Methods of targeting of PAC p (pancreatic alpha cell protein) for diabetes therapy and detection

Abstract

The present disclosure generally relates to methods, assays, devices and kits for measuring Pancreatic Alpha Cell Protein, detecting early Type 1 diabetes mellitus (T1D), and treating T1D.

Description

ACESO.002WO PATENTMETHODS OF TARGETING OF PAC P (PANCREATIC ALPHA CELL PROTEIN) FOR DIABETES THERAPY AND DETECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application 63 / 702410, filed October 02, 2024, which is hereby expressly incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled ACESO002WOSEQLIST.xml, which was created and last modified on September 29, 2025, which is 32,833 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.FIELD

[0003] The present disclosure generally relates to methods, assays, devices and kits for measuring Pancreatic Alpha Cell Protein, detecting early Type 1 diabetes mellitus (T1D), and treating T1D.SUMMARY

[0004] The instant disclosure provides methods, kits, assays, and devices for measuring PAC P in a sample for use in T1D detection and T1D therapies.

[0005] Some embodiments provided herein are described by way of the following provided embodiments and also provided as possible combinations or overlapping embodiments:

[0006] Some embodiments herein are directed to methods of inhibiting, ameliorating, or treating Type 1 diabetes (T1D) in a subject, including for example, identifying a patient in need of T1D therapy, wherein the identification includes assessing PAC Pexpression in a biological sample obtained from the subject; identifying the subject as one having need of T1D therapy when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering a T1D therapy to the subject.

[0007] Some embodiments herein are directed to methods of identifying a subject in need of insulin therapy, including, for example, assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as one having need of insulin therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering insulin therapy to the subject.

[0008] Some embodiments herein are directed to methods that include, for example, assessing PAC P expression in a sample from a subject at risk or suspected of being at risk for developing Type 1 diabetes (T1D). In some embodiments, the assessing of PAC P expression in a sample from the subject comprises: selecting a subject at risk or suspected of being at risk for developing T1D; obtaining, or having obtained, a biological sample from the subject, assessing PAC P expression from the biological sample; determining a probability whether the subject has pre-symptomatic T1D, or may develop T1D from the assessment of the PAC P expression of the sample from the subject; and administering T1D therapy to the subject when the subject has pre-symptomatic T1D, or may develop T1D.

[0009] Some embodiments herein are directed to methods for delaying or preventing the onset of Type 1 diabetes (T1D), that may include, for example, selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject.

[0010] Some embodiments herein are directed to methods of identifying a subject in need of pancreatic islet repair or regeneration, including, for example, assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subject.

[0011] Some embodiments herein are directed to methods for delaying or preventing the onset of Type 1 diabetes (T1D), including, for example, selecting a subjectsuspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subject.

[0012] Some embodiments herein are directed to kits for measuring the expression of PAC P in a biological sample via a PCR reaction, including, for example, a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; reagents and buffers for the PCR reaction; a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable probe; and software to convert a PCR product measured result to a result when compared to a previously established cut-off value.

[0013] Some embodiments herein are directed to assays for measuring PAC P expression from a biological sample obtained from a subject, including, for example, isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product. In some embodiments, the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20- 24.

[0014] Some embodiments herein are directed to assays for detection of pre- symptomatic T1D from a biological sample obtained from a subject, including, for example, isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product. In some embodiments, the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, expression of PAC P above a previously established cut-off value is indicative of pre-symptomatic T1D.

[0015] Some embodiments herein are directed to methods of inhibiting, ameliorating, or treating Type 1 diabetes (T1D), or sequela thereof, in a subject, the method including, for example, identifying a patient in need of T1D therapy. In some embodiments,the identification comprises assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as one having need of T1D therapy when the PAC P expression is identified at levels in excess of a cut-off value in the biological sample; and administering T1D therapy to the subject. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA. In some embodiments, the T1D therapy comprises insulin therapy.

[0016] Some embodiments herein are directed to methods for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA. In some embodiments, the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, antithymocyte globulin, mycophenolate, or any combination thereof.

[0017] Some embodiments herein are directed to methods of identifying a subject in need of pancreatic islet repair or regeneration, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subject. In some embodiments, the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA. In some embodiments, the therapy to reduce PAC P expression comprises administering a siRNA to the subject. In some embodiments, the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30. In some embodiments, the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36. In some embodiments, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30 and the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.

[0018] Some embodiments herein are directed to methods for delaying or preventing the onset of Type 1 diabetes (T1D), including, for example, selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subject. In some embodiments, the therapy to reduce PAC P expression includes gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay includes at least oneforward primer comprising or consisting of any one of SEQ ID NOs: 1 -7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 g RNA. In some embodiments, the therapy to reduce PAC P expression comprises administering a siRNA to the subject. In some embodiments, the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30. In some embodiments, the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36. In some embodiments, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30 and the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.

[0019] In some embodiments, a double-stranded short interfering ribonucleic acid (siRNA) molecule for suppressing expression of pancreatic alpha cell protein is provided, comprising: an antisense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 25-30, and a sense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 31-36, which is complementary to a portion of the preproglucagon gene.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 depicts some embodiments of an alternative causal model of T1D and how early diagnosis provides an opportunity for early therapeutic intervention.

[0021] FIG. 2 depicts some embodiments of the current landscape of T1D management.

[0022] FIG. 3 depicts some embodiments of series of proposed analytical studies for a PAC P assay described herein.

[0023] FIG. 4A-4B depicts some non-limiting examples of primer set pairs.

[0024] FIG. 5A depicts a small interfering (si)RNA construct comprising an antisense strand and a sense strand.

[0025] FIG. 5B depicts a siRNA construct and a small hairpin (sh)RNA construct.

[0026] FIG. 6 depicts the steps of a jetPEI® transfection method.

[0027] FIG. 7 provides an illustration demonstrating the effectiveness of PAC P in preventing hyperglycemia in pre-onsetNOD mice treated with an PAC P-siRNA construct, (a) NOD mice were placed into three groups; (1) pre-onset mice (glucose below 100 mg / dl) receiving scrambled construct, as controls (open circles; 72=4); (2) post-onset mice (glucose above 250 mg / dl) receiving PAC P-siRNA (open diamonds; zz=5), and (3) pre-onset mice receiving PAC P-siRNA (open squares; 77=5). Arrows indicate PAC P-siRNA injection and data point shape identify treatment group.

[0028] FIG. 8A depicts the cellular processes in the development of the T1D disease state when there is no intervention.

[0029] FIG. 8B depicts the cellular processes when there is intervention in the development of the T1D disease state to suppress PAC P over-expression.

[0030] FIG. 9 depicts some non-limiting examples of siRNA antisense strands and siRNA sense strands.DETAILED DESCRIPTION

[0031] In some embodiments, the instant disclosure provides methods for the detection and treatment of Type 1 diabetes (T1D). In some embodiments, detection of T1D comprises measuring expression levels of Pancreatic Alpha Cell Protein (PAC P) in a biological sample obtained from a subject. In some embodiments, measuring PAC P expression allows for the early detection of T1D in presymptomatic subjects. In some embodiments, early diagnosis of T1D via PAC P assessment is coupled with therapeutic intervention to prevent or delay the onset of T1D. Also provided in some embodiments are kits, assays, and devices for measuring PAC P expression in biological samples obtained from a subject.Terms

[0032] All terms have their ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.

[0033] As used herein, “Type 1 diabetes mellitus”, or “Type 1 diabetes” (T1D) has its plain and ordinary meaning as understood in light of the specification and refers to anautoimmune disease characterized by the destruction of pancreatic (B-cells and histologically characterized by islet cell inflammation.

[0034] As used herein, “Pancreatic Alpha Cell Protein” (PAC P) has its plain and ordinary meaning as understood in light of the specification and refers to a protein formally identified as islet homeostasis protein (IHoP), which was shown to effect glucagon synthesis and secretion within the pancreatic a-cell.

[0035] As used herein, “pre-symptomatic” has its plain and ordinary meaning as understood in light of the specification and refers to not yet displaying symptoms of an illness or disease.

[0036] As used herein, “early detection” has its plain and ordinary meaning as understood in light of the specification and refers to detection of a disease, such as T1D, before the onset of advanced stages of disease. In some embodiments, “early detection” comprises detection of a disease, such as T1D, in pre-symptomatic subjects.

