate. (FIG. 1A), The bacterial system used for the screening of activities of genetic variants of human metabolic enzymes. (FIG. IB), Growth of the E. coli phosphoglucose isomerase (encoded by pgi) and glucose-6-phosphate dehydrogenase (encoded by zwf) double-knockout strain is dependent on human glucose-6-phosphate isomerase (GPI) activity. EMPP: the Embden-Meyerhof-Pamas pathway. HMP shunt: hexose monophosphate shunt. G6P: glucose-6-phosphate. 6PG: 6-phosphogluconate. F6P: fructose-6-phosphate. FBP: fructose- 1,6-bisphosphate. TCA cycle: tricarboxylic acid cycle. (FIG. 1C), Activities of human GPI variants were represented by growth rates of the humanized E. coli for GPI. Data are presented as mean values + / - SD. Error bars indicate SD of ten replicated cultures. Numbers are relative differences of growth rates compared to the wild-type (WT) enzyme; in percentage, ns, no significant difference. * / ?-value=0.022. ** / ?-values=0.002 (Fukuoka), 0.000 (Matsumoto), and 0.000 (R347H) (two-sided Welch’s t-test with Bonferroni correction). Circles indicate ten independent cultures. (FIG. ID), Growth rates of the E. coli with human gene swap shows high linear correlation with previously reported activities of recombinant enzymes (Pearson’s R2of 0.96; dashed line)14. Data are presented as mean values + / - SD. Error bars indicate SD of biological replicates (n=10). Specific activities of recombinant enzymes were pulled from a previous report14. (FIG. IE), Growth of the double knockout strain expressing human G6PD is governed by activity of the human G6PD. (FIG. IF), Growth rates of humanized A. coli for G6PD and catalytic constants (kcat) of recombinant enzymes25 30had high correlation (Pearson’s R2of 0.84; dashed linear regression line). Data are presented as mean values + / - SD. Error bars indicate SD of five replicated cultures. WT: wild-type. For G6PD-Volendam, the specific -7- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710activity was used31, because kcat is not available. (FIG. 1G), Activities of a few less-characterized human G6PD variants were represented by growth rates of the humanized E. coli. Data are presented as mean values + / - SD. Error bars indicate SD of five replicated cultures. Circles indicate independent data points.

[0022] FIGS. 2A - 2E: Effect of small molecules on glucose 6-phosphate dehydrogenase. (FIG. 2A), Effect of a G6PD activator AG1 on growth rate of humanized E. coli. Half-maximal effective concentration (ECso) is calculated from the dose-response curve (dashed lines). Circles are individual data points (n=10). (FIG. 2B), Growth rates of humanized E. coli in LEICA with different compounds. Vehicle: untreated control (1% DMSO). Tc: tetracycline. PPC: proparacaine. 6 AN: 6-aminonicotinamide. RRx-001: brom onitrozi dine. BMN: brimonidine. DHEA: dehydroepiandrosterone. G6PDi-l: inhibitor of G6PD 1. MMZ: metamizol. All compounds were treated with the final concentration of 100 pM, except for tetracycline (10 pg / ml). ND: no observable growth was detected. Data are presented as mean values + / - SD. Error bars show SD of three replicates. Dots are individual data points, ns: no significant difference. *p-values=0. 025 (BMN) and 0.0135 (G6PDi-l) (two-sided Welch’s t-test with Bonferroni correction, compared to the untreated control), c-e, Dose-response validation of inhibitory effect of three primary hits G6PDi-l (FIG. 2C), DHEA (FIG. 2D), and brimonidine (FIG. 2E). Half-maximal inhibitory concentration (ICso) was calculated from a Hill curve (four parameters; dashed line) fitted to the dose response curve. Dots are individual data points (n=5). * / ?-value < 0.05. ** / ?-value < 0.005. *** - value < 0.001 (two-sided Welch’s t-test with Bonferroni correction, compared to the untreated control).

[0023] FIGS. 3A - 3C: Drug library screening using LEICA. (FIG. 3A), Various compounds are supplemented to LEICA and growth is monitored using a plate reader.Change in G6PD activity in response to chemical compounds is reported as the growth difference of the humanized E. coli at a fixed concentration compared to untreated control (primary hits). Primary hits are further validated by dose-response assay, resulting in discovery of lead compounds. (FIG. 3B), Growth rates of E. coli expressing human G6PD compared to that of E. coli expressing endogenous G6PD. Seven compounds induced significant ( - value < 0.001; two-sided Welch’s / -test, compared to the untreated control) -8- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710growth difference, while inducing no significant difference on E. coli expressing endogenous G6PD. All compounds were treated with the final concentration of 100 pM. Data are presented as mean values + / - SD. Error bars show SD of three replicates. (FIG. 3C), Effect of primary hit compounds at different concentrations on humanized A. coli for G6PD WT. Half-maximal effective concentration (IC50) is calculated from the dose-response curve (dashed lines). Circles are individual data points (n=5).

[0024] FIGS. 4A - 4C: Expanding LEICA for screening human argininosuccinate lyase (ASL). (FIG. 4A), Schematic representation of the human urea cycle. BC: bicarbonate. CP: carbamoyl phosphate. CIT: citrulline. ASA: argininosuccinate. ORN: ornithine. Fum: fumarate. (FIG. 4B), Arginine auxotrophy induced by argH knockout is complemented by human argininosuccinate lyase (ASL). (FIG. 4C), Growth rates of wildtype (WT) E. coli, argH knockout strain, and argH knockout strain expressing WT and variants of human ASL. in vitro activities of the recombinant enzymes12,52are indicated above. ND: no observable growth was detected. Data are presented as mean values + / - SD. Error bars show SD of five replicates. Dots are individual data points. *** / ?-value=0.000 (two-sided Welch’s / -test with Bonferroni correction, compared to the WT).

[0025] FIGS. 5A - 5G: Different sensitivities of individual mutants against different compounds. (FIG. 5A), LEICA of human G6PD variants screens for different compound sensitivities, b-d, Growth profiles of humanized E. coli in LEICA with different variants under dehydroepiandrosterone (DHEA) (FIG. 5B), inhibitor of G6PD 1 (G6PDi-l) (FIG. 5C), and brimonidine (BMN) (FIG. 5D) treatments. All compounds were treated with the final concentration of 100 pM. Data are presented as mean values + / - SD. Error bars show SD of five replicates. (FIGS 5E - 5G), The growth rates of humanized E. coli with treatments compared to the untreated controls represent residual G6PD activities. Variants have different levels of inhibition to DHEA (FIG. 5E), G6PDi-l (FIG. 5F), and BMN (FIG.5G) Data are presented as mean values + / - SD. Error bars show SD of five replicates. Dots are individual data points (n=5). * / ?-value < 0.05. ** / ?-value < 0.005. *** / ?-value < 0.001 (two-sided Welch’s / -test of log-transformed ratios with Bonferroni correction, the residual activities were inferred by comparing treated samples with the untreated samples).-9- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0026] FIG. 6: Growth profiles of E. coli carrying human glucose-6-phosphate isomerase (GPI) or its mutants in M9 glucose medium. WT : wild-type human GPI. Data are presented as mean values + / - SD. Error bars indicate SD of ten replicated cultures.1’0027] FIGS. 7A-7B: Growth of E. coli lacking phosphoglucose isomerase (pgi) or glucose-6-phosphate dehydrogenase (zwf) in M9 glucose medium. (FIG. 7A) Growth profiles of E. coli Apgi and Apgi Azwf strains in M9 glucose medium. Human glucose-6-phosphate dehydrogenase (G6PD) rescued glucose utilization of Apgi Azwf knockout strain. Data are presented as mean values + / - SD. Error bars show SD of five replicates. (FIG. 7B) Growth rates of Apgi and Apgi Azwf strains. E. coli utilizing glucose with its endogenous G6PD (Zwf; E. coli Apgi) or human G6PD exhibited no significant difference in growth rate. Data are presented as mean values + / - SD. Error bars show SD of five replicates. ND, no growth was detected, ns, no significant statistical difference (p-value = 0.346, two-tailed Welch’s t-test). Dots represent individual data points.10028] FIGS. 8A-8B: LEICA monitors activities of G6PD variants as growth rates are contingent on G6PD activity. (FIG. 8A, right and center panels) Growth profiles of humanized E. coli for G6PD expressing well-characterized variants. (FIG. 8B) Growth profiles of humanized E. coli for G6PD expressing or less-characterized variants. Data are presented as mean values + / - SD. Error bars show SD of five replicates.

[0029] FIG. 9. Effect of various compounds on humanized E. coli for G6PD. Growth profiles of humanized E. coli for G6PD (E coli 20.71 Apgi Azwf + HsaG6PD) strain under different drug treatments, veh: untreated control (1% DMSO). Tc: tetracycline. PPC: proparacaine. 6AN: 6-aminonicotinamide. RRx-001: bromonitrozidine. BMN: brimonidine. DHEA: dehydroepiandrosterone. G6PDi-l: inhibitor of G6PD 1. MMZ: metamizol. All compounds were treated with the final concentration of 100 pM, except for tetracycline (10 pg / ml). Data are presented as mean values + / - SD. Error bars show SD of three replicates.

[0030] FIGS. 10A-10B. Effect of various compounds on E. coli. (FIG. 10A) Growth profiles of E. coli Apgi knockout strain under different drug treatments, veh: untreated control (1% DMSO). Tc: tetracycline. PPC: proparacaine. 6AN: 6-aminonicotinamide. RRx-001: bromonitrozidine. BMN: brimonidine. DHEA: dehydroepiandrosterone. G6PDi-l: inhibitor-10- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710of G6PD 1. MMZ: metamizol. All compounds were treated with the final concentration of 100 pM, except for tetracycline (10 pg / ml). Data are presented as mean values + / - SD. Error bars show SD of three replicates. (FIG. 10A) Growth rates of the strain treated with the treatments. Data are presented as mean values + / - SD. Error bars show SD of three replicates. Dots represent individual data points. No significant difference in growth rate was observed (two-tailed Welch’s t-test with Bonferroni correction).FIG. 11: Growth profiles of wild-type (WT) E. coli, AargH knockout strain, and AargH knockout strain expressing WT and variants of human ASL. Data are presented as mean values + / - SD. Error bars show SD of five replicates.DETAILED DESCRIPTIONDefinitions

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0032] Throughout and within this application technical and patent literature are referenced by a citation. For certain of these references, the identifying citation is found at the end of this application immediately preceding the claims. All publications are incorporated by reference into the present disclosure to more fully describe the state of the art to which this disclosure pertains.

[0033] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology -11- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710(Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rdedition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).[00341 All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0035] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.[0036J As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable -12- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0037] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and / or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.

[0038] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0039] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases -13- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710(or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.[0040| An equivalent or biological equivalent nucleic acid, polynucleotide or oligonucleotide or peptide is one having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to the reference nucleic acid, polynucleotide, oligonucleotide or peptide.

[0041] “Detectable label”, “label”, “detectable marker” or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein.[0042| Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0043] In some embodiments, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl-14- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0044] As used herein, a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a polyhistidine (His) tag, calmodulin binding protein (CBP), or maltose-binding protein (MBP)), or a fluorescent tag.

[0045] The term “propagates” or “expand” means to grow a cell or population of cells. The term “growing” also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.

[0046] The term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0047] A population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and / or genotype. A substantially homogenous population of cells is a population having at least 70 %, or alternatively at least 75 %, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90 %, or alternatively at least 95 %, or alternatively at least 98% identical phenotype, as measured by pre-selected markers.

[0048] A “composition” is intended to mean a combination of active polypeptide, polynucleotide or antibody and another compound or composition, inert (e.g. a detectable label) or active (e.g. a gene delivery vehicle).

[0049] A “carrier” intends any material, substance, or medium that supports, stabilizes, transports, or delivers biological components, reagents, or products in life science and biotechnological processes, including solid supports (such as resins, membranes, scaffold for -15- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710cell growth, fermentation broth, and preservative or stabilizer solutions), biological carriers (such as cells, vectors, plasmids), hybrid materials (nanoparticles or hydrogels for targeted or sustained delivery), gas-phase carriers ( such as oxygen / nitrogen mixtures) for use in bioreactors to transport and sustain living cells, and pharmaceutically acceptable carriers.

[0050] A “pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0051] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents, as well as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (MackPubl. Co., Easton).

[0052] A “subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human. Besides being useful for human treatment, the present invention is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like which is susceptible to neurodegenerative disease. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent or infant under the age of eighteen years of age.

[0053] “Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. In one aspect of “treatment”, the term does not include prevention or prophylaxis.-16- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0054] The term “suffering” as it related to the term “treatment” refers to a patient or individual who has been diagnosed with or is predisposed to a disease such as cancer or an infection or a disease incident to infection. A patient may also be referred to being “at risk of suffering” from a disease because of active or latent infection. This patient has not yet developed characteristic disease pathology.

[0055] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to inhibit RNA virus replication ex vivo, in vitro or in vivo.

[0056] As used herein, the term “contacting” means direct or indirect binding or interaction between two or more molecules or other entities. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo.Contacting in vivo can be referred to as administering, or administration.-17- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0057] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), intracranial, or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The invention is not limited by the route of administration, the formulation or dosing schedule.

[0058] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0059] The term “cell” as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

[0060] “Eukaryotic cells” all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human, e.g., HEK293 cells and 293T cells.

[0061] “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like-18- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0062] The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0063] A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

[0064] A “variant thereof’ when used herein in the context of genetic or protein variants of the enzymes as described herein, includes those molecules with naturally occurring, laboratory-adapted, or genetically produced variation in the primary nucleotide or amino acid sequence but as a polynucleotide, encodes a protein that catalyzes the same reaction as the reference polynucleotide. The term includes mutant versions of the wild-type polynucleotide, polypeptide or protein. A “variant” of a protein or polypeptide also refers to a protein or polypeptide that catalyzes the same enzymatic reaction as the reference protein or polypeptide. A “variant” of a protein or polypeptide also includes post-translational modifications to the protein or polypeptide that catalyzes the same reaction as the reference protein or polypeptide. In some embodiments, a “variant” is described as a polynucleotide, protein, or polypeptide have a percent identity to the reference polynucleotide, protein, or polypeptide, respectively, as the reference polynucleotide, protein, or polypeptide, but still catalyze the same reaction.

[0065] As used herein, the term “heterologous gene” refers to a nucleotide sequence that originates from a source differing from that of the host organism, cell, or genetic context into which it is introduced. A heterologous gene may be derived from a different species, strain, or variant, or may be a synthetic, modified, or codon-optimized version of a gene, irrespective of whether the encoded product is naturally present in the host. The term-19- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710encompasses genes inserted via recombinant DNA technology, synthetic biology, or other genetic engineering methods, for purposes including, without limitation, expression of a protein, peptide, RNA molecule, or other functional product in an assay system or engineered cell line. Examples include, but are not limited to, expression of a human enzyme or cytokine gene in Saccharomyces cerevisiae. insertion of a bacterial enzyme gene into a mammalian cell line or bacterial or yeast cell lines as described herein.[0066J “Under transcriptional control” is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, this invention provides promoters operatively linked to the downstream sequences.

[0067] As used herein, the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a specified effect.

[0068] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising, or alternatively consisting essentially of, or yet further consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.[00691 The term "wild-type" refers to a gene or gene product having characteristics of that gene or gene product when isolated from a naturally occurring source. In some embodiments, the wild type genes or gene products, even for one viral strain, contain slight different sequences.

[0070] The term "mutant" refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product or the gene or gene product from other mutant strain(s). Examples of mutants are provided herein.-20- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710[00711 The term “mutation” refers to a DNA sequence variation from a wild type or other mutant strain (s). A mutation produces or does not produce a function property in an organism. There are multiple types of mutations, including but not limited to an insertion, a deletion, a truncation, a frameshift, a substitution, or a point mutation.

[0072] The term “point mutation” refers to a mutation with a single nucleotide base change, insertion, or deletion of the genetic material, DNA or RNA.[00731 “Deletion” refers to a mutation in which a part of chromosome or a sequence of DNA is missing.

[0074] “Frameshift” refers to a mutation caused by indels (insertions or deletions) of a number of nucleotides in a DNA sequence that is not divisible by three.

[0075] “ Substitution” refers to a mutation with a substitution of one or a few nucleotides of a gene.

[0076] “ Truncation” refers to a mutation with elimination of the N- or C-terminal portion of a protein by proteolysis or manipulation of the structural gene, or premature termination of protein elongation due to the presence of a termination codon in its structural gene as a result of a nonsense mutation.

[0077] In some embodiment, the mutation is a nonsynonymous mutation. The term “nonsynonymous mutation” refers to a mutation that alters the amino acid sequence of a protein, which is contrasted with a synonymous mutation that do not alter amino acid sequences.

[0078] The term “promoter” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a P-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, an -21- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710EFl promoter, a chicken P-actin (“CBA”) promoter, a HCMV IRS1 gene under control of the CMV IE promoter in the UL3 / UL4 intergenic region, or an EGR1 promoter.

[0079] Additional non-limiting exemplary promoters with certain target specificity are provided herein below including but not limited to modified retroviral promoter region (“MND”), CMV, EFla, SV40, PGK1 (human or mouse), P5, Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, and Alpha- 1 -antitrypsin. Synthetically-derived promoters may be used for ubiquitous or tissue specific expression. Further, virus-derived promoters, some of which are noted above, may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters. In some embodiments, the promoter is coupled to an enhancer to increase the transcription efficiency.

[0080] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer / promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter.[0081 j As used herein, the term “metabolic modulator” refers to a compound, whether a small molecule, biological agent, or other chemical entity, that alters the catalytic activity of a target enzyme involved in a metabolic pathway, including but not limited to human enzymes expressed in the engineered bacterial cells of the present disclosure. Metabolic modulation may occur through direct interaction with the enzyme’s active site or regulatory interfaces, by influencing enzyme stability or oligomerization, or indirectly through modulation of metabolic cofactor levels.

[0082] As used herein, the term “bacterial cell” refers to a prokaryotic microorganism from any suitable bacterial genus capable of genetic modification to replace an endogenous-22- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710metabolic enzyme with a human ortholog, and in which growth can be rendered dependent on catalytic activity of the human enzyme by deletion or inactivation of endogenous genes in the same metabolic pathway and, optionally, in intersecting pathways, thereby isolating the human enzyme’s activity. Suitable bacterial cells include, without limitation, gram-negative and gram-positive species, including laboratory model strains, industrial strains, and clinical isolates, provided they can be cultivated in defined media and are amenable to genetic engineering by methods such as recombination, transformation, or transduction. In certain embodiments, the bacterial cell is selected from species of Escherichia, Salmonella, Klebsiella, Shigella, Citrobacter, Pseudomonas, Vibrio, Bacillus, Lactococcus, and Streptomyces. The bacterial cell can, for example, be Escherichia coli (including but not limited to K-12 MG1655, BL21, W3110, and derivatives thereof) or other entericGram-negative bacteria, which share central metabolic pathways suitable for the dependency-coupling design, or Gram-positive organisms such as Bacillus subtilis, Pseudomonas aeruginosa, Salmonella enterica, Lactococcus lactis, or metabolically versatile hosts such as Pseudomonas putida or Vibrio natriegens. These bacterial species and strains are available from public repositories and culture collections such as the American Type Culture Collection (ATCC), DSMZ-German Collection of Microorganisms and Cell Cultures, NCIMB, BEI Resources, and commercial suppliers including New England Biolabs, Thermo Fisher Scientific, Invitrogen, and Sigma-Aldrich, as well as institutional repositories under appropriate material transfer agreements. Skilled artisans will recognize that the choice of bacterial cell can be varied according to experimental needs without departing from the inventive concept, and that substitutions among these listed genera and species constitute obvious modifications based on the present disclosure.

[0083] As used herein, the term “yeast” refers to unicellular fungi belonging to the phylum Ascomycota or Basidiomycota, capable of growth in aqueous or semi-solid media, and reproducing primarily by budding or fission. The term encompasses naturally occurring, laboratory-adapted, and genetically modified yeast species and strains suitable for use in biotechnology, including but not limited to the expression or production of proteins, peptides, metabolites, or other biomolecules. Representative examples include, without limitation, Saccharomyces cerevisiae, Pichia pastoris (syn. Komagataella phaffr), Hansenula-23- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710polymorpha, Yarrowia lipolytica, and Kluyveromyces lactis, as well as derivatives, mutants, and engineered variants thereof. Further examples include Saccharomyces cerevisiae strain S288C, strain VL6-48, and separately, Schizosaccharomyces pombe. The yeast are available from public repositories and culture collections such as the American Type Culture Collection (ATCC).

[0084] As used herein, the term “kit” refers to a packaged combination of one or more components, which may be physically associated or assembled together, intended for use in performing a biological, biochemical, or biotechnological procedure. Such components may include, without limitation, reagents (e.g., enzymes, buffers, media, substrates), biological materials (e.g., host cells, vectors, probes), consumables (e.g., tubes, plates, membranes), and / or instructions for use, whether in printed or electronic form. A kit may further comprise containers, labels, or packaging configured to maintain stability, sterility, or separation of components, and may be provided in any format suitable for storage, transport, or direct application to an experimental, diagnostic, manufacturing, or therapeutic protocol. The term encompasses ready -to-use kits, multi-component kits requiring user assembly, and kits provided with auxiliary devices or instruments for facilitating the intended use.

[0085] As used herein, the term “a glucose-6-phosphate dehydrogenase (G6PD)-related disorder” intends a disease or pathological condition that is caused by, associated with, or exacerbated by altered activity, expression, stability, or regulation of a glucose-6-phosphate dehydrogenase enzyme, including reduced or enhanced enzymatic function in host cells or pathogenic organisms. Such disorders may arise from genetic variants of G6PD, pharmacological modulation of G6PD activity, or metabolic dependence on the pentose phosphate pathway. Non-limiting examples include malaria, favism, oxidative stress-related anemia, and autoimmune disease. Non-limiting examples of autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease.

[0086] As used herein, the term “a cancer characterized by elevated pentose phosphate pathway activity” intends a malignancy in which increased flux through the pentose phosphate pathway contributes to tumor growth, survival, proliferation, redox homeostasis, or resistance to oxidative stress. Such cancers are characterized by increased dependence on -24- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710NADPH production and ribose-5-phosphate synthesis for nucleotide biosynthesis and antioxidant defense and are therefore susceptible to modulation of enzymes within the pentose phosphate pathway, including glucose-6-phosphate dehydrogenase.

[0087] As used herein, the term “chronic non-spherocytic hemolytic anemia caused by a pathogenic G6PD variant intends a hereditary hemolytic disorder resulting from a genetic variant in the G6PD gene that leads to persistently reduced or unstable G6PD enzyme activity in red blood cells, causing chronic hemolysis in the absence of spherocyte formation. Such variants impair the ability of red blood cells to maintain redox balance under physiological or oxidative stress conditions.

[0088] As used herein, the term “hemolytic episodes” intends acute or subacute events characterized by accelerated destruction of red blood cells, resulting in decreased hemoglobin levels, elevated bilirubin, hemoglobinuria, or related clinical manifestations. Such episodes may be triggered by oxidative stress, infection, ingestion of certain foods, or exposure to specific drugs, particularly in individuals with underlying red blood cell enzymopathies such as G6PD deficiency.

[0089] As used herein, the term “pathogenic variant” intends a genetic variant, mutation, or allele that causes or contributes to the onset, severity, or progression of a disease or disorder by producing a measurable deleterious effect on protein function, stability, expression, or regulation. In the context of enzymatic activity, a pathogenic variant is one that results in reduced or aberrant catalytic activity relative to a corresponding wild-type enzyme, as evidenced by biochemical assays, cellular assays, or growth-based surrogate readouts as described herein.

[0090] Modes For Carrying Out the Disclosure

[0091] The human genome sequence varies between individuals. Everyone typically harbors between 250 to 300 loss-of-function sequence variations relative to the reference human genome1. These variations often contribute to or cause inherited disorders, making the identification of causal mutations an essential pursuit in human biology and personalized medicine. Recent strides in genome editing offer promising avenues for treating conditions like eye and liver disease.-25- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0092] Traditionally, the identification of genotypes associated with genetic disease has relied on direct patient analysis and case reports. Once genetic variations linked to specific disorders are mapped, they are studied through various methodologies. One common approach involves in vitro studies with patient-derived materials or recombinant proteins to characterize mutant proteins. However, this approach is constrained by sample availability and necessitates labor-intensive purification processes. Moreover, it often requires specialized assay systems tailored to individual proteins, alongside time-consuming analytical methods and instrumentation. Additionally, dilute in vitro conditions do not replicate crowded and dense gel-like physiological conditions, necessitating an in vivo system for an accurate biochemical assay.

[0093] Alternatively, large-scale computational surveys, such as genome-wide association studies (GWAS) based on population sequencing datasets, effectively identify potential associations between genetic changes and pathological conditions. However, such associations do not establish causality, particularly given the many uncharacterized variations and confounding factors of zygosity and genetic linkage. Furthermore, variations that induce severe pathogenicity from early onset are rare in the human population, making it challenging to obtain samples or detect such variations in population databases. As a result, pathogenic mutations remain practically undetectable, while detected mutations are more likely to have minor impact on enzyme activity.

[0094] Applicant provides herein an alternative approach to investigate the activities of human mutant enzymes by developing a live Escherichia cpli assay (LEICA). Specifically, Applicant focused on glucose-6-phosphate isomerase (GPI) and glucose-6-phosphate dehydrogenase (G6PD), which are associated with the most common human hereditary enzymopathies.

[0095] Engineered, Non-naturally Occurring Bacterial and Yeast Cells

[0096] The present disclosure provides a bacterial or yeast cell comprising a heterologous gene encoding an enzyme that is substituted for the endogenous bacterial or yeast counterpart gene. The bacterial or yeast cell can further comprise the deletion or inactivation of at least one or all endogenous bacterial counterpart genes capable of catalyzing the same biochemical-26- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710reaction as the counterpart heterologous enzyme or enzymes, and in one aspect couples cell growth to the activity of the human enzyme or enzymes.

[0097] In certain embodiments, the heterologous gene is an animal gene, a mammalian gene, a human gene, a gene from a pathogen, or a gene from a parasite. In some embodiments, the bacterial or yeast cell is genetically modified to delete or inactivate the heterologous gene that is substituted for the endogenous bacterial or yeast counterpart gene. In one embodiment, the least one or all endogenous bacterial or yeast genes capable of catalyzing the same biochemical reaction as the heterologous enzyme or enzymes are deleted or inactivated, thereby coupling cell growth to the activity of the heterologous enzyme or enzymes.

[0098] In further embodiments, the bacterial or yeast cell further lacks or inactivates additional bacterial or yeast genes in the same or intersecting metabolic pathway, thereby isolating the heterologous enzyme’s catalytic activity from alternative metabolic flux routes. In some embodiments, the heterologous gene is a human enzyme selected from the group consisting of human glucose 6-phosphate isomerase (GPI), human glucose 6-phosphate dehydrogenase (G6PD), and human argininosuccinate lyase (ASL), and variants thereof. Examples of such genes are provided herein.[009 1 Examples of such G6PD genes include polynucleotides encoding proteins from the group of SEQ IDNOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence, and polynucleotides from the group of SEQ ID NOS: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence. Examples of such GPI genes include polynucleotides encoding proteins from the group of SEQ ID NOS: 39, 41, 43, 45, 47, 49, and 51, and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence and polynucleotides from the group of SEQ ID NOS: 38, 40, 42, 44, 46, 48, and 50, and variants thereof that retain the modified nucleotide(s) as noted in the respective polynucleotide sequence. Examples of such ASL genes include polynucleotides encoding proteins from the group of SEQ ID NOS: 53, 55, 57, 59, and variants thereof that retain the modified amino acid(s) as noted in the respective protein sequence and polynucleotides from-27- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710the group of SEQ ID NOS: 52, 54, 56, 58, and variants thereof that retain the modified nucleotide(s_ as noted in the respective polynucleotide sequence.

[0100] In particular embodiments, the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose 6-phosphate dehydrogenase (zwf) gene and comprises a human GPI gene inserted into the chromosome, into the pgi locus, or expressed by a plasmid DNA. In other embodiments, the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose 6-phosphate dehydrogenase (zwf) gene and comprises a human G6PD gene inserted into the chromosome or expressed by a plasmid DNA. Examples of such genes are provided herein.

[0101] The disclosure also encompasses bacterial cells knocked out for the argH gene required for arginine biosynthesis, wherein the heterologous gene comprises a human ASL gene inserted into the chromosome, into the chromosomal locus of the argH gene, or expressed by a plasmid DNA. In some embodiments, the bacterial cell is an Escherichia coli cell, optionally E. coli K-12 MG1655 and derivatives thereof. Examples of such genes are provided herein.[01021 In some aspects, the disclosure provides a bacterial cell including a human gene that is substituted for the endogenous bacterial counterpart gene. In one aspect, the human gene is detectably labeled. In one aspect, bacterial cell comprises the deletion or inactivation of at least one or all endogenous bacterial genes capable of catalyzing the same biochemical reaction as the counterpart human enzyme or enzymes, that are optionally detectably labeled. In one embodiment, the disclosure provides a bacterial cell, wherein growth of the bacterial cell in a defined culture medium is dependent upon catalytic activity of the human gene product of the one or more human genes. In several aspects, the bacterial cell further comprises a detectable marker.

[0103] In other aspects, the disclosure provides a bacterial cell, wherein the bacterial cell is genetically modified to delete or inactivate all endogenous bacterial genes that catalyze the same biochemical reaction as the counterpart human enzyme or enzymes, thereby coupling cell growth to the activity of the counterpart human enzyme or enzymes. In one embodiment, the bacterial cell further lacks additional bacterial genes in the same or intersecting metabolic-28- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710pathway, thereby isolating the counterpart human enzyme or enzyme's catalytic activity from alternative metabolic flux routes.

[0104] Also provided herein is a bacterial cell as described above, wherein the human enzyme is selected from human glucose-6-phosphate isomerase (GPI), human glucose-6-phosphate dehydrogenase (G6PD), and human argininosuccinate lyase (ASL), or a variant thereof. In some aspects of the bacterial cell, the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate dehydrogenase (zwf) gene and includes a human GPI gene inserted into the pgi locus.

[0105] In some embodiments of this disclosure, the bacterial cell is knocked out for the argH gene required for arginine biosynthesis and includes a human ASL gene inserted into the chromosomal locus of the argH gene. In one aspect, the human gene is detectably labeled.

[0106] In some aspects, the bacterial cell. Suitable bacterial cells include, without limitation, gram-negative and gram-positive species, including laboratory model strains, industrial strains, and clinical isolates, provided they can be cultivated in defined media and are amenable to genetic engineering by methods such as recombination, transformation, or transduction. In certain embodiments, the bacterial cell is selected from species of Escherichia, Salmonella, Klebsiella, Shigella, Citrobacter, Pseudomonas, Vibrio, Bacillus, Lactococcus, and Streptomyces. The bacterial cell can, for example, be Escherichia coli (including but not limited to K-12 MG1655, BL21, W3110, and derivatives thereof) or other enteric Gram-negative bacteria, which share central metabolic pathways suitable for the dependency-coupling design, or Gram-positive organisms such as Bacillus subtilis, Pseudomonas aeruginosa, Salmonella enterica, Lactococcus lactis, or metabolically versatile hosts such as Pseudomonas putida or Vibrio natriegens. These bacterial species and strains are available from public repositories and culture collections such as the American Type Culture Collection (ATCC), DSMZ-German Collection of Microorganisms and Cell Cultures, NCIMB, BEI Resources, and commercial suppliers including New England Biolabs, Thermo Fisher Scientific, Invitrogen, and Sigma-Aldrich, as well as institutional repositories under appropriate material transfer agreements. Skilled artisans will recognize that the choice of bacterial cell can be varied according to experimental needs without-29- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710departing from the inventive concept, and that substitutions among these listed genera and species constitute obvious modifications based on the present disclosure.

[0107] Populations and Cultures

[0108] The disclosure further provides a population of bacterial or yeast cells as described herein, optionally further comprising a carrier or culture medium suitable for growing the bacterial or yeast cells. In one aspect, the cells are grown under conditions where growth is dependent on the catalytic activity of the heterologous gene. For example, the heterologous gene is an animal gene, a mammalian gene, or a human gene encoding an enzyme or variant thereof.10109] In some embodiments, the disclosure provides an in vitro culture comprising bacterial or yeast cells as described herein, or the population as defined above.

[0110] In one aspect, the cells or population are bacterial cells provided in a composition that further includes a carrier or culture medium suitable for growing the bacterial cells under conditions where growth is dependent on the catalytic activity of the human enzyme or variant. In one aspect, the population of bacterial cells are provided in vitro culture comprising the bacterial cells as described herein, that optionally includes a carrier or culture medium.[0111 [ Methods of Assaying Enzyme Activity

[0112] The disclosure also provides a method of assaying the catalytic activity of a heterologous enzyme or enzyme variant, comprising: (a) providing a bacterial or yeast cell, population, or culture as described herein; (b) culturing the bacterial or yeast cell, population, or culture under conditions in which growth is dependent on the catalytic activity of the enzyme; and(c) determining the growth rate of the bacterial or yeast cell, wherein growth rate is used as a measure of in vivo catalytic activity of the enzyme or enzyme variant, optionally wherein the heterologous enzyme or enzymes is a human enzyme, enzymes or variants of each thereof.

[0113] In some embodiments, the bacterial or yeast cell is engineered such that the catalytic activity of the heterologous enzyme is the sole variable affecting growth, by deletion of-30- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710redundant bacterial enzymes and constant expression of the human enzyme under a stable promoter.

[0114] In certain embodiments, growth rate determination is performed by optical density measurements from about 400 nm to about 800 nm, optionally at about 600 nm in a plate reader. In other embodiments, the determined growth rate correlates linearly with an in vitro kinetic constant (fcat or specific activity) for the human enzyme or variant, the correlation having a Pearson R2of about >0.80.

[0115] Applicant further provides methods of assaying the catalytic activity of a human enzyme or enzyme variant, comprising, or consisting essentially of, or consisting of: providing a bacterial cell, population or culture according as described above wherein the bacterial cell is engineered such that catalytic activity of the human enzyme is the sole variable affecting growth, by deletion of redundant bacterial enzymes and constant expression of the human enzyme under a stable promoter; culturing the bacterial cell as provided herein under conditions in which growth is dependent on the catalytic activity of the human enzyme; and determining growth rate of the bacterial cell, wherein growth rate is used as a measure of in vivo catalytic activity of the human enzyme or variant.

[0116] In some aspects of the method, the growth rate determination is performed by optical density measurements at from about 400 to about 800nm, or alternatively from about 500 to about 700nm, or about 550 to about 625nm, or alternatively about 600 nm in a plate reader. Alternatively, the determined growth rate correlates linearly with an in vitro kinetic constant (k cat or specific activity) for the human enzyme or variant, the correlation having a Pearson R2at about > 0.80, or alternatively > 0.80.

[0117] In some embodiments of these methods, the bacterial cell expresses a human enzyme variant associated with a metabolic disorder, and the growth rate is compared to that of a bacterial cell expressing wild-type human enzyme to determine whether the variant is pathogenic. As used herein, the term “pathogenic” refers to a human genetic variant, allele, or mutation that causes or contributes to the onset, manifestation, or progression of a disease or disorder, particularly a metabolic enzymopathy, by producing a measurable deleterious effect on enzyme catalytic activity, stability, or regulation under physiological conditions. In the-31- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710context of the present application, a pathogenic variant is one that, when expressed in the engineered bacterial cell in place of the endogenous bacterial enzyme, produces a growth rate reduction that correlates with decreased enzymatic activity relative to a wild-type human enzyme control, and which is supported by clinical or molecular evidence of disease association in human subjects. Pathogenicity can be determined from literature reports, clinical databases (e.g., ClinVar), or directly inferred from the growth-coupled assay described herein, in which severe or moderate reductions in growth rate are indicative of deficiency. Non-limiting examples provided herein include without limitation: for glucose-6-phosphate isomerase (GPI), variants such as D539N (Fukuoka), T224M (Iwate), T5I (Matsumoto), and R347H, each associated with hereditary hemolytic anemia and producing significantly reduced growth in the LEICA assay; for glucose-6-phosphate dehydrogenase (G6PD), variants such as Canton (R459L), Union (R454C), Volendam (P172S), Sumare (V431G), Santiago (R198P), Omiya (Q307H), and Puerto Limon (E398K), all linked to chronic or acute non- spherocytic hemolytic anemia and demonstrating reduced growth corresponding to lower catalytic constants; and for argininosuccinate lyase (ASL), variants such as R12Q, Q286R, and D87G, each causative for argininosuccinic aciduria and exhibiting reduced or absent complementation of the arginine auxotrophy in the bacterial assay. In certain embodiments, pathogenic encompasses “likely pathogenic” variants where there is strong but not definitive clinical evidence coupled with functionally significant growth and activity reduction in the disclosed assay.

[0118] Methods of Screening Metabolic Modulators

[0119] Applicant also provides a method of screening for a metabolic modulator, comprising: (a) contacting a first population of bacterial or yeast cells as described herein with a potential metabolic modulator; (b) determining the growth rate of the first population; (c) determining the growth rate of a second population of bacterial or yeast cells as described herein grown in the absence of the potential modulator; and (d) comparing the growth rates, wherein a higher or lower growth rate of the first population indicates that the potential metabolic modulator modulates catalytic activity of the human enzyme or variant.

[0120] In some embodiments, the bacterial or yeast cells in both populations are assayed in parallel with control bacterial or yeast cells expressing the endogenous bacterial enzyme, and -32- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710a potential modulator is identified as a hit only if it alters growth of the humanized bacterial or yeast cells without significantly affecting growth of the control bacterial or yeast cells.10121] The potential metabolic modulator may be selected from a biological agent or a small molecule, optionally from a compound library. In additional embodiments, the method further comprises validating the potential metabolic modulator by performing a doseresponse analysis to determine an ECso or ICso value[01221 The disclosure also provides embodiments wherein the bacterial or yeast cell expresses a human enzyme variant associated with a metabolic disorder, and the growth rate is compared to that of a bacterial or yeast cell expressing wild-type human enzyme to determine whether the variant is pathogenic.

[0123] In some aspects, Applicant provides method of screening for a metabolic modulator, comprising, or consisting essentially of, or yet further consisting of contacting a first population of bacterial cells as described herein with a potential metabolic modulator; determining the growth rate of the first population; determining the growth rate of a second population of bacterial cells as described herein, grown in the absence of the potential modulator; and comparing the growth rates, wherein a higher or lower growth rate of the first population indicates that the potential metabolic modulator modulates catalytic activity of the human enzyme or variant.