[0037] The term “treating” or “treatment” of a condition can refer to preventing the condition, slowing the onset and / or rate of development of the condition, reducing the risk of developing the condition, preventing and / or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. The term “prevent” does not require the absolute prohibition of the disorder or disease.

[0038] A “therapeutically effective amount” or a “therapeutically effective dose” is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating a target condition, delaying the onset of the disorder and / or symptoms, and / or alleviating symptoms associated with the condition. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, biodistribution and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and / or the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly,given the present disclosure. For additional guidance, see Remington: The Science and Practice of Pharmacy 21stEdition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0039] The terms “subject,” “patient,” and “individual” interchangeably refer to an entity that is being examined and / or treated. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys), humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0040] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0041] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (for example, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, M. A. et al., “Enhanced evolutionary PCR using oligonucleotides with inosine atthe 3 ’-terminus, ’’ Nucleic Acid Res., Vol. 19, No. 18, pp. 5081, 1991; Ohtsuka, E. et al., “An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions,” J. Biol. Chem., Vol. 260, No. 5, pp. 2605-2608, 1985; Rossolini, G. M. et al., “Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information,” Mol. Cell. Probes, Vol. 8, No. 2, pp. 91-98, 1994).

[0042] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxy glutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, for example, an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0043] The term “percentage of sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (for example, a polypeptide of the present disclosure), which does not include additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0044] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manualalignment and visual inspection. Some embodiments provided herein provide polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. Optionally, the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, for example, amino acid sequences of 20 or fewer amino acids, in some embodiments, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

[0045] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0046] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.Type 1 Diabetes

[0047] Type 1 diabetes (T1D), or Type 1 diabetes mellitus, is currently characterized as an autoimmune condition in which the body is not able to produce insulin. T1D is an endocrine disorder characterized by a deficiency of insulin due to autoimmune destruction of pancreatic beta cells; this results in hyperglycemia and potential complications such as ketoacidosis, cardiovascular disease, nephropathy, and retinopathy. See, e.g., Diabetes US Report Card - Centers for Disease Control and Prevention: National Diabetes Statical Report. 2023, CDC. p. 1. Thus, insulin therapy is currently the only effective treatment forT1D. According to the CDC, approximately 1.8 million people have T1D including 306,000 children and adolescents in the United States. See, e.g., Nye, J., Heritability of Type 1 Diabetes Higher for Pediatric- vs Adult-Onset Disease. Endocrinology Advisor, 2024: p. 1-6. The overall burden in cost and suffering to these individuals and to society is large. Because of insulin dependence and generally earlier onset, although some adults do develop T1D, individuals with T1D have a 2-3x in life costs than the general. See, e.g., Nye, J., Heritability of Type 1 Diabetes Higher for Pediatric- vs Adult-Onset Disease. Endocrinology Advisor, 2024: p. 1-6; Simone, J., et al., Healthcare resource utilization and cost among patients with type 1 diabetes in the United Slates. J Manag Care Spec. Pharm, 2020. 26(11): p. 1399-1410.

[0048] In some embodiments, T1D is considered to progress through several sequential stages, as described in (Diabetes Care 2015; 38: 1964-1974). In pre-stage 1, T1D arises from both genetic and environmental factors that eventually lead to immune-mediated destruction of pancreatic 0-cells and loss of -cell function. Stage 1 represents individuals who have developed two or more type 1 diabetes-associated islet autoantibodies but are normoglycemic. Stage 2 represents individuals with two or more islet autoantibodies but whose disease has now progressed to the development of glucose intolerance, or dysglycemia, from loss of functional 0-cell mass. Stage 3 represents manifestations of the typical clinical symptoms and signs of diabetes, which may include polyuria, polydipsia, weight loss, fatigue, diabetic ketoacidosis (DKA), and others. Thus, pre-stage 1, stage 1, and stage 2 are considered pre-symptomatic, and stage 3 is considered symptomatic.Assessment of P AC P in diabetes detection

[0049] In some embodiments, assessment of PAC P expression enables early detection of pre-diabetic changes within the pancreatic islet of a subject. See, e.g., Oh, S., et al., Suppression of islet homeostasis protein thwarts diabetes mellitus progression. Laboratory Investigation, 2017. 97: p. 577-590. In some embodiments, PAC P can be found in samples from subjects with T1D but not nondiabetic subjects. In some embodiments, PAC P is overexpressed in samples from subjects pre-symptomatic for T1D. In some embodiments, PAC P is overexpressed in samples from subjects pre-symptomatic for T1D. In some embodiments, PAC P is overexpressed in the pre-onset stage of T1D. See, e.g., Oh, S., et al., Characterization of a novel functional protein in the pancreatic islet: IHoP regulation of glucagon synthesis in alpha-cells. Pancreas, 2012. 41(1): p. 22-30. In some embodiments, the methods, assays, kits,and devices, described herein can diagnose patients pre-symptomatic for T1D based upon detection of PAC P. In some embodiments, PAC P levels positively correlate with blood glucose levels. In some embodiments, the detection of the PAC P in serum is a biomarker of patients for pre-onset of T1D. In some embodiments, presence of PAC P in a sample obtained from a subject is a potential indicator of the metabolic protein dysfunction that leads to P-cell destruction and T1D. In some embodiments, subjects in need of pancreatic islet repair or regeneration are subjects in the pre-onset stage of T1D. In some embodiments, subjects in need of pancreatic islet repair or regeneration are subjects presymptomatic for T1D. The methods, assays, kits, and devices of assessing PAC P described herein for diagnostics of T1D may confer a benefit to a subject suspected of having T1D.Test for Measuring PAC P expression

[0050] In some embodiments, early diagnostic methods for T1D are provided. In some embodiments, early diagnostic tests for T1D may comprise any of the tests, methods, kits, assays, and / or devices disclosed herein. In some embodiments, the methods comprise an assay for measuring Pancreatic Alpha Cell Protein (PAC P) levels in a sample obtained, or having been obtained from a subject. In some embodiments, the methods may comprise an assay for measuring PAC P RNA levels from a biological sample. In some embodiments, the methods may comprise an assay for measuring PAC P protein levels from a biological sample. In some embodiments, the methods comprise a polymerase chain reaction (PCR) assay for PAC P RNA. In some embodiments, the methods comprise a reverse transcriptase (RT)-PCR assay for PAC P RNA. In some embodiments, the methods comprise an ELISA assay for PAC P protein. In some embodiments, tests, kits, or devices for performing the assay or methods for measuring PAC P as described herein are provided. In some embodiments, the tests, kits or devices include the primers, reagents, positive and negative controls including no-template control along with the necessary software to convert the measured result to a result when compared to the previously established cut-off value, or any combination thereof. In some embodiments, the tests, kits or devices further comprise use of a PCR machine, or thermocycler to perform the reaction. In some embodiments, tests, kits or devices are used with a sample that is obtained, or has been obtained from a patient. In some embodiments, the biological sample from the subject comprises one or more cells isolated from the subject. In some embodiments, the biological sample from the subject comprises a biological fluid. In someembodiments, the biological fluid comprises blood, serum, plasma, or a combination thereof. In some embodiments, the biological sample is a serum sample.

[0051] In some embodiments, the subject has known risk factors for T1D. In some embodiments, a subject suspected of having T1D, suspected of being presymptomatic for T1D, at risk for T1D, or suspected of being at risk for T1D, has one or more known risk factors for T1D. In some embodiments, the subject is selected because the subject has known risk factors for T1D. In some embodiments, the subject does not have known risk factors for T1D. In some embodiments, the subject has risk factors for T1D comprising HLA genetic risk factors, for example HLA class II haplotypes DRBl *0301-DQBl*0201 (DR3-DQ2) and DRBl*0401- DQB 1*0302 (DR4-DQ8); non-HLA genetic risk factors such as INS, PTPN22, CTLA4, and IL2RA genes; first- or second-degree relatives of individuals with T1D. In some embodiments, the subject is normoglycemic and positive for two or more TID-associated islet autoantibodies. In some embodiments, the subject is dysglycemic and positive for two or more TID-associated islet autoantibodies. In some embodiments, the islet autoantibodies recognize targets comprising insulin (IAA), glutamic acid decarboxylase (GAD A), protein phosphatase- like IA-2 (IA-2A), zinc transporter 8 (ZnT8A), or islet cell cytoplasmic antigen (ICA). In some embodiments, the subject is selected for PAC P expression assessment because the subject exhibits any combination of the T1D risk factors described herein.Performing the PCR Assay for PAC P

[0052] In some embodiments, diagnostic tests, assays, or kits for detecting presymptomatic T1D are provided comprising an assessment of PAC P expression in a biological sample obtained from a subject. In some embodiments, the diagnostic kits, assays, or tests for T1D comprise a PCR reaction. In some embodiments, the diagnostic kits, assays, or tests for T1D comprise a RT-PCR reaction. In some embodiments, RNA is isolated from a biological sample from a subject. In some embodiments, an amount of isolated RNA is used for cDNA synthesis via reverse transcription. In some embodiments, the RT-PCR reaction further comprises a step where the synthesized cDNA is used as a template for exponential amplification using PCR, comprising specific primers and a DNA polymerase. In some embodiments, Ct values of the PCR product below a specific cut-off value indicate the presence of the PAC P; thus may be an indicator of pre-onset of, or presymptomatic T1D. In some embodiments, PAC P expression, as determined by RT-PCR, in excess of a cut-off valuemay be an indicator of pre-onset of, or presymptomatic T1D. In some embodiments, the presence of PAC P, as determined by RT-PCR, may be an indicator of pre-onset of, or presymptomatic T1D.

[0053] In some embodiments, the amount of isolated RNA used for cDNA synthesis is, is about, or is at least 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 pg RNA. In some embodiments, the amount of isolated DNA used for cDNA synthesis is 2.0 pg RNA.

[0054] In some embodiments, the PCR Assay for detection of PAC P described herein comprises RNA primers having RNA sequences that are, that are about, or that are at least 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or that are 100%, or a percentage in a range defined by any two of the preceding values (e g., 75- 100%, 80-99%, 85-95%, 80-98%, etc.) identical to SEQ ID NOs: 1-24.

[0055] In some embodiments, the PCR Assay for detection of PAC P described herein comprises the forward primer comprising a sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to any one of SEQ ID NOs: 1-7 and 15-19.

[0056] In some embodiments, the PCR Assay for detection of PAC P described herein comprises the forward primer comprising a sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to any one of SEQ ID NOs: 1-7 and 15-19.

[0057] In some embodiments, the PCR Assay for detection of PAC P described herein comprises the reverse primer comprising a sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to any one of SEQ ID NOs: 8-14 and 20-24.

[0058] In some embodiments, the PCR Assay for detection of PAC P described herein comprises the reverse primer comprising a sequence having between about 70%-100%,70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to any one of SEQ ID NO: 8-14 and 20-24.

[0059] In some embodiments, kits are provided. In some embodiments, the kit is for performing a PCR reaction to detect PAC P in a biological sample from a subject In some embodiments, the kit is for performing a RT-PCR reaction to detect PAC P in a biological sample from a subject. In some embodiments, the kit includes the primers, reagents, positive and negative controls including no-template control along with the necessary software to convert the measured result to a result when compared to the previously established cut-off value, or any combination thereof, as described herein. In some embodiments, the kit includes at least one forward primer and at least one reverse primer as described herein. In some embodiments, the kit includes a heat stable polymerase. In some embodiments, the kit includes at least one reverse transcriptase. In some embodiments, the kit includes a deoxynucleotide triphosphate (dNTP) mix. In some embodiments, the kit includes at least one detectable probe, for example, a fluorescent probe. In some embodiments, the kit includes buffers. In some embodiments, the kit includes positive controls, for example target protein, or fragments thereof. In some embodiments, the kit includes negative controls, for example a solution that is substantially free of the target. In some embodiments, the kit includes packaging.

[0060] In some embodiments, the kit is used with a device. In some embodiments, the device comprises a thermal cycler, computer, optics for fluorescence for excitation and emission collection, data acquisition and analysis software; or any combination thereof. In some embodiments, data acquisition and analysis software are provided within the kit. Type 1 diabetes (T1D) Treatment and Monitoring

[0061] The methods, assays, kits, and devices described herein may be employed in combination therapies with other agents to achieve an improved therapeutic outcome for the subject. In some embodiments, the methods, assays, kits, and devices described herein may be used to diagnose T1D. In some embodiments, the methods, assays, kits, and devices described herein may be used to identify patients who would benefit from treatment for T1D. In some embodiments, methods, assays, kits, and devices described herein further comprise treatment for T1D. In some embodiments, treatment of T1D includes administration of insulin therapy.In some embodiments, the insulin is administered as basal insulin. In some embodiments, the insulin is administered as prandial (bolus or meal-time) insulin. In some embodiments, the treatment of T1D includes both basal and bolus insulin administration. In some embodiments, the treatment of T1D includes: rapid-acting insulin, for example, insulin lispro, insulin aspart, or insulin glulisine (genetically engineered insulin analogs); short-acting insulin, for example, regular (soluble) insulin; intermediate-acting insulin, for example, NPH (isophane); long- acting insulin, for example, insulin glargine, insulin detemir, and insulin degludec (genetically engineered insulin analogs); premixed insulin; inhaled insulin; insulin biosimilars, for example Basaglar, Basalin, or Semglee; or any combination thereof. In some embodiments, the insulin is administered subcutaneously via injection, insulin pen, or insulin pump. In some embodiments, T1D treatment includes non-insulin therapies including Pramlintide, metformin, Sodium Glucose Cotransporter 2 (SGLT2) inhibitors, incretin-based therapies, or any combination thereof.

[0062] The methods, assays, kits, and devices described herein may be employed in monitoring the development or progression of T1D. In some embodiments, monitoring of T1D includes self-monitoring of blood glucose (SMBG). In some embodiments, SMBG includes subject use of blood glucose monitoring systems (BGMS). In some embodiments, monitoring of T1D includes continuous glucose monitoring (CGM). In some embodiments, CGM includes measurement of glucose concentrations in the interstitial fluid (ISF). In some embodiments, T1D therapy includes measurement of A1C. In some embodiments, A1C measurements can be performed approximately every 3 months. In some embodiments, the glycemic target for an adult can include a target A1C of <7%. In some embodiments, fructosamine can be considered a substitute for A1C in assessing a patient’s glycemic control. Delay or prevention of T1D onset

[0063] In some embodiments, the methods, assays, kits, and devices described herein further include methods of preventing, delaying the onset, and / or ameliorating T1D in a subject found to be pre-symptomatic for T1D or being at risk for developing T1D. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D can include treatment of a subject found to be pre-symptomatic for T1D with immunosuppressive therapy comprising immunosuppressive agents. In some embodiments, treatment with immunosuppressive agents can include depletion of T lymphocytes in a subject. In someembodiments, treatment with immunosuppressive agents can include depletion of B lymphocytes in a subject. In some embodiments, depletion of T and / or B lymphocytes can include treatment with one or more monoclonal antibodies. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T ID in a subject can include treatment with anti-CD3 monoclonal antibody. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D in a subject can include treatment with anti-CD20 monoclonal antibody. In some embodiments, treatment with immunosuppressive agents can include impairing effector immune cell signaling or proliferation. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D in a subject can include treatment with anti-IL2 receptor a monoclonal antibody. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D in a subject can include treatment of a subject found to be pre- symptomatic for T1D with cyclosporin, prednisone, azathioprine, antithymocyte globulin, mycophenolate, or any combination thereof. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T ID in a subject can include induction of immune tolerance in a subject found to be pre- symptomatic for T1D by vaccination with peptides targeted by immune cells during T1D, such as, for example GAD65 protein, or DiaPeP227. In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T ID in a subject can include any of the methods described herein.

[0064] In some embodiments, delaying the onset of T1D can comprise increasing the amount of time until T1D symptoms appear in a subject relative to a subject that does not receive therapy to delay the onset of T1D. In some embodiments, delaying the onset of T1D can comprise increasing the amount of time until pancreatic islet damage occurs in the subject relative to a subject that does not receive therapy to delay the onset of T1D. In some embodiments, delaying the onset of T1D can comprise delaying onset until T1D symptoms appear for at least, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, 48 months, 51 months, 54 months, 57 months, or 60 months. In some embodiments, delaying the onset of T1D can comprise delaying onset until T1D symptoms appear for at least, 6 months-1 year, 1-2 years, 2-3 years, 3-4 years, 4-5 years, 5-6 years, 6-7 years, 7-8 years, 8- 9 years, 9-10 years. In some embodiments, onset of T1D is prevented. In some embodiments, delaying the onset of T1D can comprise delaying onset until pancreatic islet damage occursfor at least, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, 48 months, 51 months, 54 months, 57 months, or 60 months. In some embodiments, delaying the onset of T1D can comprise delaying onset until pancreatic islet damage occurs for at least, 6 months-1 year, 1-2 years, 2-3 years, 3-4 years, 4-5 years, 5-6 years, 6-7 years, 7-8 years, 8-9 years, 9-10 years. In some embodiments, onset of T1D is prevented.