[0124] As used herein, the term “metabolic modulator” refers to a compound, whether a small molecule, biological agent, or other chemical entity, that alters the catalytic activity of a target enzyme involved in a metabolic pathway, including but not limited to human enzymes expressed in the engineered bacterial cells of the present disclosure. Metabolic modulation may occur through direct interaction with the enzyme’s active site or regulatory interfaces, by influencing enzyme stability or oligomerization, or indirectly through modulation of metabolic cofactor levels. In the embodiments described in this application, metabolic modulators were identified by screening a 160-compound human metabolism modulator library in a Live Escherichia coli Assay (LEICA) format, using bacterial strains whose growth is dependent upon the catalytic activity of human glucose-6-phosphate dehydrogenase (G6PD). The screen yielded seven primary hit compounds that significantly inhibited growth-33- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710of the humanized strain while not affecting the control strain expressing endogenous bacterial G6PD. These included previously known modulators such as the selective G6PD inhibitor G6PDi-l, the dihydrofolate reductase inhibitor methotrexate (which also shows reported G6PD inhibitory effects and antimalarial activity), and gossypol (a polyphenolic aldehyde with known oxidoreductase inhibition and antimalarial properties). The remaining four compounds had no prior reported association with G6PD activity but were identified in this assay as novel modulators, three of which exhibited potent inhibition with ICso values ranging from 15.6 to 109 pM. Additional examples of metabolic modulators tested in targeted experiments included the G6PD activator AG1, brimonidine (BMN), dehydroepiandrosterone (DHEA), and inhibitors such as RRx-001, 6-aminonicotinamide (6 AN), and metamizole (MMZ), with differing effects on bacterial growth that reflected their specific actions on the human enzyme. These results illustrate the ability of the platform not only to rediscover known modulators but also to identify new chemical entities that affect human metabolic enzymes under physiologically relevant intracellular conditions.

[0125] In one aspect, the bacterial cells in both populations are assayed in parallel with control bacterial cells expressing the endogenous bacterial enzyme, and a potential modulator is identified as a hit only if it alters growth of the humanized bacterial cells without significantly affecting growth of the control bacterial cells.

[0126] Non-limiting examples of a potential metabolic modulator are selected from a biological agent or a small molecule, optionally from a compound library.

[0127] In further aspects of these methods, the methods further include validating the potential metabolic modulator by performing a dose-response analysis to determine an ECso or ICso value.10128] Kits10129] This disclosure further provides a kit for assaying the catalytic activity of a heterologous enzyme or variant thereof, comprising: (a) a bacterial or yeast cell as described herein;(b) culture medium suitable for growing said bacterial or yeast cells under conditions where growth is dependent on the catalytic activity of the heterologous enzyme or variant; and -34- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710(c) instructions for performing the assay by determining growth rate as an indicator of enzyme activity.

[0130] In some embodiments, the kit further comprises one or more control bacterial cells expressing a wild-type human enzyme or endogenous bacterial enzyme, and optionally one or more potential metabolic modulators or a compound library for screening, and instructions for comparative growth assays between humanized and control bacterial or yeast cells to identify enzyme-specific modulators

[0131] Also provided are kits for assaying the catalytic activity of a cell or population of cells as described herein, the kits comprising culture medium suitable for growing the bacterial cell or population thereof under conditions where growth is dependent on the catalytic activity of the human enzyme or variant and instructions for performing the assay by determining growth rate as an indicator of enzyme activity, optionally further including one or more control bacterial cells expressing a wild-type human enzyme or endogenous bacterial enzyme.

[0132] In some respects, the kit further includes one or more potential metabolic modulators or a compound library for screening, and instructions for comparative growth assays between humanized and control bacterial cells to identify enzyme-specific modulators.

[0133] Therapeutic Methods and Uses

[0134] The metabolic modulators identified through the present LEICA screening platform demonstrate clear potential for therapeutic use in diseases where modulation of human glucose-6-phosphate dehydrogenase (G6PD) activity results in clinical benefit. G6PDi-l, methotrexate, and gossypol, each exhibiting inhibitory effects on G6PD activity under in vivo-like bacterial assay conditions, are candidates for use in reducing pentose phosphate pathway flux in human cells and in pathogens reliant on host G6PD activity. Literature reports have established that reduction of G6PD activity can impair malaria parasite survival and slow tumor growth in cancers that depend on elevated nucleotide synthesis and redox control. The LEICA growth inhibition data correspond with published in vitro IC50ranges for these compounds, linking assay results to disease-relevant doses.-35- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0135] In cases of inherited G6PD deficiency, pathogenic variants such as G6PD-Canton or G6PD-Union result in reduced catalytic activity and increased susceptibility to oxidative stress-induced hemolysis. The LEICA platform confirmed that the G6PD activator AG1 increased growth rate in strains expressing the Canton variant to levels comparable with wildtype enzyme activity, indicating effective restoration of function. Literature supporting AGl’s mechanism of stabilizing G6PD oligomerization and boosting NADPH production in deficient red blood cells further reinforces its therapeutic value. Such activators are therefore suitable for treating or preventing hemolytic anemia and related disorders in G6PD-deficient individuals, with dosing informed by ECso values obtained in the LEICA assay.

[0136] The therapeutic applications of novel modulators discovered in the LEICA library screen are likewise supported by their observed potency in selectively reducing or enhancing enzyme-dependent bacterial growth. For inhibitors with micromolar ICso values, such compounds present leads for development into drugs targeting diseases with a metabolic dependence on G6PD activity, including parasitic infections and proliferative disorders. For activators, their ability to restore variant enzyme activity in a controlled in vivo-like setting facilitates translation to precision medicine strategies dependent on a patient’s genetic makeup. By correlating LEICA growth rates to known biochemical properties and clinical literature, the platform not only enables identification of modulators but also provides a mechanistic and quantitative rationale for their use in disease treatment.

[0137] The disclosure includes a method of treating a glucose 6-phosphate dehydrogenase (G6PD)-related disorder in a subject in need thereof, comprising administering to the subject an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, linezolid (CAS 2645-32-1), auranofin (CAS 34031-32-8), PR-619 (CAS 2645-32-1), 6-mercaptopurine (CAS 6112-76-1), and a novel G6PD inhibitor identified in the LEICA screen, thereby inhibiting G6PD activity in the subject, optionally wherein the subject is a mammal or a human patient.

[0138] In other embodiments, the disorder is selected from malaria, favism, oxidative stress-related anemia, and autoimmune disease. In some embodiments, the subject is administered the inhibitor in combination with an antimalarial agent.-36- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0139] In one aspect, the subject is administered the inhibitor in combination with an antimalarial agent. In a further aspect, the effective amount is determined based on ECso or ICso values measured in the LEICA assay for the specific variant.|0140] Also provided are methods of treating a parasitic or infectious disease in a subject in need thereof, comprising, or consisting essentially of, or consisting of administering to the subject an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, and a novel G6PD inhibitor identified in the LEICA screen, thereby reducing pentose phosphate pathway flux in the pathogen or host cells to inhibit infection, optionally wherein the subject is a mammal or a human patient. In a further aspect, the effective amount is determined based on EC so or ICso values measured in the LEICA assay for the specific variant. Examples of these disorders are provided herein.[01411 In another aspect, methods of treating a cancer characterized by elevated pentose phosphate pathway activity, comprising, or consisting essentially of, or consisting of administering to a subject in need thereof an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, and a novel G6PD inhibitor identified in the LEICA screen to suppress nucleotide biosynthesis and redox balance in the cancer cells, optionally wherein the subject is a mammal or a human patient. In a further aspect, the effective amount is determined based on EC so or ICso values measured in the LEICA assay for the specific variant.

[0142] Applicant further provides methods of treating chronic non-spherocytic hemolytic anemia caused by a pathogenic G6PD variant, comprising, or consisting essentially of, or consisting of administering to the subject an effective amount of a G6PD activator identified in the LEICA screen, thereby increasing catalytic activity of the variant enzyme, optionally wherein the subject is a mammal or a human patient. In a further aspect, the effective amount is determined based on EC so or ICso values measured in the LEICA assay for the specific variant.

[0143] Another embodiment provides methods of preventing or reducing hemolytic episodes in a G6PD-deficient subject, comprising, or consisting essentially of, or consisting of administering to the subject an effective amount of AG1 or another G6PD activator, thereby-37- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710stabilizing enzyme oligomerization and restoring NADPH production, optionally wherein the subject is a mammal or a human patient. In one aspect, the G6PD activator improves variant enzyme activity by at least 10% relative to baseline in red blood cells. In another aspect, the activating compound is administered prophylactically prior to exposure to oxidative stress. In a further aspect, the effective amount is determined based on ECso or ICso values measured in the LEICA assay for the specific variant.[0144| Further provided are one or more of: G6PDi-l for use in the treatment of malaria in a subject in need thereof; methotrexate for use in the treatment of malaria in a subject, wherein said use comprises inhibiting human G6PD activity; gossypol for use in the treatment of Trypanosoma infection by inhibiting human G6PD activity; AG1 for use in the treatment of chronic non- spherocytic hemolytic anemia caused by a pathogenic G6PD variant; and a G6PD activator identified in the LEICA assay for use in preventing oxidative hemolysis in a G6PD-deficient patient.10145] As the growth rate of E. coli is contingent upon glycolytic flux, it serves as a surrogate measure for the activity of heterologously expressed human enzymes. Harnessing the ease of genetic manipulation in E. coli, Applicant replicated human mutations in these enzymes. E. coli strains carrying different mutants exhibited distinct growth rates, reflecting variations in enzyme activity induced by mutations. Notably, the growth rates demonstrated a high linear correlation with enzyme activities determined via recombinant protein assays. This live bacterial cell assay provides an accurate and rapid means of screening for enzyme activity change resulting from human mutations, offering insights into the causality of genetic disorders attributable to genetic variations. Applicant also expanded LEICA to screen argininosuccinate lyase (ASL), a key urea cycle enzyme whose deficiency results in argininosuccinic aciduria. Complementation of arginine auxotrophy in E. coli lacking ASL with human equivalents demonstrates LEICA’ s broader applicability across diverse enzymes.]0146| Applicant’s disclosure relates in part to bacterial strains in which at least one or all endogenous bacterial genes capable of catalyzing the same biochemical reaction as the human enzyme are deleted or inactivated, thereby rendering bacterial growth under defined conditions solely dependent on the catalytic activity of the human enzyme or variant. For example, in one embodiment, the cell comprises a double knockout (Apgi Azwf) bacterial cell -38- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710where glycolytic flux from glucose proceeds only through the human GPI inserted into the pgi locus. Similarly, the human argininosuccinate lyase (ASL) uses a bacterial cells that comprises a knockout of argH (arginine biosynthesis) requiring complementation solely via human ASL. Applicant’s methods and cells are distinct from prior art methods and cells that do not delete all parallel / bypass pathways to enforce total metabolic dependence. Without this architecture, growth rate inherently reflects activities of both the inserted human enzyme and residual bacterial enzymes, making it unsuitable for direct quantitative assessment of variant activity. This dependency design is a novel technical arrangement and non-obvious over the cited art.

[0147] Applicant’s methods also comprise evaluation of bacterial growth rate obtained under the dependency conditions correlates linearly (Pearson R2> 0.80) with in vitro catalytic constants (k cat or specific activity) of the corresponding human enzymes or variants. This correlation is a surprising technical result, as in vivo growth depends on numerous variables (expression level, protein folding, stability, metabolic interactions) that could confound linearity. The specification demonstrates such correlation for multiple unrelated human enzymes, GPI, G6PD, and ASL, across pathogenic, benign, and uncertain variants. This converts a complementation assay into a quantitative diagnostic platform, enabling accurate ranking of variants without individualized biochemical assay development.

[0148] Applicant’s cells and methods also incorporate the step of parallel screening against a control bacterial strain expressing the endogenous bacterial enzyme. Thus, compounds can be identified as modulators of the human enzyme only if they alter growth of the humanized strain without significantly affecting growth of the control strain. This comparative architecture filters out false positives due to general antibacterial toxicity, a critical safeguard unique to this platform. While cell-based drug screens exist in the art and to the best of Applicant’s knowledge, none combine: the dependency-coupled humanized bacterial strain; the same metabolic locus in native bacterial form for control purposes; and a direct growth rate readout in the same culture conditions to enable specific detection of human enzyme modulators. This combination of structural and functional elements yields a platform that solves a long-standing technical problem in the art, i.e., the inability to rapidly and-39- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710quantitatively characterize human enzyme variants and identify modulators under physiological intracellular conditions.

[0149] Experimental|0150] EXPERIMENTAL RESULTS

[0151] Growth of E. coli with human GPI reflects enzyme activity: In a previous study, applicant demonstrated the successful replacement of E. coli pgi, which encodes for GPI, with its human ortholog8. During adaptive laboratory evolution, optimal human GPI function in live bacteria required enhanced gene expression rather than mutations in the protein sequence. Furthermore, various in vitro studies use recombinant human enzymes expressed from E. coli lysates14,15, indicating E. coli as a potential host for functional expression of human enzymes.

[0152] In both humans and E. coli, glucose is mainly utilized by glycolysis. Thus, applicant hypothesized that replacing a glycolytic enzyme in E. coli with its human counterpart would reflect human enzyme activity (FIG. 1A). To specifically assess human GPI activity, applicant disengage the hexose monophosphate (HMP) shunt by deleting zwf encoding G6PD. Applicant thus knocked out zwf from the E. coli strain 20.71 — an evolved E. coli K-12 MG1655 strain16carrying in-frame pgi swap to human GPI and promoter mutations (FIG. IB). In the resulting strain — a humanized E. coli for GPI — growth in a glucose-containing medium is solely dependent on GPI activity (FIG. 6).10153] Applicant examined genetic variations in GPI associated with hemolytic anemia, selecting six mutations including two benign and four pathogenic ones (Table I)14. Two benign mutations occurred with population frequencies ranging from 1 in 49 to 1 in 4,400 individuals, while four pathogenic variations ranged from 1 in 57,000 to 1 in 390,000 (Genome Aggregation Database; gnomeAD17). Some were reported in patients with hemolysis18 20.

[0154] To evaluate the live bacterial assay, Applicant replaced wild-type (WT) human GPI in the humanized E. coli for GPI with the mutant GPIs to investigate activity differences.Strains expressing different GPI variants showed growth rate changes ranging from -13.1 to +2.2% compared to the WT control (FIG. 1C). Growth rates of strains carrying pathogenic -40- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710mutations were significantly lower than those with WT GPI, while two benign mutants displayed either a mild decrease in growth (-5.6%) or growth indistinguishable from the WT control.

[0155] Comparison of biochemically determined properties of recombinant mutants14with the live bacterial assay revealed a high linear correlation between enzyme activity and the growth rate, demonstrating the assay’s capability to infer human enzyme activities (FIG. ID). The live Escherichia coli assay (LEICA) gives an impetus for rapidly screening genetic variants causing enzyme deficiencies. Unlike conventional biochemical assays using red blood cell (RBC) hemolysates or recombinant enzymes that require specialized systems and laborious purification21, LEICA calls for only a single gene replacement (and variants therein) with a straightforward growth measurement, as all the necessary substrates and cofactors are present in live bacteria.

[0156] Diagnosing human G6PD deficiency using LEICA: Having demonstrated the concept of LEICA analyzing human enzyme activity, applicant sought to explore another human enzyme, G6PD. G6PD deficiency is the most common cause of RBC enzymopathy, affecting 300-400 million people worldwide22. G6PD plays a critical metabolic role in RBCs as the HMP shunt provides NADPH needed for redox homeostasis23. Hereditary G6PD deficiency is caused by sequence variations in G6PD. which is highly polymorphic with over 230 variants identified6,10, due to its association with malaria resistance24. Despite many variants identified through populational sequencing, clinical or molecular characterizations remain limited6.

[0157] To assess G6PD variations using LEICA, Applicant first examined whether human G6PD could functionally express in E. coli (FIG. IE). Since the E. coli 20.71 Epgi Ezwf strain is unable to utilize glucose as a carbon source (FIG. 6), growth in a glucose medium directly reflects heterologous G6PD activity, creating a humanized E. coli for G6PD. The double knockout strain expressing human G6PD exhibited comparable growth to the E. coli Epgi single knockout strain, whose glycolytic flux is supported by its endogenous G6PD (Zwf) (FIG. 7). As demonstrated with the GPI gene replacement, human G6PD proved functional in E. coli.-41- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0158] Next, applicant selected 13 human G6PD sequence variations associated with varying severities of G6PD deficiency for LEICA application (Table 2)6. Strains expressing G6PD variants showed markedly different growth profiles to those carrying the WT enzyme (FIGS.8A & 8B). Specifically, the humanized A. coli carrying G6PD-Volendam, a class I G6PD variant associated with chronic hemolysis, grew at half the rate of the WT-expressing strain (FIG. IF) Growth of the relatively mild deficiency variant, G6PD-Mahidol (class III) was also apparent (84% of WT, / ?-value of 1.412* 10'4; Welch’s / -test), indicating high dynamic range of the growth assay. Moreover, growth rates of humanized E. coli carrying different G6PD variants correlated linearly with biochemical properties of the corresponding recombinant enzymes (Pearson’s R2of 0.84, FIG. IF)25 33. This result further illustrates LEICA’ s ability to rapidly screen human genetic variants.

[0159] Given the linear correlation observed, applicant sought to test less-characterized variants with no reported biochemical properties using LEICA. We constructed humanized E. coli strains carrying two predicted benign variants, one pathogenic variant, and one variant with uncertain clinical significance (Table 2). LEICA detected marginal growth rate reductions in the two predicted benign variants (FIG. 1G and FIG. 8B), indicating a mild decrease in enzyme activity. However, the association of these variants with disease should be considered carefully, as LEICA primarily screens for enzyme activity. If these mutations are revealed to be clinically associated with diseases, it may imply that pathogenicity arises from factors other than enzyme activity decrease, such as enzyme stability or expression level. Additionally, a variant with insufficient evidence to determine its role in disease (N426K) displayed similar results to the benign variants, suggesting N426K induces no significant enzyme deficiency.

[0160] In contrast, the G6PD-Sumare variant exhibited a notable reduction in growth (86% of WT, / ?-value of 6.736* 10'5, Welch’s / -test), comparable to that of class III variants (A+, Mahidol, and Murcia) associated with mild anemia34. Although the biochemical properties have never been studied, the results from LEICA, along with reduced blood G6PD activity in patients carrying this variation, indicate a possible association of the G6PD-Sumare variant with disease35.-42- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0161] Incorporating G6PD and GPI into LEICA enables accurate measurement of catalytic activities of human enzymes in an intracellular environment. Unlike an in vitro assay, the bacterial host provides the necessary substrates and physiological conditions for enzyme activity. This approach allows for an in vivo enzyme assay by simple growth measurement, independent of specialized chemical and reporter systems. Human genetic disorders result from a complex interplay of biological factors, including variations in catalytic activity, expression levels, and zygosity, posing challenges in identifying causality. LEICA insulates enzyme activity from such biological factors by mimicking the homozygous state and providing stable expression levels, offering a rapid means of assessing only catalytic activity as the primary variable.[01621 Small-molecules modulate G6PD in humanized E. coli: Human G6PD is functional only as a dimer or tetramer, and some mutations hamper oligomerization36. A small-molecule activator of G6PD, AG1, promotes dimer formation and activates mutants with reduced oligomerization, such as G6PD-Canton13 15. AG1 improved G6PD-Canton activity up to 1.7-fold with half maximal effective concentration (ECso) of 3.4 pM and increased WT G6PD activity by approximately 20% in previous study15. Thus, Applicant examined the effect of AG1 using LEICA to assess its suitability for screening small molecules.