[0065] In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D in a subject can include cell therapy. In some embodiments, the cell therapy can comprise stem cells, transplantation of donor islet cells, transplantation of in vitro- differentiated islet-like cells, transplantation of stem cells-derived insulin producing cells, or any combination thereof. In some embodiments, the cell therapy is combined with any of the methods of preventing, delaying the onset, and / or ameliorating T1D in a subject described herein.

[0066] In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D in a subject can include targeting the expression of PAC P in a subject. In some embodiments, targeting the expression of PAC P includes suppressing the expression of PAC P in a subject to the levels found in a subject not considered to have T1D or be at risk of T1D, such as, for example, a healthy subject. In some embodiments, suppressing the expression of PAC P in a subject can include gene silencing methods such as RNAi, siRNA, or CRISPR. In some embodiments, targeting the expression of PAC P in a subject is combined with any of the methods of preventing, delaying the onset, and / or ameliorating T ID in a subject described herein.

[0067] In some embodiments, methods of preventing, delaying the onset, and / or ameliorating T1D in a subject can include administration of any of the treatments described herein as a composition, therapeutic composition, or pharmaceutical composition.

[0068] In some embodiments, the assays, kits, or devices described herein are applied to a sample in vitro. In some embodiments, the sample is freshly harvested from a subject, for example a biopsy or blood collection. In some embodiments, the sample is fixed. In some embodiments the sample includes a whole organ and / or tissue. In some embodiments, the sample includes one or more whole cells. In some embodiments the sample is from cell extracts, for example lysates. In some embodiments, elements of the assay, kit, or device, insolution is added to a sample that does not contain a solution, for example a lyophilized sample, thus reconstituting the sample. In some embodiments, lyophilized elements of the assay, kit, or device can be added to a sample that contains solution, thus reconstituting the elements of the assay, kit, or device.Therapeutic agents and Compositions

[0069] siRNA constructs are useful compositions in preventing onset of T1D. siRNA is a synthetic RNA duplex designed to target a specific mRNA for degradation. While siRNA provides the opportunity to induce gene knockdown in a variety of cell lines, its utility is limited to cells that are amenable to the transfection of synthetic oligonucleotides. Since siRNAs achieve transient silencing, experiments are limited to relatively short time frames on the order of 2-4 days. siRNAs can also be used for the knockdown of non-coding genes, such as long non-coding RNAs (IncRNA).

[0070] siRNAs consist of two RNA strands, an antisense (or guide) strand and a sense (or passenger) strand, which form a duplex 19 to 25 bp in length with 3' dinucleotide overhangs (see, e.g., FIG. 5A and 5B).

[0071] Synthetic siRNAs are most commonly generated through solid-phase chemical synthesis methods, which provide highly pure, stable, and readily modified siRNAs. Small double-strand siRNAs are transfected into cells where the guide strand is loaded into RISC. This activated protein and nucleic acid complex can then elicit gene silencing by binding, through perfect complementarity, to a single target mRNA sequence, thereby targeting it for cleavage and degradation.

[0072] siRNA constructs are transfected into cells using either cationic lipid or polymer-based transfection reagents, electroporation (physical delivery via plasma membrane holes created by an electrical field), or chemical modifications to the duplex to aid in cellular uptake. See, e.g., FIG. 6.

[0073] The success of RNAi experiments depends on the efficiency of gene knockdown. Early work on siRNA design established conventional guidelines for siRNA structural attributes that led to reasonable functional knockdown in specific cases.

[0074] The properties of potent siRNAs are further refined through large-scale functional studies, which have defined thermodynamic and sequence-based rules for rational siRNA design. These design algorithms significantly improved the reliability of identifyingpotent siRNA sequences. While research continually strives to identify molecules with greater activity and specificity, siRNAs designed using jetPEI® transfection reagent is a linear polyethylenimine derivative, free of components of animal origin, ideal for automated or manual High Throughput Screening in adherent and suspension cells.

[0075] Although the sequence complementarity-based mechanism underlying RNAi allows for target-specific gene knockdown, the exact mechanism can result in unintended knockdown of genes that are not directly targeted. Several strategies have been developed to mitigate these so-called “off-target” effects and ensure on-target activity. No secondary effects from off-target knockdowns were observed.

[0076] Chemical modifications to the siRNA have been used successfully to promote preferential loading of the intended antisense (guide) strand into the RISC complex and reduce sense (passenger) strand loading and activity. Furthermore, to mitigate the risk of the siRNA guide strand seed region causing off-target effects, design algorithms can incorporate filters that exclude high-frequency seed sequences from known mammalian microRNAs. Finally, the strategy of pooling several independent siRNAs that target a single gene has been shown to reduce the total number of non-specific gene targets and the frequency of off-target phenotypes, while preserving potent knockdown of the target gene. When combined, these strategies work efficiently to reduce off-targeting and achieve potent, specific silencing, ensuring a successful RNAi experiment.

[0077] siRNAs are widely used to assess the individual contributions of genes to various cellular phenotypes, including cytokinesis, apoptosis, insulin signaling, and cell differentiation. siRNA screens have been used to identify novel pathways and have had a significant impact on validating targets for various cellular processes and diseases, including cancer, HIV infection, and hepatitis. Moreover, in vivo RNAi has been utilized for target validation studies in animal disease models and holds potential for therapeutic applications, where disease-causing genes are selectively targeted and suppressed.

[0078] Experiments were conducted to demonstrate the effectiveness of PAC P in preventing hyperglycemia in pre-onset NOD mice treated with a PAC P-siRNA construct. The results of the experiments are presented in FIG. 7 : (a) NOD mice were placed into three groups; (1) pre-onset mice (glucose below 100 mg / dl) receiving scrambled construct, as controls (open circles; w=4); (2) post-onset mice (glucose above 250 mg / dl) receiving PAC P-siRNA (open diamonds; n=5), and (3) pre-onset mice receiving PAC P-siRNA (open squares; n=5). Arrows indicate PAC P-siRNA injection and data point shape identify treatment group. Treated animals (a single treatment with PAC P-siRNA) survived 35 weeks with normal glycemic levels. Animals receiving a scrambled PAC P-siRNA as a control treatment lead to T1D and death. After onset of T1D, the treatment could not repair the damage or reverse T1D development. The data demonstrate that suppression of PAC P in pre-diabetic mice led to 25 weeks survival with normal blood glucose level. However, mice treated after the mice became diabetic were not able to overcome the progression of the disease and eventually died.

[0079] FIGS. 8A and 8B illustrate the effects of no intervention versus intervention by suppressing PAC P in the development of T1D. Healthy pancreatic islets are in cellular and hormonal homeostasis. An increase in PAC P expression disrupts the balance within an islet, leading to increased glucose levels and the onset of pre-onset disease. Without intervention, levels of glucagon and PAC P continue to rise, and type 1 diabetes develops, resulting in islet destruction (FIG. 8A). However, if PAC P is suppressed at the pre-onset stage, homeostasis is restored, and the islet can regenerate through the activation of the PDX1 pathway by Reg3y (FIG. 8B). The dashed line represents an arbitrary point in the progression of the disease; this point can be likened to a point of no return. Based on the data, it is believed that if treatment is administered before this point of no return, the progression of the disease can be altered and the animals can survive with normal glycemic levels. However, if treatment is given after this point, the disease progresses toward the full onset of T1D. Once the disease is considered in full onset, the damage has been done, and PAC P’s role is insufficient to rescue the islet.

[0080] The methods provided herein for measuring PAC P expression can be used to identify a patient in the pre-onset stage of T1D. Administration to such a patient of a PAC P-siRNA construct will suppress PAC P over-expression, effectively preventing full onset of T1D. FIG. 9 provides antisense strand and sense strand sequences for siRNA duplexes that can be employed to suppress PAC P over-expression. In certain embodiments, a doublestranded short interfering ribonucleic acid (siRNA) molecule for suppressing expression of pancreatic alpha cell protein can be employed. The siRNA can comprise an antisense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 25-30. The siRNA can comprise a sense strand comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 31-36. The siRNA can be complementary to a portion of the preproglucagon gene.