[0163] Exposure to AG1 had no effect on the growth of E. coli with endogenous G6PD (E. coli 20.71 Epgi) ruling out its impact on bacterial metabolism (FIG. 2). In humanized E. coli, AG1 improved the growth rates of strains expressing WT G6PD and Canton variant up to 12.8% and 20.1% at 0.3 pM, respectively (FIG. 2A); a result that is consistent with a previous study15. Activated G6PD-Canton supported growth of humanized E. coli better than untreated WT and there was no significant difference in the activities when maximally activated ( - value of 0.055; Welch’s / -test, FIG. 2A). Notably, LEICA required lower AG1 concentrations than in vitro and cell-based screens15, likely due to differences in reaction constituent concentrations (e.g., substrates, cofactors, and enzymes) or compound bioavailability across bacterial and mammalian membranes. Nonetheless, LEICA successfully reflected G6PD activation by AG1 without the need for specialized reporterbased assay system.-43- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0164] Given that LEICA can measure the effect of AG1 on human G6PD, Applicant aimed to evaluate its ability to screen various compounds that target G6PD. As G6PD deficient patients face a high risk of hemolysis when exposed to certain drugs, specific foods, or infections37, it is important to evaluate the impact of drugs on G6PD when devising treatment strategies for G6PD-deficient individuals. Additionally, drugs that act on G6PD are active areas of clinical research, as it is linked to antimalarials, immune response, and tumors38-41.

[0165] Seven compounds with known effects on G6PD were chosen for screening. These include: (1) the local anesthetic proparacaine (PPC), (2) the non-selective inhibitor of NADP-dependent enzymes 6-aminonicotinamide (6AN), (3) the energetic compound, radiosensitizer RRx-001, (4) the 012 adrenergic agonist brimonidine (BMN), (5) the steroid hormone dehydroepiandrosterone (DHEA), (6) the small molecule G6PD inhibitor G6PDi-l, and (7) the pyrazolonic analgesic metamizol (MMZ). Previous reports indicate that PPC has no significant effect on G6PD42, while G6PDi-l41, DHEA43, BMN42, and MMZ44exhibit inhibitory effects. RRx-001 and 6 AN are non-selective inhibitors45,46, either generating reactive radicals or non- selectively inhibiting NADP-dependent enzymes. Each compound was supplemented to culture media at a concentration of 100 pM and 10 pg / ml tetracycline (Tc) was used as a positive control for bacterial growth inhibition.|0166] LEICA treated with PPC exhibited no difference in growth profile compared to the untreated control (FIG. 2B and FIG. 9), aligning with previous observations42. Treatment with Tc, RRx-001, and 6 AN completely halted the growth of humanized A. coli for G6PD in LEICA (FIG. 2B). However, the growth of E. coli Epgi was also suppressed by RRx-001 and 6AN (FIG. 10), indicating their toxicity to E. coli and prompting us to exclude them as hits.10167] The growth of humanized E. coli for G6PD (WT) was significantly inhibited by G6PDi-l, DHEA, and BMN (FIGS. 2B, FIG. 2C and 9), with no effect observed onE. coli Epgi (FIG. 10), underscoring the inhibitory effect of these compounds on human G6PD, consistent with the previous reports41-43. However, MMZ showed no discernible effect. This is likely attributable to the high inhibitory concentration of MMZ (IC50 of 17 mM) required to inhibit G6PD activity44, in contrast to G6PDi-l and BMN, whose ICsos are in the sub-millimolar range (0.07-30 pM).-44- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0168] Two compounds — G6PDi-l and BMN — which exhibited inhibitory effects in the primary screen, underwent further examination in dose-response assays to validate the screening results. Applicant also included DHEA that has been reported to have varying G6PD inhibitory effect depending on the assay format. LEICA was performed with each compound at concentrations ranging from 5 to 300 pM, revealing a dose-dependent response with ICso of 111, 138, and 1309 pM, respectively (FIGS. 2C-2E). These IC50 values differed from those obtained in in vitro assays where the IC50 of G6PDi-l, DHEA, and BMN were reported as 0.07, 9, and 30 pM, respectively41,42. However, IC50 values measured from LEICA are more closely aligned with results from cell-based assays, where the IC50 value for G6PDi-l ranged from 13 to 31 pM depending on the cell type. Interestingly, DHEA exhibited an inhibitory effect in LEICA, contrary to the lack of effect in cell-based assays41. Previous reports indicate the antiproliferative effects of DHEA on various cells may stem from indirect effects that are not fully understood47. Thus, the behavior of LEICA in this context resembles that of a recombinant assay. Despite these discrepancies, LEICA effectively screened compounds with comparable IC50 values, making it a suitable format for high-throughput screening applications.|0169] Drug screening identifies potential lead compounds for G6PD: Having demonstrated the sensitivity of LEICA with known compounds that modulate G6PD activity, applicant screened a drug compound library to identify lead compounds with potential effects on G6PD. The screening involved two stages: (1) a single-dose growth assay using LEICA, and (2) validation of primary hits through dose-response assays (FIG. 3A). Of the 160 human metabolism modulators screened, 7 compounds (primary hits) were identified that inhibited the growth of E. coli expressing human G6PD. while showing no effect on the strain with endogenous E. coli G6PD (FIG. 3B).[017(>| The screening successfully re-discovered G6PDi-l as a primary hit, confirming the assay’s robustness. Two other primary hits, methotrexate and gossypol, have also been previously linked to G6PD. Methotrexate, a dihydrofolate reductase inhibitor, has reported G6PD inhibitory effects (IC50 of 114 pM)48and antimalarial activity49. Gossypol, known for its antimalarial properties, inhibits oxidoreductases, including G6PD in Trypanosoma cruzT'’ . Given that G6PDi-l was originally discovered as an inhibitor of T. cruzi G6PD51, it logically -45- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710follows that gossypol, with its reported effects on T. cruzi, might also inhibit human G6PD. These three previously known compounds detected in the screen validate the sensitivity and reliability of the assay.10171] The four remaining compounds have no previously reported association with G6PD activity; however, three completely inhibited the growth of E. coli expressing human G6PD. Further characterization using dose-response assays revealed reliable dose-dependent effects from all the primary hits (FIG. 3C). Among the four identified compounds, three displayed higher potency with ICsos ranging from 15.6 to 109 pM, compared to G6PDi-l, methotrexate, and gossypol (ICsos of 111-148 pM). This result suggests that these identified compounds are candidate leads for antimalarial drugs as potent G6PD inhibitors. The drug screening demonstrates LEICA’ s potential as a versatile high-throughput screening tool capable of identifying compounds that influence human enzymes within a bacterial context.

[0172] Expanding LEICA for non-glycolytic enzymes: To broaden LEICA’ s applicability to enzymes outside glycolysis, Applicant focused on argininosuccinate lyase (ASL), a critical enzyme in the urea cycle (FIG. 4A)52. Although E. coli does not have a urea cycle, it contains an ASL enzyme encoded by the argH gene, which is involved in arginine biosynthesis. Since knockout of argH results in arginine auxotrophy, it creates a suitable platform for assaying human ASL activity by growing cells in arginine-free conditions (FIG.4B). Applicant found that human ASL could complement the arginine auxotrophy in an E. coli argH knockout strain, indicating successful functional expression of human ASL in E. coli (FIG. 11)

[0173] Applicant then examined various ASL sequence variants that are known to cause argininosuccinic aciduria (ASA), with residual enzyme activity ranging from 0 to 6.09% (Table 3)1252The humanized E. coli for ASL whose arginine biosynthesis is supported by different pathogenic ASL variants exhibited significant growth reductions (ranging from 16 to 100%) compared to cells expressing WT human ASL (FIGS. 4C and 11). Specifically, the growth rates of humanized E. coli aligned with the residual activities of recombinant ASL variants: D87G, which results in a complete loss of activity, could not complement the arginine auxotrophy, while the least severe R12Q variant supported growth to 84% of WT levels.-46- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0174] The ASL example demonstrates LEICA’ s versatility in screening enzymes from diverse metabolic pathways. Its expandability suggests LEICA could be used as a platform to study a wide range of human metabolic enzymes, further broadening the potential applications in identifying pathogenic sequence variations and therapeutic compounds for metabolic disorders.

[0175] DISCUSSION

[0176] High-throughput sequencing has revolutionized applicant understanding of the human genome, providing vast amounts of data on genetic variations. However the key challenge lies in deciphering the functional significance of these variations, particularly those associated with genetic disorders. GWAS have identified mutations linked to various disorders, but understanding the causality of these mutations remains a significant hurdle.

[0177] To address this, applicant developed LEICA, a live bacteria assay to measure activities of heterologously expressed human enzymes. Unlike traditional in vitro assays, LEICA leverages bacterial metabolism to rapidly characterize genetic variations associated with enzyme deficiencies. By coupling bacterial growth with enzyme activity, LEICA provides a simpler, cost-effective alternative that aligns well with prior reports. Its ability to screen pathogenic variations through this coupling makes it a distinct improvement over traditional methods, by rapidly providing insights into the functional consequences of genetic variations.

[0178] Beyond its use in studying genetic variations, LEICA proves effective for screening drug effects on human enzymes. By directly assessing the impact of various compounds on enzyme activity in an intracellular environment, LEICA bridges the gap between in vitro and cell-based assays. While offering advantages such as simplicity, speed, and costeffectiveness, it also presents some limitations, such as its inability to screen compounds with antimicrobial properties. However, incorporating a pairwise comparisons between humanized E. coli and the strain carrying the endogenous bacterial gene effectively prevents false-positives, as demonstrated with 6AN treatment (FIG. 2B, FIG. 3B, and 10). Also, applicant acknowledge that bacterial cells may have different permeability to chemical compounds compared to human cells, which could affect the assay’s representation of drug-47- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710efficacy in human cells. However, E. coli cells are non-selectively permeable to small molecules (<600 Da)53, and LEICA’ s demonstrated robustness in identifying seven effective compounds from the drug library supports its reliability as a screening tool. Nonetheless, LEICA’ s ability to operate under intracellular conditions makes it a valuable approach for compound screening. Looking ahead, LEICA holds significant promise for high-throughput screening and personalized drug testing.[0179| In conclusion, LEICA represents an alternative approach for studying genetic enzymopathies, screening drug effects, and advancing precision medicine. By leveraging bacterial metabolic machinery, LEICA offers insights into the functional consequences of genetic variations and facilitates the rapid screening of drug libraries. Moving forward, continued research and innovation in this field holds the potential to impact the identification of lead compounds for human targets that can be manifested in E. coli.[0180J METHODS

[0181] Bacterial strains and culture conditions

[0182] E. coli strain 20.71, which is an adaptively evolved strain with an in-frame substitution of its endogenous phosphoglucose isomerase with human homolog, was constructed in a previous study8. The E. coli 20.71 Epgi strain was constructed by substituting the human GPI with the sacB-cat dual-selection cassette using lambda recombination54. The E. coli 20.71 Epgi zwf double knockout strain was constructed by substituting the zwf gene with a kanamycin resistance cassette on the E. coli 20.71 lpgi background using lambda recombination. Humanized E. coli carrying either the wildtype (WT) GPI or GPI variants were constructed by introducing human WT GPI or variant genes into the double knockout strain using lambda recombination. Human GPI variants were constructed by assembling split human GPI fragments (upstream and downstream of the variation) amplified by primers containing genetic variations. Full GPI constructs were assembled using Overlap Extension PCR (OE PCR). Briefly, 5 ng each of the fragments were first ligated by 15 cycles of the following PCR reaction: 98°C for 30 s, 68°C for 30 s, and 72°C for 90 s. Ligated products were amplified in the same tube by adding two outermost primers (HsaGPI F and HsaGPI R) with 20 cycles of PCR reaction: 98°C for 30-48- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710s, 70°C for 30 s, and 72°C for 90 s. The E. coli t^argH strain was constructed by substituting the argH of E. coli strain K-12 substrain MG1655 with the npt-II kanamycin resistance cassette (amplified from pKD13) using lambda recombination. The kanamycin cassette was subsequently removed by flippase-mediated recombination with a helper plasmid pCP2054. Q5 High-Fidelity DNA Polymerase (NEB) was used to perform PCR as instructed by the manufacturer. Primer sequences are summarized in Table 4. Successful recombinants were screened by sacB counterselection on salt-free LB-agar plate (1% tryptone, 0.5% yeast extract, and 1.5% agar) containing 10% sucrose. Insertion and mutations of the human GPI gene were confirmed by Sanger sequencing. Cells were propagated in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl). For growth assays, M9 glucose medium (4 g / 1 glucose, 47.75 mMNaiHPOi, 22.04 mMKH2PO4, 8.56 mMNaCl, 18.70 mMNH4Cl, 2 mM MgSO4, 0.1 mM CaCh, and trace elements) was used. Trace elements were prepared in 2000* concentrated solution (100 mM FeC13, 9.54 mM ZnCh, 8.41 mM CoCh, 8.27 mM Na2MoO4, 0.75 mM CaCh, 0.91 mM CuCl2, and 0.5 mM H3BO3 in 3.7% (w / w) hydrochloric acid solution). Kanamycin (50 pg / ml) was added when screening humanized E. coli for GPI. To screen humanized E. coli for G6PD, 50 pg / ml kanamycin, 100 pg / ml carbenicillin, and 1 mM isopropyl P-d-1 -thiogalactopyranoside (IPTG) were added to media. To screen humanized E. coli for ASL, 100 pg / ml carbenicillin and 0.01 mM IPTG were added to media. Growth profiles of E. coli strains were monitored using a Tecan Infinite 200 Pro microplate reader (Tecan) operated by i-Control software (v3.7.3.0, Tecan). Cells were incubated in 96-well microplates at 37°C with a culture volume of 100 pl. Microplates were sealed with Breathe-Easy gas-permeable sealing membranes (Diversified Biotech). Optical density (A600nm) was monitored every 15-60 min.

[0183] Cloning G6PD variants and construction of humanized G6PD E. coli model

[0184] The human G6PD gene was chemically synthesized (IDT, sequence provided herein) and cloned into the pTrcHis2A plasmid (Invitrogen) using Gibson assembly (NEBuilder HiFi DNA Assembly Kit, NEB). Briefly, 5 firnol pTrcHis2A plasmid backbone (linearized by PCR) and 20 fmol G6PD gene fragment were mixed in 6 pl reaction followed by incubation at 50°C for 15 min. Different G6PD variants were constructed by assembling split human G6PD fragments (upstream and downstream of the variation) amplified by primers-49- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710containing genetic variations. Full G6PD constructs were assembled by OE PCR as follows: 5 ng each of the fragments were ligated by 15 cycles of the following PCR reaction: 98°C for 30 s, 68°C for 30 s, and 72°C for 90 s. Ligated products were amplified in the same tube by adding two outermost primers (HsaG6PD_F and HsaG6PD_R) with 20 cycles of PCR reaction: 98°C for 30 s, 67°C for 30 s, and 72°C for 90 s. The assembled constructs were cloned into pTrcHis2A using the aforementioned method. Then, plasmid carrying each G6PD variant was introduced into the E. coli 20.71 Epgi tzwf double knockout strain to construct humanized G6PD E. coli model. Glycolytic flux was solely supported by the HMP shunt in E. coli 20.71 pgi strain or 20.71 pgi ^zwf\\a.\r\n^ human G6PD, which induces a slow growth rate, as reported elsewhere55. Thus, Applicant monitored the growth of these strains for up to 60 hrs. Primer sequences are summarized in Table 4.

[0185] Measuring effects of small molecules using LEICA

[0186] The G6PD activator AG1, RRx-001, brimonidine (BMN), 6-aminonicotinamide (6 AN), proparacaine hydrochloride (PPC) were purchased from MedChemExpress (USA). Tetracycline hydrochloride, metamizole (di pyrone) monohydrate (MMZ), and G6PDi-l, were purchased from Sigma-Aldrich (USA). Dehydroepiandrosterone (DHEA) was bought from ApexBio (USA). RRx-001 was prepared as a 100 mM dimethyl sulfoxide (DMSO) solution. AG1, G6PDi-l, and DHEA were prepared as 30 mM DMSO solutions. BMN, PPC, MMZ, and 6AN were prepared as 10 mM aqueous solutions. Tetracycline was prepared as a 10 mg / ml aqueous solution. Compounds were treated at appropriate concentrations with a final DMSO concentration of 1%. DMSO was treated (1%) as an untreated vehicle control.

[0187] Drug library screening

[0188] Cellular Metabolism Screening Library (Cayman Chemical, Cat #33705, Batch #0609421) was used for drug library screening. 1.2 pl of 10 mM compound solutions (DMSO) were dispensed to 96-well microplates. E. coli 20.71 Epgi and E. coli 20.71 Epgi tzwf carrying pTrc_G6PD-WT was incubated in 3 ml LB medium containing 50 pg / ml kanamycin, 25 pg / ml chloramphenicol, and 1 mM IPTG. 100 pg / ml carbenicillin was added to cells carrying plasmid. 1 ml of overnight cultures were washed with 1 ml of M9 glucose medium to remove excess nutrients of LB. The washed resuspensions were inoculated into 3-50- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710ml fresh M9 glucose medium (with initial OD of 0.03) containing 50 pg / ml kanamycin, 25 pg / ml chloramphenicol, and 1 mM IPTG. 100 pg / ml carbenicillin was added to cells carrying plasmid. Then cultures were Incubated at 37°C for 3 hrs and 120 pl of cultures were transferred to 96-well microplates having compounds resulting in 100 pM final concentration. Each plate comprises three replicated cultures of 15 compounds and vehicle control (1% DMSO) for both E. coli 20.71 pgi and E. coli 20.71 Apgi Szwf carrying pTrc_G6PD-WT. Cell growth was monitored every 60 min in a BioTek LogPhase 600 microplate reader (Agilent; set at 37°C with 800 rpm shaking) operated by LogPhase 600 App (vl.08, Agilent).

[0189] Cloning ASL variants and construction of humanized ASL E. coli model

[0190] The human ASL ORF clone was obtained from GenScript (Clone ID OHu22033, Acc. NM_001024943.2) and cloned into the pTrcHis2A plasmid using Gibson assembly. Briefly, 5 firnol pTrcHis2A plasmid backbone (linearized by PCR) and 20 firnol ASL gene fragments were mixed in 6 pl reaction followed by incubation at 50°C for 15 min. Different ASL variants were constructed by assembling split human ASL fragments (upstream and downstream of the variation) amplified by primers containing genetic variations. Full ASL constructs were assembled by OE PCR as follows: 5 ng each of the fragments were ligated by 15 cycles of the following PCR reaction: 98°C for 30 s, 68°C for 30 s, and 72°C for 90 s. Ligated products were amplified in the same tube by adding two outermost primers (HsaASL F and HsaASL R) with 20 cycles of PCR reaction: 98°C for 30 s, 67°C for 30 s, and 72°C for 90 s. The assembled constructs were cloned into pTrcHis2A using the aforementioned method. Primer sequences are summarized in Table 4.

[0011] Statistical analysis

[0192] To compare the difference of means, a two-sided Welch’s / -test was used with Bonferroni correction for multiple hypothesis testing. Dose-response curve was fitted to the Hill curve to estimate ECso or ICso. For drug library screening, 15 compounds were first compared with untreated control using a two-sided Welch’s / -test corrected for multiple hypothesis testing using Bonferroni method. Compounds that induced a significant growth difference (p- value < 0.001) in E. coli 20.71 Spgi Szwf carrying pTrc_G6PD-WT strain,-51- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710while not inducing a significant difference ( - value > 0.001) for E. coli 20.71 pgi were selected as primary hits.

[0193] Data Availability

[0194] Allele frequencies of SNPs in human population were obtained from the Genome Aggregation Database (gnomAD v4.1.0) accessed through gnomAD browser (https: / / gnomad.broadinstitute.org / ). All compounds in the drug library (n=160) were anonymized using proxy identifiers.

[0195] Comparing effects of AG1 on hu-G6PD-Canton and in vitro experiment.

[0196] According to a previous report, AG1 maximally activated recombinant G6PD-Canton by 1.7-fold56. Using LEICA, Applicant reverse-estimated the activity of G6PD-Canton from the measured growth rate of humanized E. coli for G6PD-Canton (FIG. 5F) when activated by AG1. Fitted linear regression function of log-enzyme activity vs. growth rate is as follows (FIG. 5F)GR = 0.0551 A + 0.0459 (Pearson's R2= 0.840)0.6117,

[0197] where GR is the growth rate and A is activity of G6PD.

[0198] Without AG1 treatment, the growth rate of humanized E. coli for G6PD-Canton was 0.0855 h'1(±0.0047, s.d.; n=10). When maximally activated (with 0.3 pM AG1), the growth rate of the same strain was 0.1028 h'1(±0.0026; s.d.; n=10). Relative activities are calculated to be 0.6914±0.0714 (0 pM AG1) and 0.9538±0.0403 (0.3 pM AG1). This gives a 1.38-fold increase, consistent with the previous observation (1.7-fold improvement).

[0199] G6PD inhibitors screened.

[0200] 1. G6PDi-l

[0201] A small molecule inhibitor of G6PDi-l was recently discovered by high-throughput screening57. Reported half-maximal inhibitory concentration (ICso) in in vitro settings is 0.07 pM. It exhibited inhibitory effect in cell-based experiments suppresses T cell (mouse CD8±)-52- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710cytokine production (at concentration of 10-50 pM) and neutrophil (bone-marrow-derived macrophages) oxidative burst at 50 pM2.

[0202] 2. Dehydroepiandrosterone (DHEA)

[0203] DHEA is a steroid hormone precursor and its G6PD inhibitory activity has been reported previously, recombinant enzymes58. However, cell-based assay indicated a limited bioavailability57and G6PD inhibition by indirect mechanisms59.

[0204] 3. RRx-001

[0205] RRx-001 is one of the interesting classes of energetic compounds. Recently, it has been highlighted as a potential radiosensitizer60. Although the mechanisms of chemo / radiosensitizing effect have not fully been understood, its inhibitory activity on G6PD may originate primarily through nitric oxide production61.

[0206] 4 and 5. Brimonidine (BMN) and proparacaine (PPC)

[0207] These two are widely used ophthalmologic drugs. Brimonidine (BMN) is a selective a2- adrenergic receptor agonist, mainly used to lower intraocular pressure in the clinical treatment of patients with glaucoma62,63. Proparacaine (PPC) is a local anesthetic for surface anesthesia in ophthalmology. In the previous study, BMN has an inhibitory effect of G6PD with ICso of 29.93 pM, while PPC has none61,64.

[0208] 6. Metamizol (MMZ; di pyrone)

[0209] Metamizol is a pain reliever that belongs to pyrazolones. Pyrazolones have been reported to potentially induce severe hemolytic anemia65. Effect of metamizole on G6PD was addressed previously, showing an inhibitory effect both in vitro and in vivo, although IC50 was relatively high (17 mM in vitro)66.

[0210] 7. 6-aminonicotinamide (6AN)

[0211] Applicant used 6AN as a positive control of assay. It is a non-selective inhibitor of NADP-dependent enzymes exhibiting antiproliferative effects on prostate cancer cells67.

[0212] LEICA in personalized medicine research.4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0213] As genome sequencing becomes increasingly accessible, precision medicine emerges as an innovative approach to treat patients by accounting for characteristics of individuals. A fundamental aspect of personalized medicine involves understanding a person’s genetic makeup to identify medical interventions that are more likely to succeed while minimizing potential side effects.

[0214] Applicant’s assay method has demonstrated the ability to measure the activities of genetic variants and their responses to drug effects, enabling an exploration of each variant’s sensitivity to drugs. Applicant subjected DHEA, G6PDi-l, and BMN, which exerted inhibitory effects on the WT G6PD, to all constructed variants to assess differences in sensitivities (FIG. 5A).

[0215] In this assay, variants exhibited different sensitivities to the same treatments (FIGS.5B - 5D). Upon DHEA treatment, G6PD-Canton, Mahidol, and Volendam variants showed activities comparable to untreated controls, indicating insensitivities to DHEA (FIG. 5E). Similarly, G6PD-Canton and Volendam were insensitive to G6PDi-l, while G6PD-A+, Mahidol, and Union exhibited similar levels of inhibition to the WT (FIG. 5F). Interestingly, G6PD-Mahidol was more sensitive to BMN than WT G6PD, exhibiting 67.9% residual activity compared to the untreated control, where WT G6PD retained 84.4% activity (FIG.5G). In contrast, the G6PD-Canton variant appeared resistant to all three compounds tested (FIGS. 5E - 5G). These differential drug sensitivities of variants may explain why different G6PD-deficient patients with various G6PD variations exhibit varying levels of hemolytic events after drug administration, such as antimalarials68. However, a detailed structural understanding of these drug bindings on G6PD is lacking, necessitating comprehensive molecular investigation into the drug-enzyme interactions.

[0216] Nucleotide sequences

[0217] Human G6PD cDNA (1548 bp) (SEQ ID NO: 1) - The following double-stranded DNA fragment was synthesized chemically (Integrated DNA Technologies, USA).

[0218] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG-54- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTAA|0219] Protein (SEQ ID NO: 3) Human G6PD (WT)

[0220] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII-55- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*

[0221] Polynucleotide (SEQ ID NO: 4) Human G6PD (Songklanagarind; T196A)

[0222] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCATCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG-56- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTAA

[0223] Protein (SEQ ID NO: 5) Human G6PD (Songklanagarind; F66I)

[0224] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTIIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*

[0225] Polynucleotide (SEQ ID NO: 6) Human G6PD (Namoru; T208C)|0226] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCCATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG-57- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTAA[0227| Protein (SEQ ID NO: 7) Human G6PD (Namoru; Y70H)[0228| MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGHARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNR.il VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*-58- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0229] Polynucleotide (SEQ ID NO: 8) Human G6PD (Murcia; A209G)

[0230] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTGTGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTAA-59- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0231] Protein (SEQ ID NO: 9) Human G6PD (Murcia; Y70C)

[0232] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGCARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*[0233{ Polynucleotide (SEQ ID NO: 10) Human G6PD (A+; A376G)

[0234] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGGAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC-60- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA

[0235] Protein (SEQ ID NO: 11) Human G6PD (A+; N126D)[0236J MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMDALHLGSQANRLFYLALPPTVYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*

[0237] Polynucleotide (SEQ ID NO: 12) Human G6PD (Mahidol; G487A)

[0238] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG-61- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAA GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA[G239] Protein (SEQ ID NO: 13) Human G6PD (Mahidol; G163S)[024(>| MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQISWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA-62- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*

[0241] Polynucleotide (SEQ ID NO: 14) Human G6PD (Plymouth; G488A)

[0242] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG ACTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT-63- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA

[0243] Protein (SEQ ID NO: 15) Human G6PD (Plymouth; G163D)

[0244] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIDWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*-64- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0245] Polynucleotide (SEQ ID NO: 16) Human G6PD (Volendam; C514T)

[0246] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGTCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA-65- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0247] Protein (SEQ ID NO: 17) Human G6PD (Volendam; P172S)[0248| MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKSFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*[02491 Polynucleotide (SEQ ID NO: 18) Human G6PD (Santiago; G593C)[0250| ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCCCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC-66- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA[0251 | Protein (SEQ ID NO: 19) Human G6PD (Santiago; R198P)

[0252] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYPIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0253] Polynucleotides (SEQ ID NO: 20) Human G6PD (Durham; A713G)

[0254] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAGGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA-68- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0255] Protein (SEQ ID NO: 21) Human G6PD (Durham; K238R)

[0256] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFREPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*

[0257] Polynucleotides (SEQ ID NO: 22) Human G6PD (Omiya; G921C)

[0258] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCACT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC-69- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA

[0259] Protein (SEQ ID NO: 23) Human G6PD (Omiya; Q307H)[0260J MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGHYVGNPDGEGEATKGYLDDPTVPRGSTTATFA AVVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRV QPNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQ MHFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTY KWVNPHKL*-70- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0261] Polynucleotide (SEQ ID NO: 24) Human G6PD (Puerto Limon; G1192A)

[0262] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACA AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA-71- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0263] Protein (SEQ ID NO: 25) Human G6PD (Puerto Limon; E398K)

[0264] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNKAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*[0265{ Polynucleotide (SEQ ID NO: 26) Human G6PD (Canton; G1376T)[0266| ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC-72- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCTTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA

[0267] Protein (SEQ ID NO: 27) Human G6PD (Canton; R459L)[0268J MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELLEAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0269] Polynucleotide (SEQ ID NO: 28) Human G6PD (Union; C1360T)

[0270] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTG1 GCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA-74- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0271] Protein (SEQ ID NO: 29) Human G6PD (Union; R454C)[0272| MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVCSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*[0273{ Polynucleotide (SEQ ID NO: 30) Human G6PD (N426K; C1278G)[0274| ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC-75- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAAGAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA

[0275] Protein (SEQ ID NO: 31) Human G6PD (N426K; N426K)[0276J MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGKRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0277] Polynucleotide (SEQ ID NO: 32) Human G6PD (Sumare; T1292G)[0278| ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGGGAAGCTC CCTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACT TCGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCA CCAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGG CCCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCAC CTACAAGTGGGTGAACCCCCACAAGCTCTGA-77- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0279] Protein (SEQ ID NO: 33) Human G6PD (Sumare; V431G)

[0280] MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNGKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*

[0281] Polynucleotide (SEQ ID NO: 34) Human G6PD (R104H; G311 A)

[0282] ATGGC AGAGC AGGTGGCCCTGAGCCGGACCC AGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCACAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC-78- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGGATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA

[0283] Protein (SEQ ID NO: 35) Human G6PD (R104H; R104H)[0284J MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFAHNSYVAG QYDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNR IIVEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIW NRDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSD DVRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFA AVVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRV QPNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQ MHFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTY KWVNPHKL*4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0285] Polynucleotide (SEQ ID NO: 36) Human G6PD (Mira d’ Aire; G1048C)

[0286] ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCG GGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATC ATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGG TGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCC GTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGC CACCCCAGAGGAGAAGCTCAAGCTGGAGGACTTCTTTGCCCGCAACTCCTATGTG GCTGGCCAGTACGATGATGCAGCCTCCTACCAGCGCCTCAACAGCCACATGAAT GCCCTCCACCTGGGGTCACAGGCCAACCGCCTCTTCTACCTGGCCTTGCCCCCGA CCGTCTACGAGGCCGTCACCAAGAACATTCACGAGTCCTGCATGAGCCAGATAG GCTGGAACCGCATCATCGTGGAGAAGCCCTTCGGGAGGGACCTGCAGAGCTCTG ACCGGCTGTCCAACCACATCTCCTCCCTGTTCCGTGAGGACCAGATCTACCGCAT CGACCACTACCTGGGCAAGGAGATGGTGCAGAACCTCATGGTGCTGAGATTTGC CAACAGGATCTTCGGCCCCATCTGGAACCGGGACAACATCGCCTGCGTTATCCTC ACCTTCAAGGAGCCCTTTGGCACTGAGGGTCGCGGGGGCTATTTCGATGAATTTG GGATCATCCGGGACGTGATGCAGAACCACCTACTGCAGATGCTGTGTCTGGTGG CCATGGAGAAGCCCGCCTCCACCAACTCAGATGACGTCCGTGATGAGAAGGTCA AGGTGTTGAAATGCATCTCAGAGGTGCAGGCCAACAATGTGGTCCTGGGCCAGT ACGTGGGGAACCCCGATGGAGAGGGCGAGGCCACCAAAGGGTACCTGGACGAC CCCACGGTGCCCCGCGGGTCCACCACCGCCACTTTTGCAGCCGTCGTCCTCTATG TGGAGAATGAGAGGTGGCATGGGGTGCCCTTCATCCTGCGCTGCGGCAAGGCCC TGAACGAGCGCAAGGCCGAGGTGAGGCTGCAGTTCCATGATGTGGCCGGCGACA TCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACG AGGCCGTGTACACCAAGATGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCG AGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCC CTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTT CGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCAC CAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGC CCCACGGAGGCAGACGAGCTGATGAAGAGAGTGGGTTTCCAGTATGAGGGCACC TACAAGTGGGTGAACCCCCACAAGCTCTGA-80- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0287] Protein (SEQ ID NO: 37) Human G6PD (Mira d’ Aire; D350H)[0288| MAEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIW WLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQ YDDAASYQRLNSHMNALHLGSQANRLFYL ALPPT VYEAVTKNIHESCMSQIGWNRII VEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWN RDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDD VRDEKVKVLKCISEVQANNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAA VVLYVENERWHGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQ PNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQM HFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYK WVNPHKL*[0289{ Polynucleotide (SEQ ID NO: 38) Human GPI (WT)[0290| ATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCGGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCATTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGACGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG-81- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCGCTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACGACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA

[0291] Protein (SEQ ID NO: 39) Human GPI (WT)

[0292] MAALTRDPQFQKLQQWYREHRSELNLRRLFD ANKDRFNHF SLTLNTNHGHI LVDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRNR SNTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSDLG PLMVTEALKP YS SGGPRVW YVSNIDGTHIAKTL AQLNPES SLFIIASKTFTTQETITNA ETAKE WFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYSLW SAIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFGCE THAMLPYDQYLHRFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNGQH AFYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEARK ELQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGIIW DINSFDQWGVELGKQLAKKIEPELDGSAQVTSHDASTNGLINFIKQQREARVQ*-82- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0293] Polynucleotide (SEQ ID NO: 40) Human GPI (I208T; T623C)

[0294] ATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCGGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCACTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGACGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCGCTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA-83- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACGACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA

[0295] Protein (SEQ ID NO: 41) Human GPI (I208T; I208T)|0296] MAALTRDPQFQKLQQWYREHRSELNLRRLFD ANKDRFNHF SLTLNTNHGHI LVDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRNR SNTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSDLG PLMVTEALKP YS SGGPRVWYVSNIDGTHIAKTL AQLNPES SLFITASKTFTTQETITNA ETAKE WFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYSLW SAIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFGCE THAMLPYDQYLHRFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNGQH AFYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEARK ELQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGIIW DINSFDQWGVELGKQLAKKIEPELDGSAQVTSHDASTNGLINFIKQQREARVQ*

[0297] Polynucleotide (SEQ ID NO: 42) Human GPI (R106Q; G317A)

[0298] ATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCAGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCATTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGACGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG-84- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCGCTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACGACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA

[0299] Protein (SEQ ID NO: 43) Human GPI (R106Q; R106Q)

[0300] MAALTRDPQFQKLQQWYREHRSELNLRRLFD ANKDRFNHF SLTLNTNHGHI LVDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRN QSNTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSD LGPLMVTEALKP YS SGGPRVWYVSNIDGTHIAKTL AQLNPES SLFIIASKTFTTQETIT NAETAKEWFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYS LWSAIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFG CETHAMLPYDQYLHRFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNG QHAFYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEA RKELQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGI IWDINSFDQWGVELGKQLAKKIEPELDGSAQVTSHDASTNGLINFIKQQREARVQ* 10301] Polynucleotide (SEQ ID NO: 44) Human GPI (Fukuoka; G1615A)-85- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0302] ATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCGGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCATTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGACGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCGCTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACAACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA-86- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0303] Protein (SEQ ID NO: 45) Human GPI (Fukuoka; D539N)

[0304] MAALTRDPQFQKLQQWYREHRSELNLRRLFDANKDRFNHFSLTLNTNHGHI LVDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRNR SNTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSDLG PLMVTEALKP YS SGGPRVW YVSNIDGTHIAKTL AQLNPES SLFIIASKTFTTQETITNA ETAKE WFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYSLW SAIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFGCE THAMLPYDQYLHRFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNGQH AFYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEARK ELQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGIIW DINSFDQWGVELGKQLAKKIEPELDGSAQVTSHNASTNGLINFIKQQREARVQ*

[0305] Polynucleotide (SEQ ID NO: 46) Human GPI (Iwate; C671T)

[0306] ATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCGGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCATTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGATGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG-87- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCGCTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACGACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA

[0307] Protein (SEQ ID NO: 47) Human GPI (Iwate; T224M)

[0308] MAALTRDPQFQKLQQWYREHRSELNLRRLFD ANKDRFNHF SLTLNTNHGHI LVDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRNR SNTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSDLG PLMVTEALKP YS SGGPRVW YVSNIDGTHIAKTL AQLNPES SLFIIASKTFTTQETITNA EMAKEWFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYSL WSAIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFGC ETHAMLPYDQYLHRFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNGQ HAFYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEAR KELQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGII WDINSFDQWGVELGKQLAKKIEPELDGSAQVTSHDASTNGLINFIKQQREARVQ*

[0309] Polynucleotide (SEQ ID NO: 48) Human GPI (Matsumoto; C14T)

[0310] ATGGCCGCTCTCATCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG-88- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCGGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCATTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGACGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCGCTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACGACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA

[0311] Protein (SEQ ID NO: 49) Human GPI (Matsumoto; T5I)

[0312] MAALIRDPQFQKLQQWYREHRSELNLRRLFDANKDRFNHFSLTLNTNHGHIL VDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRNRS-89- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710NTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSDLGP LMVTEALKP YS SGGPRVWYVSNIDGTHIAKTL AQLNPES SLFIIASKTFTTQETITNAE TAKEWFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYSLWS AIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFGCET HAMLPYDQYLHRFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNGQHA FYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEARKE LQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGIIWD INSFDQWGVELGKQLAKKIEPELDGSAQVTSHDASTNGLINFIKQQREARVQ* |0313] Polynucleotide (SEQ ID NO: 50) Human GPI (R347H; G1040A)