[0081] In some embodiments, the methods, assays, kits, and devices described herein further comprise administration of a composition to a subject. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition can also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier can be a liquid or solid fdler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier is “pharmaceutically acceptable” in that it is compatible with the other ingredients of the formulation. It is also suitable for contact with any tissue, organ, or portion of the body that it can encounter, meaning that, ideally it will not carry a significant risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0082] In some embodiments, the methods, assays, kits, and devices described herein further comprise administration of a composition to a subject. The pharmaceutical, or therapeutic, compositions described herein can be administered by any suitable route of administration. A route of administration can refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. “Transdermal” administration can be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, sublingual, transmucosal, or transtracheal. In some embodiments, the antigen binding construct can be delivered intraoperatively as a local administration during an intervention or resection.

[0083] The pharmaceutical, or therapeutic, compositions described herein can be administered by any suitable route of administration. A route of administration can refer to anyadministration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. “Transdermal” administration can be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracap sul ar, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subcapsular, sublingual, subcutaneous, transmucosal, or transtracheal. In some embodiments, the antigen binding construct can be delivered intraoperatively as a local administration during an intervention or resection.Methods of detecting the presence or absence of the target molecule

[0084] The methods, assays, kits, and devices described herein, can be used to detect the presence or absence of the target molecule in vitro. Accordingly, some embodiments include methods of detecting the presence or absence of the target.

[0085] Methods of detecting the presence or absence of the target molecule are provided herein. It will be appreciated that the processes below can be performed in any sequence, and / or can be optionally repeated and / or eliminated, and that additional steps can optionally be added to the method. In some embodiments, the methods, kits, assays, and devices, as described herein can be applied to a sample. In some embodiments, the methods, kits, assays, and devices, as described herein comprise a PCR reaction. In some embodiments, the PCR reaction is a RT-PCR reaction. In some embodiments, the RT-PCR reaction can be performed as a one step or as a two-step RT-PCR reaction.

[0086] In some embodiments, the presence or absence of the target molecule is detected. In some embodiments, the presence or absence of the target can be determined based upon the threshold cycle (ct) value of the PCR product in the sample. For example, in some embodiments, ct values below a specific cut-off value can indicate the presence of the target, while ct values above a specific cut-off value can indicate the absence of the target.

[0087] In some embodiments, methods of inhibiting, ameliorating, or treating Type 1 diabetes (T1D) in a subject are provided. In some embodiments, the methods comprise identifying a patient in need of T1D therapy, wherein the identification comprises assessingPAC P expression in a biological sample obtained from the subject, selecting the subject as one having need of T1D therapy when the PAC P expression is determined to be present at levels in excess of a cut-off value in the biological sample. In some embodiments, the method further comprises administering T1D therapy to the subject. In some embodiments, the T1D therapy is any one of the T1D treatments described herein.

[0088] In some embodiments, methods of selecting a subject in need of insulin therapy are provided. In some embodiments, the methods comprise assessing PAC P expression in a biological sample obtained from the subject; and selecting the subject as one having need of insulin therapy, when the PAC P expression is determined to be present at levels in excess of a cut-off value in the biological sample; and administering insulin therapy to the subj ect.

[0089] In some embodiments, methods comprising assessing PAC P expression in a sample from a subject suspected of being at risk for developing Type 1 diabetes (T1D) are provided. In some embodiments, the assessing of PAC P expression in a sample from the subject comprises: selecting a subject suspected of being at risk for developing T1D; obtaining, or having obtained, a biological sample from the subject, assessing PAC P expression from the biological sample; determining a probability whether the subject has T1D, or may develop T1D from the assessment of the PAC P expression of the sample from the subject; administering T1D therapy to the subject.

[0090] In some embodiments, methods for delaying or preventing the onset of Type 1 diabetes (T1D) are provided, comprising: selecting a subject from a patient population suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; selecting the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject.

[0091] In some embodiments, methods of identifying a subject in need of pancreatic islet repair or regeneration are provided, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subject.

[0092] In some embodiments, methods for delaying or preventing the onset of Type 1 diabetes (T1D) are provided, comprising: selecting a subject suspected to be pre- symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subject.

[0093] In some embodiments, the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof.

[0094] In some embodiments, kits for measuring the expression of PAC P in a biological sample via a reverse transcriptase PCR reaction are provided, comprising: a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; reagents and buffers for the PCR reaction; a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable target; and software to convert a PCR product measured result to a result when compared to the previously established cut-off value.

[0095] In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 1 and SEQ ID NO: 8. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 2 and SEQ ID NO: 9. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 3 and SEQ ID NO: 10. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 4 and SEQ ID NO: 11. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 5 and SEQ ID NO: 12. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 6 and SEQ ID NO: 13. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 7 and SEQ ID NO: 14. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 15 and SEQ ID NO: 20. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 16 and SEQ ID NO: 21. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 17 and SEQ ID NO: 22. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 18 and SEQ ID NO: 23. In some embodiments, the PCR assay comprises a primer pair comprising SEQ ID NO: 19 and SEQ ID NO: 24. In someembodiments, the method, kit, assay, or device comprises at least one of the primer pairs described herein. In some embodiments, the method, kit, assay, or device comprises one of the primer pairs described herein. In some embodiments, the method, kit, assay, or device comprises multiple primer pairs described herein.

[0096] In some embodiments, kits for measuring the expression of PAC P in a biological sample via a reverse transcriptase (RT) PCR reaction are provided, comprising: a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; reagents and buffers for the RT-PCR reaction; a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable target; and software to convert a PCR product measured result to a result when compared to the previously established cut-off value.

[0097] In some embodiments, kits for detection of pre-symptomatic Type 1 diabetes (T1D) from a biological sample are provided, comprising: a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; reagents and buffers for the PCR reaction; a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable target; and software to convert a PCR product measured result to a result when compared to the previously established cut-off value.

[0098] In some embodiments, kits for detection of pre-symptomatic Type 1 diabetes (T1D) from a biological sample are provided, comprising: a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; reagents and buffers for the RT-PCR reaction; a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable target; and software to convert a PCR product measured result to a result when compared to the previously established cut-off value.

[0099] In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 1 and SEQ ID NO: 8. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 2 and SEQ ID NO: 9. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 3 and SEQ ID NO: 10. Insome embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 4 and SEQ ID NO: 11. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 5 and SEQ ID NO: 12. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 6 and SEQ ID NO: 13. In some embodiments, the RT- PCR assay comprises a primer pair comprising SEQ ID NO: 7 and SEQ ID NO: 14. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 15 and SEQ ID NO: 20. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 16 and SEQ ID NO: 21. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 17 and SEQ ID NO: 22. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 18 and SEQ ID NO: 23. In some embodiments, the RT-PCR assay comprises a primer pair comprising SEQ ID NO: 19 and SEQ ID NO: 24. In some embodiments, the method, kit, assay, or device comprises at least one of the primer pairs described herein. In some embodiments, the method, kit, assay, or device comprises one of the primer pairs described herein. In some embodiments, the method, kit, assay, or device comprises multiple primer pairs described herein.

[0100] In some embodiments, devices for use with the kits described herein are provided. In some embodiments, the devices can comprise any combination of a thermal cycler, a computer, optics for fluorescence for excitation and emission collection, data acquisition and analysis software.

[0101] In some embodiments, assays for measuring PAC P expression from a biological sample obtained from a subject are provided, comprising isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product. In some embodiments, the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.

[0102] In some embodiments, assays for measuring PAC P expression from a biological sample obtained from a subject are provided, comprising isolating total RNA from the biological sample obtained from a subject; performing a reverse transcriptase (RT-PCR) reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product. In some embodiments, the RT-PCR reaction comprises a forward primercomprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the RT-PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.

[0103] In some embodiments, assays for detection of pre-symptomatic T1D from a biological sample obtained from a subject are provided, comprising isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product. In some embodiments, the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, expression of PAC P above a previously established cut-off value is indicative of pre-symptomatic T1D.

[0104] In some embodiments, assays for detection of pre-symptomatic T1D from a biological sample obtained from a subject are provided, comprising isolating total RNA from the biological sample obtained from a subject; performing a reverse transcriptase (RT-PCR) reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product. In some embodiments, the RT-PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the RT-PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, expression of PAC P above a previously established cut-off value is indicative of pre-symptomatic T1D.

[0105] In some embodiments, the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, anti-thymocyte globulin, mycophenolate, or any combination thereof.