[0314] ATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAAGCTGCAGCAATGGTA CCGCGAGCACCGCTCCGAGCTGAACCTGCGCCGCCTCTTCGATGCCAACAAGGA CCGCTTCAACCACTTCAGCTTGACCCTCAACACCAACCATGGGCATATCCTGGTG GATTACTCCAAGAACCTGGTGACGGAGGACGTGATGCGGATGCTGGTGGACTTG GCCAAGTCCAGGGGCGTGGAGGCCGCCCGGGAGCGGATGTTCAATGGTGAGAAG ATCAACTACACCGAGGGTCGAGCCGTGCTGCACGTGGCTCTGCGGAACCGGTCA AACACACCCATCCTGGTAGACGGCAAGGATGTGATGCCAGAGGTCAACAAGGTT CTGGACAAGATGAAGTCTTTCTGCCAGCGTGTCCGGAGCGGTGACTGGAAGGGG TACACAGGCAAGACCATCACGGACGTCATCAACATTGGCATTGGCGGCTCCGAC CTGGGACCCCTCATGGTGACTGAAGCCCTTAAGCCATACTCTTCAGGAGGTCCCC GCGTCTGGTATGTCTCCAACATTGATGGAACTCACATTGCCAAAACCCTGGCCCA GCTGAACCCCGAGTCCTCCCTGTTCATCATTGCCTCCAAGACCTTTACTACCCAG GAGACCATCACGAATGCAGAGACGGCGAAGGAGTGGTTTCTCCAGGCGGCCAAG GATCCTTCTGCAGTGGCGAAGCACTTTGTTGCCCTGTCTACTAACACAACCAAAG TGAAGGAGTTTGGAATTGACCCTCAAAACATGTTCGAGTTCTGGGATTGGGTGGG AGGACGCTACTCGCTGTGGTCGGCCATCGGACTCTCCATTGCCCTGCACGTGGGT TTTGACAACTTCGAGCAGCTGCTCTCGGGGGCTCACTGGATGGACCAGCACTTCC GCACGACGCCCCTGGAGAAGAACGCCCCCGTCTTGCTGGCCCTGCTGGGTATCTG GTACATCAACTGCTTTGGGTGTGAGACACACGCCATGCTGCCCTATGACCAGTAC CTGCACCACTTTGCTGCGTACTTCCAGCAGGGCGACATGGAGTCCAATGGGAAAT ACATCACCAAATCTGGAACCCGTGTGGACCACCAGACAGGCCCCATTGTGTGGG-90- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GGGAGCCAGGGACCAATGGCCAGCATGCTTTTTACCAGCTCATCCACCAAGGCA CCAAGATGATACCCTGTGACTTCCTCATCCCGGTCCAGACCCAGCACCCCATACG GAAGGGTCTGCATCACAAGATCCTCCTGGCCAACTTCTTGGCCCAGACAGAGGC CCTGATGAGGGGAAAATCGACGGAGGAGGCCCGAAAGGAGCTCCAGGCTGCGG GCAAGAGTCCAGAGGACCTTGAGAGGCTGCTGCCACATAAGGTCTTTGAAGGAA ATCGCCCAACCAACTCTATTGTGTTCACCAAGCTCACACCATTCATGCTTGGAGC CTTGGTCGCCATGTATGAGCACAAGATCTTCGTTCAGGGCATCATCTGGGACATC AACAGCTTTGACCAGTGGGGAGTGGAGCTGGGAAAGCAGCTGGCTAAGAAAATA GAGCCTGAGCTTGATGGCAGTGCTCAAGTGACCTCTCACGACGCTTCTACCAATG GGCTCATCAACTTCATCAAGCAGCAGCGCGAGGCCAGAGTCCAATAA[03151 Protein (SEQ ID NO: 51) Human GPI (R347H; R347H)[0316| MAALTRDPQFQKLQQWYREHRSELNLRRLFDANKDRFNHFSLTLNTNHGHI LVDYSKNLVTEDVMRMLVDLAKSRGVEAARERMFNGEKINYTEGRAVLHVALRNR SNTPILVDGKDVMPEVNKVLDKMKSFCQRVRSGDWKGYTGKTITDVINIGIGGSDLG PLMVTEALKP YS SGGPRVW YVSNIDGTHIAKTL AQLNPES SLFIIASKTFTTQETITNA ETAKE WFLQAAKDPSAVAKHFVALSTNTTKVKEFGIDPQNMFEFWDWVGGRYSLW SAIGLSIALHVGFDNFEQLLSGAHWMDQHFRTTPLEKNAPVLLALLGIWYINCFGCE THAMLPYDQYLHHFAAYFQQGDMESNGKYITKSGTRVDHQTGPIVWGEPGTNGQH AFYQLIHQGTKMIPCDFLIPVQTQHPIRKGLHHKILLANFLAQTEALMRGKSTEEARK ELQAAGKSPEDLERLLPHKVFEGNRPTNSIVFTKLTPFMLGALVAMYEHKIFVQGIIW DINSFDQWGVELGKQLAKKIEPELDGSAQVTSHDASTNGLINFIKQQREARVQ*[0317| Polynucleotide (SEQ ID NO: 52) Human ASL (WT)

[0318] ATGGCCTCGGAGAGTGGGAAGCTTTGGGGTGGCCGGTTTGTGGGTGCAGT GGACCCCATCATGGAGAAGTTCAACGCGTCCATTGCCTACGACCGGCACCTTTGG GAGGTGGATGTTCAAGGCAGCAAAGCCTACAGCAGGGGCCTGGAGAAGGCAGG GCTCCTCACCAAGGCCGAGATGGACCAGATACTCCATGGCCTAGACAAGGTGGC TGAGGAGTGGGCCCAGGGCACCTTCAAACTGAACTCCAATGATGAGGACATCCA CACAGCCAATGAGCGCCGCCTGAAGGAGCTCATTGGTGCAACGGCAGGGAAGCT GCACACGGGACGGAGCCGGAATGACCAGGTGGTCACAGACCTCAGGCTGTGGAT-91- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710GCGGCAGACCTGCTCCACGCTCTCGGGCCTCCTCTGGGAGCTCATTAGGACCATG GTGGATCGGGCAGAGGCGGAACGTGATGTTCTCTTCCCGGGGTACACCCATTTGC AGAGGGCCCAGCCCATCCGCTGGAGCCACTGGATTCTGAGCCACGCCGTGGCAC TGACCCGAGACTCTGAGCGGCTGCTGGAGGTGCGGAAGCGGATCAATGTCCTGC CCCTGGGGAGTGGGGCCATTGCAGGCAATCCCCTGGGTGTGGACCGAGAGCTGC TCCGAGCAGAACTCAACTTTGGGGCCATCACTCTCAACAGCATGGATGCCACTAG TGAGCGGGACTTTGTGGCCGAGTTCCTGTTCTGGGCTTCGCTGTGCATGACCCAT CTCAGCAGGATGGCCGAGGACCTCATCCTCTACTGCACCAAGGAATTCAGCTTCG TGCAGCTCTCAGATGCCTACAGCACGGGAAGCAGCCTGATGCCCCAGAAGAAAA ACCCCGACAGTTTGGAGCTGATCCGGAGCAAGGCTGGGCGTGTGTTTGGGCGGT GTGCCGGGCTCCTGATGACCCTCAAGGGACTTCCCAGCACCTACAACAAAGACTT ACAGGAGGACAAGGAAGCTGTGTTTGAAGTGTCAGACACTATGAGTGCCGTGCT CCAGGTGGCCACTGGCGTCATCTCTACGCTGCAGATTCACCAAGAGAACATGGG ACAGGCTCTCAGCCCCGACATGCTGGCCACTGACCTTGCCTATTACCTGGTCCGC AAAGGGATGCCATTCCGCCAGGCCCACGAGGCCTCCGGGAAAGCTGTGTTCATG GCCGAGACCAAGGGGGTCGCCCTCAACCAGCTGTCACTGCAGGAGCTGCAGACC ATCAGCCCCCTGTTCTCGGGCGACGTGATCTGCGTGTGGGACTACGGGCACAGTG TGGAGCAGTATGGTGCCCTGGGCGGCACTGCGCGCTCCAGCGTCGACTGGCAGA TCCGCCAGGTGCGGGCGCTACTGCAGGCACAGCAGGCCTAATAA

[0319] Protein (SEQ ID NO: 53) Human ASL (WT)[0320| MASESGKLWGGRFVGAVDPIMEKFNASIAYDRHLWEVDVQGSKAYSRGLEK AGLLTKAEMDQILHGLDKVAEEWAQGTFKLNSNDEDIHTANERRLKELIGATAGKL HTGRSRNDQVVTDLRLWMRQTCSTLSGLLWELIRTMVDRAEAERDVLFPGYTHLQ RAQPIRWSHWILSHAVALTRDSERLLEVRKRINVLPLGSGAIAGNPLGVDRELLRAEL NFGAITLNSMDATSERDFVAEFLFWASLCMTHLSRMAEDLILYCTKEFSFVQLSDAY STGSSLMPQKKNPDSLELIRSKAGRVFGRCAGLLMTLKGLPSTYNKDLQEDKEAVFE VSDTMSAVLQVATGVISTLQIHQENMGQALSPDMLATDLAYYLVRKGMPFRQAHE ASGKAVFMAETKGVALNQLSLQELQTISPLFSGDVICVWDYGHSVEQYGALGGTAR S S VDWQIRQ VRALLQ AQQ A*

[0321] Polynucleotide (SEQ ID NO: 54) Human ASL (R12Q; G35A)-92- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0322] ATGGCCTCGGAGAGTGGGAAGCTTTGGGGTGGCCAGTTTGTGGGTGCAGT GGACCCCATCATGGAGAAGTTCAACGCGTCCATTGCCTACGACCGGCACCTTTGG GAGGTGGATGTTCAAGGCAGCAAAGCCTACAGCAGGGGCCTGGAGAAGGCAGG GCTCCTCACCAAGGCCGAGATGGACCAGATACTCCATGGCCTAGACAAGGTGGC TGAGGAGTGGGCCCAGGGCACCTTCAAACTGAACTCCAATGATGAGGACATCCA CACAGCCAATGAGCGCCGCCTGAAGGAGCTCATTGGTGCAACGGCAGGGAAGCT GCACACGGGACGGAGCCGGAATGACCAGGTGGTCACAGACCTCAGGCTGTGGAT GCGGCAGACCTGCTCCACGCTCTCGGGCCTCCTCTGGGAGCTCATTAGGACCATG GTGGATCGGGCAGAGGCGGAACGTGATGTTCTCTTCCCGGGGTACACCCATTTGC AGAGGGCCCAGCCCATCCGCTGGAGCCACTGGATTCTGAGCCACGCCGTGGCAC TGACCCGAGACTCTGAGCGGCTGCTGGAGGTGCGGAAGCGGATCAATGTCCTGC CCCTGGGGAGTGGGGCCATTGCAGGCAATCCCCTGGGTGTGGACCGAGAGCTGC TCCGAGCAGAACTCAACTTTGGGGCCATCACTCTCAACAGCATGGATGCCACTAG TGAGCGGGACTTTGTGGCCGAGTTCCTGTTCTGGGCTTCGCTGTGCATGACCCAT CTCAGCAGGATGGCCGAGGACCTCATCCTCTACTGCACCAAGGAATTCAGCTTCG TGCAGCTCTCAGATGCCTACAGCACGGGAAGCAGCCTGATGCCCCAGAAGAAAA ACCCCGACAGTTTGGAGCTGATCCGGAGCAAGGCTGGGCGTGTGTTTGGGCGGT GTGCCGGGCTCCTGATGACCCTCAAGGGACTTCCCAGCACCTACAACAAAGACTT ACAGGAGGACAAGGAAGCTGTGTTTGAAGTGTCAGACACTATGAGTGCCGTGCT CCAGGTGGCCACTGGCGTCATCTCTACGCTGCAGATTCACCAAGAGAACATGGG ACAGGCTCTCAGCCCCGACATGCTGGCCACTGACCTTGCCTATTACCTGGTCCGC AAAGGGATGCCATTCCGCCAGGCCCACGAGGCCTCCGGGAAAGCTGTGTTCATG GCCGAGACCAAGGGGGTCGCCCTCAACCAGCTGTCACTGCAGGAGCTGCAGACC ATCAGCCCCCTGTTCTCGGGCGACGTGATCTGCGTGTGGGACTACGGGCACAGTG TGGAGCAGTATGGTGCCCTGGGCGGCACTGCGCGCTCCAGCGTCGACTGGCAGA TCCGCCAGGTGCGGGCGCTACTGCAGGCACAGCAGGCCTAATAA

[0323] Protein (SEQ ID NO: 55) Human ASL (R12Q; R12Q)|0324] MASESGKLWGGQFVGAVDPIMEKFNASIAYDRHLWEVDVQGSKAYSRGLE KAGLLTKAEMDQILHGLDKVAEEWAQGTFKLNSNDEDIHTANERRLKELIGATAGK LHTGRSRNDQVVTDLRLWMRQTCSTLSGLLWELIRTMVDRAEAERDVLFPGYTHLQ-93- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710RAQPIRWSHWILSHAVALTRDSERLLEVRKRINVLPLGSGAIAGNPLGVDRELLRAEL NFGAITLNSMDATSERDFVAEFLFWASLCMTHLSRMAEDLILYCTKEFSFVQLSDAY STGSSLMPQKKNPDSLELIRSKAGRVFGRCAGLLMTLKGLPSTYNKDLQEDKEAVFE VSDTMSAVLQVATGVISTLQIHQENMGQALSPDMLATDLAYYLVRKGMPFRQAHE ASGKAVFMAETKGVALNQLSLQELQTISPLFSGDVICVWDYGHSVEQYGALGGTAR S S VDWQIRQ VRALLQ AQQ A*[0325| Polynucleotide (SEQ ID NO: 56) Human ASL (D87G; A260G)

[0326] ATGGCCTCGGAGAGTGGGAAGCTTTGGGGTGGCCGGTTTGTGGGTGCAGT GGACCCCATCATGGAGAAGTTCAACGCGTCCATTGCCTACGACCGGCACCTTTGG GAGGTGGATGTTCAAGGCAGCAAAGCCTACAGCAGGGGCCTGGAGAAGGCAGG GCTCCTCACCAAGGCCGAGATGGACCAGATACTCCATGGCCTAGACAAGGTGGC TGAGGAGTGGGCCCAGGGCACCTTCAAACTGAACTCCAATGATGAGGGCATCCA CACAGCCAATGAGCGCCGCCTGAAGGAGCTCATTGGTGCAACGGCAGGGAAGCT GCACACGGGACGGAGCCGGAATGACCAGGTGGTCACAGACCTCAGGCTGTGGAT GCGGCAGACCTGCTCCACGCTCTCGGGCCTCCTCTGGGAGCTCATTAGGACCATG GTGGATCGGGCAGAGGCGGAACGTGATGTTCTCTTCCCGGGGTACACCCATTTGC AGAGGGCCCAGCCCATCCGCTGGAGCCACTGGATTCTGAGCCACGCCGTGGCAC TGACCCGAGACTCTGAGCGGCTGCTGGAGGTGCGGAAGCGGATCAATGTCCTGC CCCTGGGGAGTGGGGCCATTGCAGGCAATCCCCTGGGTGTGGACCGAGAGCTGC TCCGAGCAGAACTCAACTTTGGGGCCATCACTCTCAACAGCATGGATGCCACTAG TGAGCGGGACTTTGTGGCCGAGTTCCTGTTCTGGGCTTCGCTGTGCATGACCCAT CTCAGCAGGATGGCCGAGGACCTCATCCTCTACTGCACCAAGGAATTCAGCTTCG TGCAGCTCTCAGATGCCTACAGCACGGGAAGCAGCCTGATGCCCCAGAAGAAAA ACCCCGACAGTTTGGAGCTGATCCGGAGCAAGGCTGGGCGTGTGTTTGGGCGGT GTGCCGGGCTCCTGATGACCCTCAAGGGACTTCCCAGCACCTACAACAAAGACTT ACAGGAGGACAAGGAAGCTGTGTTTGAAGTGTCAGACACTATGAGTGCCGTGCT CCAGGTGGCCACTGGCGTCATCTCTACGCTGCAGATTCACCAAGAGAACATGGG ACAGGCTCTCAGCCCCGACATGCTGGCCACTGACCTTGCCTATTACCTGGTCCGC AAAGGGATGCCATTCCGCCAGGCCCACGAGGCCTCCGGGAAAGCTGTGTTCATG GCCGAGACCAAGGGGGTCGCCCTCAACCAGCTGTCACTGCAGGAGCTGCAGACC-94- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710ATCAGCCCCCTGTTCTCGGGCGACGTGATCTGCGTGTGGGACTACGGGCACAGTG TGGAGCAGTATGGTGCCCTGGGCGGCACTGCGCGCTCCAGCGTCGACTGGCAGA TCCGCCAGGTGCGGGCGCTACTGCAGGCACAGCAGGCCTAATAA|0327] Protein (SEQ ID NO: 57) Human ASL (D87G; D87G)

[0328] MASESGKLWGGRFVGAVDPIMEKFNASIAYDRHLWEVDVQGSKAYSRGLEK AGLLTKAEMDQILHGLDKVAEEWAQGTFKLNSNDEGIHTANERRLKELIGATAGKL HTGRSRNDQVVTDLRLWMRQTCSTLSGLLWELIRTMVDRAEAERDVLFPGYTHLQ RAQPIRWSHWILSHAVALTRDSERLLEVRKRINVLPLGSGAIAGNPLGVDRELLRAEL NFGAITLNSMDATSERDFVAEFLFWASLCMTHLSRMAEDLILYCTKEFSFVQLSDAY STGSSLMPQKKNPDSLELIRSKAGRVFGRCAGLLMTLKGLPSTYNKDLQEDKEAVFE VSDTMSAVLQVATGVISTLQIHQENMGQALSPDMLATDLAYYLVRKGMPFRQAHE ASGKAVFMAETKGVALNQLSLQELQTISPLFSGDVICVWDYGHSVEQYGALGGTAR S S VDWQIRQ VRALLQ AQQ A*

[0329] Polynucleotide (SEQ ID NO: 58) Human ASL (Q286R; A857G)

[0330] ATGGCCTCGGAGAGTGGGAAGCTTTGGGGTGGCCGGTTTGTGGGTGCAGT GGACCCCATCATGGAGAAGTTCAACGCGTCCATTGCCTACGACCGGCACCTTTGG GAGGTGGATGTTCAAGGCAGCAAAGCCTACAGCAGGGGCCTGGAGAAGGCAGG GCTCCTCACCAAGGCCGAGATGGACCAGATACTCCATGGCCTAGACAAGGTGGC TGAGGAGTGGGCCCAGGGCACCTTCAAACTGAACTCCAATGATGAGGACATCCA CACAGCCAATGAGCGCCGCCTGAAGGAGCTCATTGGTGCAACGGCAGGGAAGCT GCACACGGGACGGAGCCGGAATGACCAGGTGGTCACAGACCTCAGGCTGTGGAT GCGGCAGACCTGCTCCACGCTCTCGGGCCTCCTCTGGGAGCTCATTAGGACCATG GTGGATCGGGCAGAGGCGGAACGTGATGTTCTCTTCCCGGGGTACACCCATTTGC AGAGGGCCCAGCCCATCCGCTGGAGCCACTGGATTCTGAGCCACGCCGTGGCAC TGACCCGAGACTCTGAGCGGCTGCTGGAGGTGCGGAAGCGGATCAATGTCCTGC CCCTGGGGAGTGGGGCCATTGCAGGCAATCCCCTGGGTGTGGACCGAGAGCTGC TCCGAGCAGAACTCAACTTTGGGGCCATCACTCTCAACAGCATGGATGCCACTAG TGAGCGGGACTTTGTGGCCGAGTTCCTGTTCTGGGCTTCGCTGTGCATGACCCAT CTCAGCAGGATGGCCGAGGACCTCATCCTCTACTGCACCAAGGAATTCAGCTTCG-95- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710TGCAGCTCTCAGATGCCTACAGCACGGGAAGCAGCCTGATGCCCCGGAAGAAAA ACCCCGACAGTTTGGAGCTGATCCGGAGCAAGGCTGGGCGTGTGTTTGGGCGGT GTGCCGGGCTCCTGATGACCCTCAAGGGACTTCCCAGCACCTACAACAAAGACTT ACAGGAGGACAAGGAAGCTGTGTTTGAAGTGTCAGACACTATGAGTGCCGTGCT CCAGGTGGCCACTGGCGTCATCTCTACGCTGCAGATTCACCAAGAGAACATGGG ACAGGCTCTCAGCCCCGACATGCTGGCCACTGACCTTGCCTATTACCTGGTCCGC AAAGGGATGCCATTCCGCCAGGCCCACGAGGCCTCCGGGAAAGCTGTGTTCATG GCCGAGACCAAGGGGGTCGCCCTCAACCAGCTGTCACTGCAGGAGCTGCAGACC ATCAGCCCCCTGTTCTCGGGCGACGTGATCTGCGTGTGGGACTACGGGCACAGTG TGGAGCAGTATGGTGCCCTGGGCGGCACTGCGCGCTCCAGCGTCGACTGGCAGA TCCGCCAGGTGCGGGCGCTACTGCAGGCACAGCAGGCCTAATAA

[0331] Protein (SEQ ID NO: 59) Human ASL (Q286R; Q286R)

[0332] MASESGKLWGGRFVGAVDPIMEKFNASIAYDRHLWEVDVQGSKAYSRGLEK AGLLTKAEMDQILHGLDKVAEEWAQGTFKLNSNDEDIHTANERRLKELIGATAGKL HTGRSRNDQVVTDLRLWMRQTCSTLSGLLWELIRTMVDRAEAERDVLFPGYTHLQ RAQPIRWSHWILSHAVALTRDSERLLEVRKRINVLPLGSGAIAGNPLGVDRELLRAEL NFGAITLNSMDATSERDFVAEFLFWASLCMTHLSRMAEDLILYCTKEFSFVQLSDAY STGSSLMPRKKNPDSLELIRSKAGRVFGRCAGLLMTLKGLPSTYNKDLQEDKEAVFE VSDTMSAVLQVATGVISTLQIHQENMGQALSPDMLATDLAYYLVRKGMPFRQAHE ASGKAVFMAETKGVALNQLSLQELQTISPLFSGDVICVWDYGHSVEQYGALGGTAR S S VDWQIRQ VRALLQ AQQ A*

[0333] Table 1. Human genetic variations in glucose-6-phosphate isomerase (GPI) and their clinical manifestations. SNV, single nucleotide variation. P, pathogenic. LP, likely pathogenic. B, benign. LB, likely benign. U, uncertain significance. HA, hemolytic anemia. RBS, red blood cells. ND, not detected, in vitro activities of variants were previously reported69.-96- 4923-5828-5447.1Atty. Dkt. No.: 114198-37104923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0334] Table 2. Human genetic variations in glucose 6-phosphate dehydrogenase (G6PD) and their clinical manifestations. P, pathogenic. LP, likely pathogenic. B, benign. LB, likely benign. U, uncertain significance. HA, hemolytic anemia. AHA, acute hemolytic anemia. CNSHA, chronic non- spherocytic hemolytic anemia. F, favism NA, not available. ND, not detected. Source data are provided as a Source Data file.-98- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710[0335 { Table 3. Human genetic variations in argininosuccinate lyase (ASL) and their clinical manifestations. SNV, single nucleotide variation. P, pathogenic. LP, likely pathogenic. B, benign. LB, likely benign. U, uncertain significance. HA, hemolytic anemia. RBS, red blood cells. ND, not detected, a: reference, b: gnomeAD. in vitro activities were previously reported70,71.[0336{ Table 4. Primers used in this study. Overlap extension (OE) PCRwas performed to assemble two DNA fragments.-99- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710-100- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710-101- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710-102- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710-103- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710-104- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0337] Experimental Summary

[0338] Applicant’s disclosure addresses the difficulty of establishing the functional consequences of human genetic variations, particularly those associated with metabolic-105- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710enzymopathies such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, glucose-6-phosphate isomerase (GPI) deficiency, and argininosuccinate lyase (ASL) deficiency. While high-throughput sequencing can identify large numbers of sequence variants in these genes, the majority of these variants are of unknown significance due to the lack of functional characterization. Traditional biochemical assays rely on patient-derived material or recombinant protein purification, which are labor-intensive, low-throughput, dependent on sample availability, and often fail to replicate in vivo biochemical milieu. Furthermore, population-genetics-based association studies can identify correlations but cannot establish causal relationships for specific variants, particularly rare or deleterious ones. Consequently, there is a gap in the art for a rapid, scalable, physiologically relevant assay capable of directly measuring the activity of human enzyme variants in a living cellular context.