[0106] In some embodiments, the T1D therapy comprises insulin therapy.

[0107] In some embodiments, PAC P expression is assessed by a PCR reaction.

[0108] In some embodiments, PAC P expression is assessed by a reverse transcriptase (RT) PCR reaction.

[0109] In some embodiments, methods for measuring the expression of PAC P in a biological sample obtained from a subject are provided, comprising isolating total RNA froma biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; assessing expression of PAC P in the PCR product. In some embodiments, the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.

[0110] In some embodiments, methods for measuring the expression of PAC P in a biological sample obtained from a subject are provided, comprising isolating total RNA from a biological sample obtained from a subject; performing a reverse transcriptase (RT) PCR reaction to amplify PAC P from the biological sample; assessing expression of PAC P in the PCR product. In some embodiments, the RT-PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19. In some embodiments, the RT-PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.[OHl] In some embodiments, RNA isolated from the biological sample can be used for cDNA synthesis.

[0112] In some embodiments, the amount of isolated RNA used for cDNA synthesis is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.

[0113] In some embodiments, the amount of isolated DNA used for cDNA synthesis is 2.0 pg RNA.

[0114] In some embodiments, the PCR reaction comprises a forward primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

[0115] In some embodiments, the RT-PCR reaction comprises a forward primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

[0116] In some embodiments, the PCR reaction comprises a forward primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

[0117] In some embodiments, the RT-PCR reaction comprises a forward primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

[0118] In some embodiments, the PCR reaction comprises a reverse primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

[0119] In some embodiments, the RT-PCR reaction comprises a reverse primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

[0120] In some embodiments, the PCR reaction comprises a reverse primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

[0121] In some embodiments, the RT-PCR reaction comprises a reverse primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

[0122] In some embodiments, the PCR reaction comprises primers having sequences having at least 80% identity to any one of SEQ ID NO: 1-7 and 15-19 and at least 80% identity to any one of SEQ ID NO: 8-14 and 20-24.

[0123] In some embodiments, the RT-PCR reaction comprises primers having sequences having at least 80% identity to any one of SEQ ID NO: 1-7 and 15-19 and at least 80% identity to any one of SEQ ID NO: 8-14 and 20-24.

[0124] In some embodiments, the subject has no first degree relative with T1D.

[0125] In some embodiments, the subject has at least one first degree relative withT1D

[0126] In some embodiments, the subject is pre- symptomatic for T1D.

[0127] In some embodiments, the subject is normoglycemic and positive for two or more T ID-associated islet autoantibodies.

[0128] In some embodiments, the subject is dysglycemic and positive for two or more T ID-associated islet autoantibodies.

[0129] In some embodiments, kits are provided for any of the methods or assays described herein.

[0130] In some embodiments, a method of inhibiting, ameliorating, or treating Type 1 diabetes (T1D), or sequela thereof, in a subject is provided, the method comprising: identifying a patient in need of T1D therapy, wherein the identification comprises assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as one having need of T1D therapy when the PAC P expression is identified at levels in excess of a cut-off value in the biological sample; and administering T1D therapy to the subject. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA. In some embodiments, the T1D therapy comprises insulin therapy.

[0131] In some embodiments, a method for delaying or preventing the onset of Type 1 diabetes (T1D) is provided, comprising: selecting a subject suspected to be pre- symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and atleast one reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA. In some embodiments, the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, antithymocyte globulin, mycophenolate, or any combination thereof.

[0132] In some embodiments, a method of identifying a subject in need of pancreatic islet repair or regeneration is provided, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subject. In some embodiments, the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.

[0133] In some embodiments, a method for delaying or preventing the onset of Type 1 diabetes (T1D) is provided, comprising: selecting a subject suspected to be pre- symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subject. In some embodiments, the therapy to reduce PACP expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof. In some embodiments, PAC P expression is assessed by a RT-PCR assay. In some embodiments, the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24. In some embodiments, the biological sample is a serum sample. In some embodiments, the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.Numbered Arrangements

[0134] Some embodiments provided herein are described by way of the following provided numbered arrangements and also provided as possible combinations or overlapping embodiments:1. A method of inhibiting, ameliorating, or treating Type 1 diabetes (T1D) in a subject, the method comprising: identifying a patient in need of T1D therapy, wherein the identification comprises assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as one having need of T1D therapy when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering T1D therapy to the subject.2. A method of identifying a subject in need of insulin therapy, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as one having need of insulin therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering insulin therapy to the subject.3. A method comprising assessing PAC P expression in a sample from a subject at risk or suspected of being at risk for developing Type 1 diabetes (T1D), wherein the assessing of PAC P expression in a sample from the subject comprises: selecting a subject at risk or suspected of being at risk for developing T1D; obtaining, or having obtained, a biological sample from the subject, assessing PAC P expression from the biological sample; determining a probability whether the subject has pre-symptomatic T1D, or may develop T1D from the assessment of the PAC P expression of the sample from the subject; administering T1D therapy to the subject when the subject has pre-symptomatic T1D, or may develop T1D.4. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject.5. A method of identifying a subject in need of pancreatic islet repair or regeneration, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subject.6. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample;administering therapy to reduce PAC P expression in the subject.7. The method of any one of arrangements 5 or 6, wherein the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof.8. The method of arrangement 7, wherein the therapy to reduce PAC P expression comprises administering a siRNA to the subject.9. The method of arrangement 8, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30.10. The method of arrangement 9, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.11. The method of arrangement 9, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30 and the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.12. The method of any one of the previous arrangements, wherein PAC P expression is assessed by polymerase chain reaction (PCR).13. The method of arrangement 12, wherein PAC P expression is assessed by a reverse transcriptase (RT) PCR reaction.14. A method for measuring the expression of PAC P in a biological sample obtained from a subject, comprising: isolating total RNA from a biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; assessing expression of PAC P in the PCR product; wherein the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; and wherein the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.15. The method of arrangement 14, wherein the PCR reaction is a RT-PCR reaction.16. The method of any of arrangements 14 or 15, wherein RNA isolated from the biological sample is used for cDNA synthesis.17. The method of arrangement 16, wherein the amount of isolated RNA used for cDNA synthesis is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.18. The method of arrangement 17, wherein the amount of isolated DNA used for cDNA synthesis is 2.0 pg RNA.19. The method of any one of the preceding arrangements, comprising a forward primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.20. The method of any one of the preceding arrangements, comprising a forward primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.21. The method of any one of the preceding arrangements, comprising a reverse primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.22. The method of any one of the preceding arrangements, comprising a reverse primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.23. The method of any one of the preceding arrangements, comprising primers having sequences having at least 80% identity to any one of SEQ ID NOs: 1 -7 and at least 80% identity to any one of SEQ ID NOs: 8-14 and 20-24.24. The method of any one of the preceding arrangements, wherein the subject has no first degree relative with T1D.25. The method of any one of the preceding arrangements, wherein the subject is selected because the subject has at least one first degree relative with T1D.26. The method of any one of the preceding arrangements, wherein the subject is pre-symptomatic for T1D.27. The method of any one of the preceding arrangements, wherein the subject is selected because the subject is normoglycemic and positive for two or more T ID-associated islet autoantibodies.28. The method of any one of the preceding arrangements, wherein the subject is selected because the subject is dysglycemic and positive for two or more TID-associated islet autoantibodies.29. The method of any one of the preceding arrangements, wherein the subject is selected because the subject has HLA or non-HLA genetic risk factors for T1D.30. The method of any one of the preceding arrangements, wherein the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, anti-thymocyte globulin, mycophenolate, or any combination thereof.31. The method of any one of the preceding arrangements, wherein the T1D therapy comprises insulin therapy.32. A kit for assessing PAC P expression of any one of the preceding arrangements.33. A kit for measuring the expression of PAC P in a biological sample via a PCR reaction, comprising: a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; reagents and buffers for the PCR reaction; a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable probe; and software to convert a PCR product measured result to a result when compared to a previously established cut-off value.34. The kit of arrangement 33, wherein the PCR reaction is a RT-PCR reaction.35. An assay for measuring PAC P expression from a biological sample obtained from a subject, comprising: isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product; wherein the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; and wherein the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.36. An assay for detection of pre-symptomatic T1D from a biological sample obtained from a subject, comprising: isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product; wherein the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; wherein the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; and wherein expression of PAC P above a previously established cut-off value is indicative of pre-symptomatic T1D.37. The assay of any of arrangements 35 or 36, wherein the PCR reaction is a RT- PCR reaction.38. A device for use with the kit of any one of arrangements 32-34, comprising any combination of: a thermal cycler, a computer, an optics for fluorescence for excitation and emission collection, and data acquisition and analysis software.39. The kit, assay, or device of any one of arrangements 32-38, comprising an amount of isolated DNA used for cDNA synthesis of at least 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, or 5.0 pg RNA.40. The kit, assay, or device of arrangement 39, wherein the amount of isolated DNA used for cDNA synthesis is 2.0 pg RNA.41. The kit, assay, or device of any one of arrangements 32-40, comprising a forward primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%- 97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.42. The kit, assay, or device of any one of arrangements 32-41, comprising a forward primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.43. The kit, assay, or device of any one of arrangements 32-42, comprising a reverse primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%- 97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.44. The kit, assay, or device of any one of arrangements 32-43, comprising a reverse primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.45. The kit, assay, or device of any one of arrangements 32-44, comprising primers having sequences having at least 80% identity to any one of SEQ ID NOs: 1-7 and 15-19 and at least 80% identity to any one of SEQ ID NOs: 8-14 and 20-24.46. A method of inhibiting, ameliorating, or treating Type 1 diabetes (T1D), or sequela thereof, in a subject, the method comprising: identifying a patient in need of T1D therapy, wherein the identification comprises assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as one having need of T1D therapy when the PAC P expression is identified at levels in excess of a cut-off value in the biological sample; andadministering T1D therapy to the subject; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-22; wherein the biological sample is a serum sample; wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA; and wherein the T1D therapy comprises insulin therapy.47. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; wherein the biological sample is a serum sample; wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA; andwherein the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, anti-thymocyte globulin, mycophenolate, or any combination thereof.48. A method of identifying a subject in need of pancreatic islet repair or regeneration, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subject; wherein the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; wherein the biological sample is a serum sample; and wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.49. The method of arrangement 48, wherein the therapy to reduce PAC P expression comprises administering a siRNA to the subject.50. The method of arrangement 49, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30.51. The method of arrangement 49, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.52. The method of arrangement 49, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36 and any one of the antisense strands consisting of SEQ ID NO: 25-30.53. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subject; wherein the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; wherein the biological sample is a serum sample; and wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.54. The method of arrangement 53, wherein the therapy to reduce PAC P expression comprises administering a siRNA to the subject.55. The method of arrangement 54, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30.56. The method of arrangement 54, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.57. The method of arrangement 54, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36 and any one of the antisense strands consisting of SEQ ID NO: 25-30.58. The method, kit, assay, or device of any one of the preceding arrangements, wherein the primers comprise the forward and reverse primers comprising any one of the primer sets consisting of:a) SEQ ID NO: 1 and SEQ ID NO: 8; b) SEQ ID NO: 2 and SEQ ID NO: 9; c) SEQ ID NO: 3 and SEQ ID NO: 10; d) SEQ ID NO: 4 and SEQ ID NO: 11; e) SEQ ID NO: 5 and SEQ ID NO: 12; f) SEQ ID NO: 6 and SEQ ID NO: 13; g) SEQ ID NO: 7 and SEQ ID NO: 14; h) SEQ ID NO: 15 and SEQ ID NO: 20; i) SEQ ID NO: 16 and SEQ ID NO: 21; j) SEQ ID NO: 17 and SEQ ID NO: 22; k) SEQ ID NO: 18 and SEQ ID NO: 23; and l) SEQ ID NO: 19 and SEQ ID NO: 24.59. A double-stranded short interfering ribonucleic acid (siRNA) molecule for suppressing expression of pancreatic alpha cell protein, comprising: an antisense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 25-30; and a sense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 31-36; which is complementary to a portion of the preproglucagon gene.

[0135] Embodiments of the present disclosure are further defined in the following Examples. It should be understood that these Examples are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the disclosure to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety, and for the disclosure referenced herein.EXAMPLESExample 1 : Analytical Performance

[0136] The methods, assays, kits, and devices described herein will be subjected to analytical testing as described in the studies illustrated in FIG. 3.

[0137] A single site precision laboratory evaluation will be performed lasting 20 days testing 2 replicates per run and 2 runs per day for each of the samples, where operators will be from intended user.

[0138] A multisite precision study will be performed at three sites (at least 2 external), testing five non-consecutive days with one run per day alternately morning and afternoon with 5 replicates resulting in 3 x 5 x 5 to provide laboratory to laboratory as well as day to day and within day (repeatability). Each site will have a different instrument and at least 2 different operators.

[0139] A product stability study will be performed including real-time and accelerated stability studies at 37°C. Precision panel samples will be tested at 3 replicates per day per level at time 0 and multiple time points including 10% longer than the claimed shelf life, where product can be evaluated in product packaging.

[0140] A product shipping study will be performed using product packaged as final kits and per specified temperature control, the product will undergo shipping stress testing to confirm that product packaging can withstand the expected shipping stress.

[0141] A specimen stability study will be performed, where specimens will be drawn and tested at time 0 (after 30 minutes allowing it to clot and spun down) and then at 4, 8, 24 hours at room temperature and refrigerated.

[0142] A limit of blank / limit of detection study will be performed using known healthy and low positive samples.

[0143] An assay cutoff study will be performed with banked samples to establish a significant cut-off value.

[0144] A linearity study will be performed testing at least 5 replicates of admixture of high serum pool and negative serum.

[0145] An interference study will be performed to test common endogenous interferents including Hgb, bilirubin, and lipids and common drugs using a spike minus blank approach and a level series if any high concentration shows potential for interference.

[0146] A cross-reactivity study will be performed, where based on in silico analysis, any potentially similar structured substances will be spiked into negative samples to test for cross reactivity.Example 2: Clinical Validity StudyPositive Samples for Clinical Sensitivity

[0147] At least 60 T1D serum samples from a wide range of ages, ethnicities and approximately split between males and females will be tested. These samples will be obtained from clinical specimen banks which have been properly handled and stored with appropriate demographic and disease information. An additional 20 confirmed T1D patients with at least half with insulin autoantibodies and / or GAD Ab (but not all) will be included. These specimens will be blind labeled to the testing personnel.Negative Samples for Clinical Specificity

[0148] At least 50 banked samples from patients with non-diabetic diseases and all known to be negative for T1D will be tested. These samples should include approximately 20 with Graves’ disease, 20 Hashimoto’s thyroiditis, and 10 rheumatoid arthritis patients. An additional 20 samples from Type 2 diabetes and 50 nominally healthy normal samples will be included.Design Overview

[0149] All samples will be obtained from reputable specimen banks and the specimen aliquots will be blind labeled for the testing personnel. Positive and negative samples will be interspersed in the testing runs. Testing personnel will not know anything about the specimens except a deidentified code. These samples will also be tested for information against the existing cleared auto antibody tests.

[0150] All specimens will be tested for use in the methods, assays, kits, and devices described herein. The data will be analyzed into a traditional 2 x 2 table based on truth of whether the sample was from a subject with T1D vs. the positive or negative result on the PAC P assay.Acceptance Criteria

[0151] The study will adhere to the following acceptance criteria: Clinical sensitivity: Lower bound of the two-sided 95% CI of >=75%, Clinical specificity: Lower bound of the two-sided 95% CI of >= 90%

[0152] Headings are included herein for reference and to aid in locating various sections. These headings are not intended to limit the scope of the concepts described with respect thereto. Such concepts may have applicability throughout the entire specification.

[0153] The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents are based on available information and are not admissions as to their accuracy or correctness.

[0154] In the foregoing description, specific details are given to provide a thorough understanding of the examples. However, it will be understood by one of ordinary skill in the art that the examples may be practiced without these specific details.

[0155] In at least some of the described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0156] With respect to the use of substantially any plural or singular terms herein, those having skill in the art can translate from the plural to the singular or from the singular to the plural as is appropriate to the context or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0157] The embodiments illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter, and use of the term “about” at the beginning of a string of values modifies each of the values. For example, a weight of “about 100 grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of the embodiments.

[0158] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0159] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0160] While preferred embodiments described herein have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the description. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments. It is intended that the following claims define the scope of embodiments provided herein and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting, ameliorating, or treating Type 1 diabetes (T1D) in a subject, the method comprising: identifying a patient in need of T1D therapy, wherein the identification comprises assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as one having need of T1D therapy when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering T1D therapy to the subject.