[0339] Prior art solutions are limited and are insufficient for bridging this gap. In vitro enzyme assays, while precise, require recombinant protein expression and purification, which is often challenging for unstable or aggregation-prone variants. Prior art methods and systems centered on cross-species gene replacement and evolutionary assimilation outcomes; it does not present the strict dependency-coupled architecture as a design requirement for quantitative functional readout. By contrast, the present work’s design purpose is to enforce growth dependence on the human enzyme activity by removing alternative metabolic routes / bypass contributions, so that growth can be used as a quantitative proxy for catalytic function. Moreover, conditions in vitro conditions in vitro do not replicate the crowded and dynamic intracellular environment where protein folding, complex assembly, and cofactor availability can markedly affect. Moreover, conditions in vitro do not replicate the crowded and dynamic intracellular environment where protein folding, complex assembly, and cofactor availability can markedly affect activity. Patient-derived cell assays can better mimic physiological contexts but are constrained by availability of samples for rare mutations, genetic background noise, zygosity effects, and low throughput. Computational prediction methods have improved but still lack experimental validation and cannot fully account for the structural and kinetic subtleties of enzyme function in vivo. As for drug screening, existing high-throughput assays for enzyme modulators often rely on artificial reporter systems or-106- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710purified proteins and thus may not accurately reflect intracellular efficacy or account for compound penetration and stability in cells.

[0340] Applicant’s disclosure solves this technical problem by establishing a Live Escherichia coli Assay (LEICA) that functionally replaces specific essential metabolic enzymes of E. coli with their human orthologs or disease-associated variants. By knocking out orthologous bacterial genes in defined pathways and complementing them with the human counterparts, bacterial growth in selective media is rendered dependent on the activity of the heterologously expressed human enzyme. As the growth rate is directly coupled to enzyme activity under physiological, intracellular conditions, a simple optical density measurement enables quantitative assessment of catalytic function. This system accommodates a wide range of genetic variants without individual assay development, allows use of standard microbiological techniques, and permits high replicate numbers with minimal cost and time. Moreover, because the bacterial platform is permeable to small molecules and supports physiologically relevant cofactor concentrations, it doubles as an intracellular drug screening platform, capable of identifying modulators that either impair or rescue activity of specific enzyme variants.

[0341] The exemplified embodiments demonstrate feasibility for multiple distinct human metabolic enzymes: GPI and G6PD, both in glycolysis / pentose phosphate pathway, and ASL in the urea cycle. The correlation between growth rate in LEICA and previously measured in vitro kinetic parameters for a variety of pathogenic and benign variants strongly supports the validity of the readout. The assay also successfully detected functional effects of smallmolecule modulators, including known inhibitors and an activator of G6PD (AG1), with dose-response characteristics broadly consistent with cell-based data. A targeted compound panel and a larger human metabolism modulator library screen further confirmed the ability of LEICA to identify both known and novel G6PD inhibitors, validating its use for high-throughput hit discovery.

[0342] These examples serve to enable the claimed embodiments because the underlying concept can be applied in other systems for which a human enzyme for which loss of function imposes a selectable growth defect in E. coli can be substituted into the corresponding bacterial metabolic node. This includes, but is not limited to, enzymes in central carbon -107- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710metabolism, amino acid biosynthesis, nucleotide metabolism, and other essential pathways where bacterial homologs exist. By appropriate gene knockout strategy and medium formulation, growth can be coupled to the activity of virtually any soluble enzyme.Furthermore, the assay conditions can be adjusted to tune sensitivity, enabling application for either gain-of-function or loss-of-function variants. The success with enzymes from both glycolysis and the urea cycle underscores that the method is not constrained to a particular class of enzyme or cofactor usage. Likewise, the drug-screening modality can extend to modulators of any enzyme expressed in the system, allowing the selection of variant-specific or pan-enzyme therapeutics. The bacterial expression context also facilitates the introduction of high-diversity variant libraries, supporting future adaptation for mutational scanning or directed evolution toward desirable biochemical profiles.

[0343] Equivalents

[0344] It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0345] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0346] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.-108- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0347] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0348] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0349] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0350] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.-109- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0351] Clauses

[0352] Clause 1. A bacterial or yeast cell comprising a heterologous gene encoding an enzyme that is substituted for the endogenous bacterial or yeast counterpart gene, and optionally, wherein the bacterial or yeast cell comprises the deletion or inactivation of at least one or all endogenous bacterial or yeast counterpart genes capable of catalyzing the same biochemical reaction as the counterpart heterologous enzyme or enzymes, and further optionally coupling cell growth to the activity of the heterologous enzyme or enzymes.

[0353] Clause 2. The bacterial or yeast cell of clause 1, the heterologous gene is an animal gene, a mammalian gene, a human gene, a gene from a pathogen, or a gene from a parasite. .

[0354] Clause 3. The bacterial or yeast cell of clause 2, wherein the bacterial or yeast cell is genetically modified to delete or inactivate the heterologous gene that is substituted for the endogenous bacterial or yeast counterpart gene, optionally wherein at least one or all endogenous bacterial or yeast genes capable of catalyzing the same biochemical reaction as the heterologous enzyme or enzymes are deleted or inactivated, thereby coupling cell growth to the activity of the heterologous enzyme or enzymes.

[0355] Clause 4. The bacterial or yeast cell of any one of clauses 1-3, wherein the bacterial or yeast cell further lacks or inactivates additional bacterial or yeast genes in the same or intersecting metabolic pathway, thereby isolating the heterologous enzyme's catalytic activity from alternative metabolic flux routes.1 356] Clause 5. The bacterial or yeast cell of any one of clauses 1-4, wherein the heterologous gene is a human enzyme selected from the group of human glucose-6-phosphate isomerase (GPI), human glucose-6-phosphate dehydrogenase (G6PD), and human argininosuccinate lyase (ASL), and variants of each thereof.

[0357] Clause 6. The bacterial cell of any one of clauses 1-5, wherein the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate dehydrogenase (zwf) gene and comprises a human GPI gene inserted into the chromosome, the pgi locus, or expressed by a plasmid DNA, or wherein the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate-110- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710dehydrogenase (zwf) gene and comprises a human G6PD gene inserted into the chromosome or expressed by a plasmid DNA.

[0358] Clause 7. The bacterial cell of any one of clauses 1-5, wherein the bacterial cell is knocked out for the argH gene required for arginine biosynthesis and the heterologous gene comprises a human ASL gene inserted into the chromosome, the chromosomal locus of the argH gene or expressed by a plasmid DNA.

[0359] Clause 8. The bacterial cell of any one of clauses 1-7, wherein the bacterial cell is an Escherichia coli cell, optionally an E. coli K-12 MG1655 and derivatives thereof.

[0360] Clause 9. A population of bacterial or yeast cells according to any one of clauses 1-5, optionally further comprising a carrier or culture medium suitable for growing the bacterial or yeast cells under conditions where growth is dependent on the catalytic activity of the heterologous gene optionally a human enzyme or variant.[0361 [ Clause 10. An in vitro culture comprising bacterial or yeast cells according to any one of clauses 1-5 or the population of claim 9.

[0362] Clause 11. A method of assaying the catalytic activity of a heterologous enzyme or enzyme variant, comprising: (a) providing a bacterial cell or yeast according to any one of clauses 1-8, the population of claim 9 or the culture of claim 10; (b) culturing the bacterial cell or yeast population or culture under conditions in which growth is dependent on the catalytic activity of the enzyme; and (c) determining growth rate of the bacterial or yeast cell, wherein growth rate is used as a measure of in vivo catalytic activity of the enzyme or enzyme variant, optionally wherein the heterologous enzyme or enzymes is a human enzyme or variant.

[0363] Clause 12. The method of clause 11, wherein the bacterial cell or yeast is engineered such that catalytic activity of the heterologous enzyme is the sole variable affecting growth, by deletion of redundant bacterial enzymes and constant expression of the human enzyme under a stable promoter.

[0364] Clause 13. The method of clause 11 or 12, wherein the growth rate determination is performed by optical density measurements from about 400 nm to about 800 nm, optionally at about 600 nm in a plate reader.-Ill- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710

[0365] Clause 14. The method of any one of clauses 11-13, wherein the determined growth rate correlates linearly with an in vitro kinetic constant (k cat or specific activity) for the human enzyme or variant, the correlation having a Pearson R2of about > 0.80.

[0366] Clause 15. The method of any one of clauses 11-14, wherein the bacterial or yeast cell expresses a human enzyme variant associated with a metabolic disorder, and the growth rate is compared to that of a bacterial or yeast cell expressing wild-type human enzyme to determine whether the variant is pathogenic.

[0367] Clause 16. A method of screening for a metabolic modulator, comprising: (a) contacting a first population of bacterial or yeast cells according to any one of clauses 1-8, the population of claim 9 or the culture of claim 10, with a potential metabolic modulator; (b) determining the growth rate of the first population; (c) determining the growth rate of a second population of bacterial or yeast cells according to any one of the bacterial or yeast cell of claims 1-8, the population of claim 9 or the culture of claim 10, grown in the absence of the potential modulator; and (d) comparing the growth rates, wherein a higher or lower growth rate of the first population indicates that the potential metabolic modulator modulates catalytic activity of the human enzyme or variant.

[0368] Clause 17. The method of clause 16, wherein the bacterial or yeast cell or cells in both populations are assayed in parallel with control bacterial or yeast cell or cells expressing the endogenous bacterial enzyme, and a potential modulator is identified as a hit only if it alters growth of the humanized bacterial or yeast cells without significantly affecting growth of the control bacterial or yeast cells.

[0369] Clause 18. The method of clause 16 or 17, wherein the potential metabolic modulator is selected from a biological agent or a small molecule, optionally from a compound library.

[0370] Clause 19. The method of any one of clauses 16-18, further comprising validating the potential metabolic modulator by performing a dose-response analysis to determine an ECso or ICso value.

[0371] Clause 20. A kit for assaying the catalytic activity of a heterologous enzyme or variant thereof, comprising: (a) a bacterial or yeast cell according to any one of clauses 1-8,-112- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710the population of claim 9 or the culture of claim 10; (b) culture medium suitable for growing said bacterial or yeast cell or cells under conditions where growth is dependent on the catalytic activity of the heterologous enzyme or variant; and (c) instructions for performing the assay by determining growth rate as an indicator of enzyme activity, optionally further comprising one or more control bacterial cells expressing a wild-type human enzyme or endogenous bacterial enzyme, optionally further comprising one or more potential metabolic modulators or a compound library for screening, and instructions for comparative growth assays between humanized and control bacterial or yeast cells to identify enzyme-specific modulators.

[0372] Clause 21. A method of treating a glucose-6-phosphate dehydrogenase (G6PD)-related disorder in a subject in need thereof, comprising administering to the subject an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, linezolid (CAS 2645-32-1), auranofin (CAS 34031-32-8), PR-619 (CAS 2645-32-1), and 6-mercaptopurine (CAS 6112-76-1), and a novel G6PD inhibitor identified in the LEICA screen, thereby inhibiting G6PD activity in the subject, optionally wherein the subject is a mammal or a human patient.

[0373] Clause 22. A method of treating a parasitic or infectious disease in a subject in need thereof, comprising administering to the subject an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, and a novel G6PD inhibitor identified in the LEICA screen, thereby reducing pentose phosphate pathway flux in the pathogen or host cells to inhibit infection, optionally wherein the subject is a mammal or a human patient.

[0374] Clause 23. A method of treating a cancer characterized by elevated pentose phosphate pathway activity, comprising administering to a subject in need thereof an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, and a novel G6PD inhibitor identified in the LEICA screen to suppress nucleotide biosynthesis and redox balance in the cancer cells, optionally wherein the subject is a mammal or a human patient.

[0375] Clause 24. A method of treating chronic non-spherocytic hemolytic anemia caused by a pathogenic G6PD variant, comprising administering to the subject an effective amount of a-113- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710G6PD activator identified in the LEICA screen, thereby increasing catalytic activity of the variant enzyme, optionally wherein the subject is a mammal or a human patient.

[0376] Clause 25. A method of preventing or reducing hemolytic episodes in a G6PD-deficient subject, comprising administering to the subject an effective amount of AG1 or another G6PD activator, thereby stabilizing enzyme oligomerization and restoring NADPH production, optionally wherein the subject is a mammal or a human patient.

[0377] Clause 26. The method of clause 21, wherein the disorder is selected from malaria, favism, oxidative stress-related anemia, and autoimmune disease.

[0378] Clause 27. The method of clause 21, wherein the subject is administered the inhibitor in combination with an antimalarial agent.

[0379] Clause 28. The method of clause 24, wherein the G6PD activator improves variant enzyme activity by at least 10% relative to baseline in red blood cells.

[0380] Clause 29. The method of clause 25, wherein the activating compound is administered prophylactically prior to exposure to oxidative stress.

[0381] Clause 30. The method of clause 21 or 24, wherein the effective amount is determined based on EC so or ICso values measured in the LEICA assay for the specific variant.

[0382] Clause 31. G6PDi-l for use in the treatment of malaria in a subject in need thereof.

[0383] Clause 32. Methotrexate for use in the treatment of malaria in a subject, wherein said use comprises inhibiting human G6PD activity.

[0384] Clause 33. Gossypol for use in the treatment of Trypanosoma infection by inhibiting human G6PD activity.

[0385] Clause 34. AG1 for use in the treatment of chronic non- spherocytic hemolytic anemia caused by a pathogenic G6PD variant.

[0386] Clause 35. A G6PD activator identified in the LEICA assay for use in preventing oxidative hemolysis in a G6PD-deficient patient. Other aspects are set forth within the following claims.-114- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710REFERENCES1. 1000 Genomes Proj ect Consortium et al. A map of human genome variation from population-scale sequencing. Nature 467, 1061-1073 (2010).2. Jang, H. et al. Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases. Nat Biomed Eng 6, 181-194 (2022).3. An, M. et al. Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nat. Biotechnol. 42, 1526-1537 (2024).4. van Eunen, K. & Bakker, B. M. The importance and challenges of in vzvo-like enzyme kinetics. Perspectives in Science 1, 126-130 (2014).5. International HapMap Consortium et al. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851-861 (2007).6. Geek, R. C., Powell, N. R. & Dunham, M. J. Functional interpretation, cataloging, and analysis of 1,341 glucose-6-phosphate dehydrogenase variants. Am. J. Hum. Genet. 110, 228-239 (2023).7. 1000 Genomes Project Consortium et al. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56-65 (2012).8. Sandberg, T. E., Szubin, R., Phaneuf, P. V. & Palsson, B. O. Synthetic cross-phyla gene replacement and evolutionary assimilation of major enzymes. Nat Ecol Evol 4, 1402- 1409 (2020).9. Kugler, W. & Lakomek, M. Glucose-6-phosphate isomerase deficiency. Baillieres. Best Pract. Res. Clin. Haematol. 13, 89-101 (2000).10. Luzzatto, L., Ally, M. & Notaro, R. Glucose-6-phosphate dehydrogenase deficiency. Blood 136, 1225-1240 (2020).11. Brockman, I. M. & Prather, K. L. J. Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites. Metab. Eng. 28, 104-113 (2015).-115- 4923-5828-5447.1Atty. Dkt. No.: 114198-371012. Sampaleanu, L. M., Vallee, F., Thompson, G. D. & Howell, P. L. Three-dimensional structure of the argininosuccinate lyase frequently complementing allele Q286R.Biochemistry 40, 15570-15580 (2001).13. Raub, A. G. et al. Small-Molecule Activators of Glucose-6-phosphate Dehydrogenase (G6PD) Bridging the Dimer Interface. ChemMedChem 14, 1321-1324 (2019).14. Kanno, H., Fujii, H. & Miwa, S. Expression and enzymatic characterization of human glucose phosphate isomerase (GPI) variants accounting for GPI deficiency. Blood Cells Mol. Dis. 24, 54-61 (1998).15. Hwang, S. etal. Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator. Nat. Commun. 9, 4045 (2018).16. Lamoureux, C. R. et al. The Bitome: digitized genomic features reveal fundamental genome organization. Nucleic Acids Res. 48, 10157-10163 (2020).17. Gudmundsson, S. etal. Variant interpretation using population databases: Lessons from gnomAD. Hum. Mutat. 43, 1012-1030 (2022).18. Jamwal, M. et al. Next-generation sequencing unravels homozygous mutation in glucose-6-phosphate isomerase, GPIc.l040G>A (p.Arg347His) causing hemolysis in an Indian infant. Clin. Chim. Acta 468, 81-84 (2017).19. Nakashima, K., Miwa, S., Oda, S., Oda, E. & Matsumoto, N. Electrophoretic and kinetic studies of glucosephosphate isomerase (GPI) in two different Japanese families with GPI deficiency. Am. J. Hum. Genet. 25, 294-301 (1973).20. Kanno, H. et al. Molecular analysis of glucose phosphate isomerase deficiency associated with hereditary hemolytic anemia. Blood 88, 2321-2325 (1996).21. Beutler, E. et al. International Committee for Standardization in Haematology: recommended methods for red-cell enzyme analysis. Br. J. Haematol. 35, 331-340 (1977).22. Nkhoma, E. T., Poole, C., Vannappagari, V., Hall, S. A. & Beutler, E. The global prevalence of glucose-6-phosphate dehydrogenase deficiency: a systematic review and metaanalysis. Blood Cells Mol. Dis. 42, 267-278 (2009).-116- 4923-5828-5447.1Atty. Dkt. No.: 114198-371023. Luzzatto, L. & Battistuzzi, G. Glucose-6-phosphate dehydrogenase. Adv. Hum. Genet. 14, 217-329, 386-8 (1985).24. Louicharoen, C. et al. Positively selected G6PD-Mahidol mutation reduces Plasmodium vivax density in Southeast Asians. Science 326, 1546-1549 (2009).25. Gomez-Manzo, S. etal. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein. Int. J. Mol. Sci. 16, 28657-28668 (2015).26. Ramirez-Nava, E. J. etal. Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant. Int. J. Mol. Sci. 18, (2017).27. Pakparnich, P. et al. Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency. Sci. Rep. 11, 24307 (2021).28. Boonyuen, U. etal. Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PDViangchan and G6PDViangchan+Mahidol:Decreased stability and catalytic efficiency contribute to the clinical phenotype. Mol. Genet. Metab. 118, 84-91 (2016).29. Huang, Y. et al. Purification and detailed study of two clinically different human glucose 6-phosphate dehydrogenase variants, G6PD(Plymouth) and G6PD(Mahidol):Evidence for defective protein folding as the basis of disease. Mol. Genet. Metab. 93, 44-53 (2008).30. Wang, X.-T., Lam, V. M. S. & Engel, P. C. Marked decrease in specific activity contributes to disease phenotype in two human glucose 6-phosphate dehydrogenase mutants, G6PD(Union) and G6PD(Andalus). Hum. Mutat. 26, 284 (2005).31. Roos, D. et al. Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections. Blood 94, 2955-2962 (1999).-117- 4923-5828-5447.1Atty. Dkt. No.: 114198-371032. Boonyuen, U. et al. A trade off between catalytic activity and protein stability determines the clinical manifestations of glucose-6-phosphate dehydrogenase (G6PD) deficiency. hit. J. Biol. Macromol. 104, 145-156 (2017).33. Cunningham, A. D., Colavin, A., Huang, K. C. & Mochly-Rosen, D. Coupling between protein stability and catalytic activity determines pathogenicity of G6PD variants. Cell Rep. 18, 2592-2599 (2017).34. WHO Working Group. Glucose-6-phosphate dehydrogenase deficiency. WHO Working Group. Bull. World Health Organ. 67, 601-611 (1989).35. van Wijk, R. et al. Distinct phenotypic expression of two de novo missense mutations affecting the dimer interface of glucose-6-phosphate dehydrogenase. Blood Cells Mol Dis 32, 112-117 (2004).36. Cohen, P. & Rosemeyer, M. A. Subunit interactions of glucose-6-phosphate dehydrogenase from human erythrocytes. Eur. J. Biochem. 8, 8-15 (1969).37. Cappellini, M. D. & Fiorelli, G. Glucose-6-phosphate dehydrogenase deficiency. Lancet 371, 64-74 (2008).38. Hong, X. etal. PTEN antagonises Tcll / hnRNPK-mediated G6PD pre-mRNA splicing which contributes to hepatocarcinogenesis. Gut 63, 1635-1647 (2014).39. Pu, H. et al. Overexpression of G6PD is associated with high risks of recurrent metastasis and poor progression-free survival in primary breast carcinoma. World J. Surg. Oncol. 13, 323 (2015).40. Mele, L. et al. A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo. Cell Death Dis. 9, 572 (2018).41. Ghergurovich, J. M. etal. A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nat. Chem. Biol. 16, 731-739 (2020).42. ah§kan, B., Oztiirk Kesebir, A., Demir, Y. & Akyol Salman, L The effect of brimonidine and proparacaine on metabolic enzymes: Glucose-6-phosphate dehydrogenase,-118- 4923-5828-5447.1Atty. Dkt. No.: 114198-37106-phosphogluconate dehydrogenase, and glutathione reductase. Biotechnol. Appl. Biochem.69, 281-288 (2022).43. Marks, P. A. & Banks, J. INHIBITION OF MAMMALIAN GLUCOSE-6-PHOSPHATE DEHYDROGENASE BY STEROIDS. Proc. Natl. Acad. Sci. U. S. A. 46, 447-452 (1960).44. Ciftgi, M., Ozmen, I., Buyiikokuroglu, M. E., Penge, S. & Kiifrevioglu, O. I. Effects of metamizol and magnesium sulfate on enzyme activity of glucose 6-phosphate dehydrogenase from human erythrocyte in vitro and rat erythrocyte in vivo. Clin. Biochem.34, 297-302 (2001).45. Oronsky, B. et al. RRx-001, a novel clinical-stage chemosensitizer, radiosensitizer, and immunosensitizer, inhibits glucose 6-phosphate dehydrogenase in human tumor cells. Discov. Med. 21, 251-265 (2016).46. Tsouko, E. et al. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth.Oncogenesis 3, el 03 (2014).47. Ho, H.-Y., Cheng, M.-L., Chiu, H.-Y., Weng, S.-F. & Chiu, D. T.-Y.Dehydroepiandrosterone induces growth arrest of hepatoma cells via alteration of mitochondrial gene expression and function. Int. J. Oncol. 33, 969-977 (2008).48. Akkemik, E., Budak, H. & Ciftci, M. Effects of some drugs on human erythrocyte glucose 6-phosphate dehydrogenase: an in vitro study. J Enzyme Inhib Med Chem 25, 871— 875 (2010).49. Imwong, M. et al. Methotrexate is highly potent against pyrimethamine-resistant Plasmodium vivax. J Infect Dis 203, 207-210 (2011).50. Gerez de Burgos, N. M., Burgos, C., Montamat, E. E., Rovai, L. E. & Blanco, A. Inhibition by gossypol of oxidoreductases from Trypanosoma cruzi. Biochem Pharmacol 33, 955-959 (1984).51. Mercaldi, G. F., Ranzani, A. T. & Cordeiro, A. T. Discovery of new uncompetitive inhibitors of glucose-6-phosphate dehydrogenase. J Biomol Screen 19, 1362-1371 (2014).-119- 4923-5828-5447.1Atty. Dkt. No.: 114198-371052. Yu, B., Thompson, G. D., Yip, P., Howell, P. L. & Davidson, A. R. Mechanisms for intragenic complementation at the human argininosuccinate lyase locus. Biochemistry 40, 15581-15590 (2001).53. Vergalli, J. et al. Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria. Nat Rev Microbiol 18, 164-176 (2020).54. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97, 6640-6645 (2000).55. Canonaco, F. et al. Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA. FEMS Microbiol Lett 204, 247-252 (2001).56. Hwang, S. et al. Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator. Nat. Commun. 9, 4045 (2018).57. Ghergurovich, J. M. et al. A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nat. Chem. Biol. 16, 731-739 (2020).58. Marks, P. A. & Banks, J. INHIBITION OF MAMMALIAN GLUCOSE-6-PHOSPHATE DEHYDROGENASE BY STEROIDS. Proc. Natl. Acad. Sci. U. S. A. 46, 447-452 (1960).59. Ho, H.-Y., Cheng, M.-L., Chiu, H.-Y., Weng, S.-F. & Chiu, D. T.-Y.Dehydroepiandrosterone induces growth arrest of hepatoma cells via alteration of mitochondrial gene expression and function. Int. J. Oncol. 33, 969-977 (2008).60. Oronsky, B. et al. RRx-001, a novel clinical-stage chemosensitizer, radiosensitizer, and immunosensitizer, inhibits glucose 6-phosphate dehydrogenase in human tumor cells. Discov. Med. 21, 251-265 (2016).61. Yalcin, O. et al. From METS to malaria: RRx-001, a multi-faceted anticancer agent with activity in cerebral malaria. Malar. J. 14, 218 (2015).62. Quigley, H. A. & Broman, A. T. The number of people with glaucoma worldwide in 2010 and 2020. Br. J. Ophthalmol. 90, 262-267 (2006).-120- 4923-5828-5447.1Atty. Dkt. No.: 114198-371063. Zhou, X., Zhang, T. & Wu, J. Brimonidine enhances inhibitory postsynaptic activity of OFF- and ON-type retinal ganglion cells in a Wistar rat chronic glaucoma model. Exp. Eye Res. 189, 107833 (2019).64. ah§kan, B., Oztiirk Kesebir, A., Demir, Y. & Akyol Salman, L The effect of brimonidine and proparacaine on metabolic enzymes: Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase. BiotechnoL AppL Biochem.69, 281-288 (2022).65. Sansone, G., Reali, S., Sansone, R. & Allegranza, F. Acute hemolytic anemia induced by a pyrazolonic drug in a child with glucose-6-phosphate dehydrogenase deficiency. Acta Haematol. 72, 285-287 (1984).66. Ciftgi, M., Ozmen, I., Biiyukokuroglu, M. E., Penge, S. & Kiifrevioglu, O. I. Effects of metamizol and magnesium sulfate on enzyme activity of glucose 6-phosphate dehydrogenase from human erythrocyte in vitro and rat erythrocyte in vivo. Clin. Biochem.34, 297-302 (2001).67. Tsouko, E. et al. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth.Oncogenesis 3, el03-el03 (2014).68. Bancone, G. & Chu, C. S. G6PD Variants and Haemolytic Sensitivity to Primaquine and Other Drugs. Front. Pharmacol. 12, 638885 (2021).69. Kanno, H., Fujii, H. & Miwa, S. Expression and enzymatic characterization of human glucose phosphate isomerase (GPI) variants accounting for GPI deficiency. Blood Cells Mol Dis 24, 54-61 (1998).70. Sampaleanu, L. M., Vallee, F., Thompson, G. D. & Howell, P. L. Three-dimensional structure of the argininosuccinate lyase frequently complementing allele Q286R.Biochemistry 40, 15570-15580 (2001).71. Yu, B., Thompson, G. D., Yip, P., Howell, P. L. & Davidson, A. R. Mechanisms for intragenic complementation at the human argininosuccinate lyase locus. Biochemistry 40, 15581-15590 (2001).-121- 4923-5828-5447.1

Claims

Atty. Dkt. No.: 114198-3710WHAT IS CLAIMED IS:

1. A bacterial or yeast cell comprising a heterologous gene encoding an enzyme that is substituted for the endogenous bacterial or yeast counterpart gene, and optionally, wherein the bacterial or yeast cell comprises the deletion or inactivation of at least one or all endogenous bacterial or yeast counterpart genes capable of catalyzing the same biochemical reaction as the counterpart heterologous enzyme or enzymes, and further optionally coupling cell growth to the activity of the heterologous enzyme or enzymes.

2. The bacterial or yeast cell of claim 1, the heterologous gene is an animal gene, a mammalian gene, a human gene, a gene from a pathogen, or a gene from a parasite.

3. The bacterial or yeast cell of claim 2, wherein the bacterial or yeast cell is genetically modified to delete or inactivate the heterologous gene that is substituted for the endogenous bacterial or yeast counterpart gene, optionally wherein at least one or all endogenous bacterial or yeast genes capable of catalyzing the same biochemical reaction as the heterologous enzyme or enzymes are deleted or inactivated, thereby coupling cell growth to the activity of the heterologous enzyme or enzymes.

4. The bacterial or yeast cell of any one of claims 1-3, wherein the bacterial or yeast cell further lacks or inactivates additional bacterial or yeast genes in the same or intersecting metabolic pathway, thereby isolating the heterologous enzyme’s catalytic activity from alternative metabolic flux routes.

5. The bacterial or yeast cell of any one of claims 1-4, wherein the heterologous gene is a human enzyme selected from the group of human glucose-6-phosphate isomerase (GPI), human glucose-6-phosphate dehydrogenase (G6PD), and human argininosuccinate lyase (ASL), and variants of each thereof.

6. The bacterial cell of any one of claims 1-5, wherein the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate dehydrogenase (zwf) gene and comprises a human GPI gene inserted into the chromosome, the pgi locus, or expressed by a plasmid DNA, or wherein the bacterial cell is a double knockout for the phosphoglucose isomerase (pgi) gene and the glucose-6-phosphate-122- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710dehydrogenase (zwf) gene and comprises a human G6PD gene inserted into the chromosome or expressed by a plasmid DNA.

7. The bacterial cell of any one of claims 1-5, wherein the bacterial cell is knocked out for the argH gene required for arginine biosynthesis and the heterologous gene comprises a human ASL gene inserted into the chromosome, the chromosomal locus of the argH gene or expressed by a plasmid DNA.

8. The bacterial cell of any one of claims 1-7, wherein the bacterial cell is an Escherichia coli cell, optionally an A. coli K-12 MG1655 and derivatives thereof.

9. A population of bacterial or yeast cells according to any one of claims 1-5, optionally further comprising a carrier or culture medium suitable for growing the bacterial or yeast cells under conditions where growth is dependent on the catalytic activity of the heterologous gene optionally a human enzyme or variant.

10. An in vitro culture comprising bacterial or yeast cells according to any one of claims 1-5 or the population of claim 9.

11. A method of assaying the catalytic activity of a heterologous enzyme or enzyme variant, comprising:(a) providing a bacterial cell or yeast according to any one of claims 1-8, the population of claim 9 or the culture of claim 10;(b) culturing the bacterial cell or yeast population or culture under conditions in which growth is dependent on the catalytic activity of the enzyme; and(c) determining growth rate of the bacterial or yeast cell,wherein growth rate is used as a measure of in vivo catalytic activity of the enzyme or enzyme variant, optionally wherein the heterologous enzyme or enzymes is a human enzyme or variant.

12. The method of claim 11, wherein the bacterial cell or yeast is engineered such that catalytic activity of the heterologous enzyme is the sole variable affecting growth, by deletion of redundant bacterial enzymes and constant expression of the human enzyme under a stable promoter.-123- 4923-5828-5447.1Atty. Dkt. No.: 114198-371013. The method of claim 11 or 12, wherein the growth rate determination is performed by optical density measurements from about 400 nm to about 800 nm, optionally at about 600 nm in a plate reader.

14. The method of any one of claims 11-13, wherein the determined growth rate correlates linearly with an in vitro kinetic constant (k cat or specific activity) for the human enzyme or variant, the correlation having a Pearson R2of about > 0.80.

15. The method of any one of claims 11-14, wherein the bacterial or yeast cell expresses a human enzyme variant associated with a metabolic disorder, and the growth rate is compared to that of a bacterial or yeast cell expressing wild-type human enzyme to determine whether the variant is pathogenic.

16. A method of screening for a metabolic modulator, comprising:(a) contacting a first population of bacterial or yeast cells according to any one of claims 1-8, the population of claim 9 or the culture of claim 10, with a potential metabolic modulator; (b) determining the growth rate of the first population;(c) determining the growth rate of a second population of bacterial or yeast cells according to any one of the bacterial or yeast cell of claims 1-8, the population of claim 9 or the culture of claim 10, grown in the absence of the potential modulator; and(d) comparing the growth rates, wherein a higher or lower growth rate of the first population indicates that the potential metabolic modulator modulates catalytic activity of the human enzyme or variant.

17. The method of claim 16, wherein the bacterial or yeast cell or cells in both populations are assayed in parallel with control bacterial or yeast cell or cells expressing the endogenous bacterial enzyme, and a potential modulator is identified as a hit only if it alters growth of the humanized bacterial or yeast cells without significantly affecting growth of the control bacterial or yeast cells.

18. The method of claim 16 or 17, wherein the potential metabolic modulator is selected from a biological agent or a small molecule, optionally from a compound library.-124- 4923-5828-5447.1Atty. Dkt. No.: 114198-371019. The method of any one of claims 16-18, further comprising validating the potential metabolic modulator by performing a dose-response analysis to determine an ECso or ICso value.

20. A kit for assaying the catalytic activity of a heterologous enzyme or variant thereof, comprising:(a) a bacterial or yeast cell according to any one of claims 1-8, the population of claim 9 or the culture of claim 10;(b) culture medium suitable for growing said bacterial or yeast cell or cells under conditions where growth is dependent on the catalytic activity of the heterologous enzyme or variant; and(c) instructions for performing the assay by determining growth rate as an indicator of enzyme activity, optionally further comprising one or more control bacterial cells expressing a wild-type human enzyme or endogenous bacterial enzyme, optionally further comprising one or more potential metabolic modulators or a compound library for screening, and instructions for comparative growth assays between humanized and control bacterial or yeast cells to identify enzyme-specific modulators.

21. A method of treating a glucose-6-phosphate dehydrogenase (G6PD)-related disorder in a subject in need thereof, comprising administering to the subject an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, linezolid (CAS 2645-32-1), auranofin (CAS 34031-32-8), PR-619 (CAS 2645-32-1), and 6-mercaptopurine (CAS 6112-76-1), and a novel G6PD inhibitor identified in the LEICA screen, thereby inhibiting G6PD activity in the subject, optionally wherein the subject is a mammal or a human patient.

22. A method of treating a parasitic or infectious disease in a subject in need thereof, comprising administering to the subject an effective amount of a G6PD inhibitor selected from G6PDi-l, methotrexate, gossypol, and a novel G6PD inhibitor identified in the LEICA screen, thereby reducing pentose phosphate pathway flux in the pathogen or host cells to inhibit infection, optionally wherein the subject is a mammal or a human patient.

23. A method of treating a cancer characterized by elevated pentose phosphate pathway activity, comprising administering to a subject in need thereof an effective amount of a G6PD-125- 4923-5828-5447.1Atty. Dkt. No.: 114198-3710inhibitor selected from G6PDi-l, methotrexate, gossypol, and a novel G6PD inhibitor identified in the LEICA screen to suppress nucleotide biosynthesis and redox balance in the cancer cells, optionally wherein the subject is a mammal or a human patient.

24. A method of treating chronic non- spherocytic hemolytic anemia caused by a pathogenic G6PD variant, comprising administering to the subject an effective amount of a G6PD activator identified in the LEICA screen, thereby increasing catalytic activity of the variant enzyme, optionally wherein the subject is a mammal or a human patient.

25. A method of preventing or reducing hemolytic episodes in a G6PD-deficient subject, comprising administering to the subject an effective amount of AG1 or another G6PD activator, thereby stabilizing enzyme oligomerization and restoring NADPH production, optionally wherein the subject is a mammal or a human patient.

26. The method of claim 21, wherein the disorder is selected from malaria, favism, oxidative stress-related anemia, and autoimmune disease.

27. The method of claim 21, wherein the subject is administered the inhibitor in combination with an antimalarial agent.

28. The method of claim 24, wherein the G6PD activator improves variant enzyme activity by at least 10% relative to baseline in red blood cells.

29. The method of claim 25, wherein the activating compound is administered prophylactically prior to exposure to oxidative stress.

30. The method of claim 21 or 24, wherein the effective amount is determined based on ECso or ICso values measured in the LEICA assay for the specific variant.

31. G6PDi-l for use in the treatment of malaria in a subject in need thereof.

32. Methotrexate for use in the treatment of malaria in a subject, wherein said use comprises inhibiting human G6PD activity.

33. Gossypol for use in the treatment of Trypanosoma infection by inhibiting human G6PD activity.-126- 4923-5828-5447.1Atty. Dkt. No.: 114198-371034. AG1 for use in the treatment of chronic non-spherocytic hemolytic anemia caused by a pathogenic G6PD variant.

35. A G6PD activator identified in the LEICA assay for use in preventing oxidative hemolysis in a G6PD -deficient patient.-127- 4923-5828-5447.1

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