2. A method of identifying a subject in need of insulin therapy, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as one having need of insulin therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering insulin therapy to the subject.

3. A method comprising assessing PAC P expression in a sample from a subject at risk or suspected of being at risk for developing Type 1 diabetes (T1D), wherein the assessing of PAC P expression in a sample from the subject comprises: selecting a subject at risk or suspected of being at risk for developing T1D; obtaining, or having obtained, a biological sample from the subject, assessing PAC P expression from the biological sample; determining a probability hether the subject has pre-symptomatic T1D, or may develop T1D from the assessment of the PAC P expression of the sample from the subject; administering T1D therapy to the subject when the subject has pre- symptomatic T1D, or may develop T1D.

4. A method for delaying or preventing the onset of Type I diabetes (T1D). comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject;identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject.

5. A method of identifying a subject in need of pancreatic islet repair or regeneration, comprising: assessing PAC P expression in a biological sample obtained from the subject; and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subj ect.

6. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subj ect.

7. The method of any one of claims 5 or 6, wherein the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof.

8. The method of claim 7, wherein the therapy to reduce PAC P expression comprises administering a siRNA to the subject.

9. The method of claim 8. wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30.

10. The method of claim 9, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.

11. The method of claim 9. wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30 and the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.

12. The method of any one of the previous claims, wherein PAC P expression is assessed by polymerase chain reaction (PCR).

13. The method of claim 12, wherein PAC P expression is assessed by a reverse transcriptase (RT) PCR reaction.

14. A method for measuring the expression of PAC P in a biological sample obtained from a subject, comprising: isolating total RNA from a biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; assessing expression of PAC P in the PCR product; wherein the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; and wherein the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.

15. The method of claim 14, wherein the PCR reaction is a RT-PCR reaction.

16. The method of any of claims 14 or 15, wherein RNA isolated from the biological sample is used for cDNA synthesis.

17. The method of claim 16, wherein the amount of isolated RNA used for cDNA synthesis is, is at least, or is not more than 0.1, 0.2, 0.3, 0.

4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.

0. 1.1, 1.2, 1.3, 1.

4. 1.5, 1.6, 1.7, 1.

8. 1.9, 2.

0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.

7. 2.8, 2.7, 2.8, 2.

9. 3.0, 3.1, 3.

2. 3.

3. 3.4, 3.5, 3.

6. 3.

7. 3.8, 3.9, 4.0, 4.

1. 4.

2. 4.3, 4.4, 4.

5. 4.

6. 4.7, 4.8, 4.

9. or 5.0 pg RNA.

18. The method of claim 17, wherein the amount of isolated DNA used for cDNA synthesis is 2.0 pg RNA.

19. The method of any one of the preceding claims, comprising a forward primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%. 95%-100%, 95%-97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

20. The method of any one of the preceding claims, comprising a forward primer comprising a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 1-7 and 15-19.

21. The method of any one of the preceding claims, comprising a reverse primer comprising a sequence having about 70%-100%. 70%-95%, 70%-90%, 70%-80%, 75%- 100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%- 97%, or 97%-100%, sequence identify to any one of SEQ ID NOs: 8-14 and 20-24.

22. The method of any one of the preceding claims, comprising a reverse primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

23. The method of any one of the preceding claims, comprising primers having sequences having at least 80% identity to any one of SEQ ID NOs: 1-7 and at least 80% identity to any one of SEQ ID NOs: 8-14 and 20-24.

24. The method of any one of the preceding claims, wherein the subject has no first degree relative with T1D.

25. The method of any one of the preceding claims, wherein the subject is selected because the subject has at least one first degree relative with T1D.

26. The method of any one of the preceding claims, wherein the subject is pre- symptomatic for T1D.

27. The method of any one of the preceding claims, wherein the subject is selected because the subject is normoglycemic and positive for two or more T1D- associated islet autoantibodies.

28. The method of any one of the preceding claims, wherein the subject is selected because the subject is dysglycemic and positive for two or more T ID-associated islet autoantibodies.

29. The method of any one of the preceding claims, wherein the subject is selected because the subject has HLA or non-HLA genetic risk factors for T ID.

30. The method of any one of the preceding claims, wherein the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, anti-thymocyte globulin, my cophenolate, or any combination thereof.

31. The method of any one of the preceding claims, wherein the T1D therapy comprises insulin therapy.

32. A kit for assessing PAC P expression of any one of the preceding claims.

33. A kit for measuring the expression of PAC P in a biological sample via a PCR reaction, comprising: a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; a reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; reagents and buffers for the PCR reaction;a deoxynucleotide triphosphate (dNTP) mix; a DNA polymerase; a reverse transcriptase; positive and negative controls; a detectable probe; and software to convert a PCR product measured result to a result when compared to a previously established cut-off value.

34. The kit of claim 33. wherein the PCR reaction is a RT-PCR reaction.

35. An assay for measuring PAC P expression from a biological sample obtained from a subject, comprising: isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product; wherein the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; and wherein the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24.

36. An assay for detection of pre-symptomatic T1D from a biological sample obtained from a subject, comprising: isolating total RNA from the biological sample obtained from a subject; performing a PCR reaction to amplify PAC P from the biological sample; determining expression of PAC P in the PCR product; wherein the PCR reaction comprises a forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19; wherein the PCR reaction comprises a reverse primer comprising or consisting of any one of SEQ ID NOs: 8-14 and 20-24; and wherein expression of PAC P above a previously established cut-off value is indicative of pre-symptomatic T1D.

37. The assay of any of claims 35 or 36, wherein the PCR reaction is a RT-PCR reaction.

38. A device for use with the kit of any one of claims 32-34, comprising any combination of: a thermal cycler, a computer. an optics for fluorescence for excitation and emission collection, anddata acquisition and analysis software.

39. The kit. assay, or device of any one of claims 32-38. comprising an amount of isolated DNA used for cDNA synthesis of at least 0.1, 0.2, 0.3, 0.

4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.

0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.

9. or 5.0 pg RNA.

40. The kit. assay, or device of claim 39, wherein the amount of isolated DNA used for cDNA synthesis is 2.0 pg RNA.

41. The kit, assay, or device of any one of claims 32-40, comprising a forward primer comprising a sequence having about 70%-100%, 70%-95%. 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

42. The kit, assay, or device of any one of claims 32-41, comprising a forward primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-7 and 15-19.

43. The kit, assay, or device of any one of claims 32-42, comprising a reverse primer comprising a sequence having about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

44. The kit, assay, or device of any one of claims 32-43, comprising a reverse primer comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-14 and 20-24.

45. The kit, assay, or device of any one of claims 32-44, comprising primers having sequences having at least 80% identity to any one of SEQ ID NOs: 1-7 and 15-19 and at least 80% identity to any one of SEQ ID NOs: 8-14 and 20-24.

46. A method of inhibiting, ameliorating, or treating Type 1 diabetes (T1D), or sequela thereof, in a subject, the method comprising: identifying a patient in need of T1D therapy, wherein the identification comprises assessing PAC P expression in a biological sample obtained from the subject:identifying the subject as one having need of T1D therapy when the PAC P expression is identified at levels in excess of a cut-off value in the biological sample; and administering T1D therapy to the subject; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-22; wherein the biological sample is a serum sample; wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4.0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.

0. 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.

0. 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.

1. 4.2, 4.3, 4.4, 4.

5. 4.6, 4.7, 4.8, 4.

9. or 5.0 pg RNA; and wherein the T1D therapy comprises insulin therapy.

47. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symplomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering immunosuppressive therapy to the subject; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; wherein the biological sample is a serum sample; wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.

4. 0.

5. 0.6, 0.7, 0.8, 0.

9. 1.0, 1.1, 1.2, 1.

3. 1.4, 1.5, 1.

6. 1.7, 1.8, 1.9, 2.

0. 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.

0. 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.

1. 4.2, 4.3, 4.4, 4.

5. 4.6, 4.7, 4.8, 4.

9. or 5.0 pg RNA; and wherein the immunosuppressive therapy comprises agents for depleting or inhibiting T cells, agents for depleting or inhibiting B cells, cyclosporin, prednisone, azathioprine, anti-thymocyte globulin, mycophenolate, or any combination thereof.

48. A method of identifying a subject in need of pancreatic islet repair or regeneration, comprising: assessing PAC P expression in a biological sample obtained from the subject: and identifying the subject as being in need of pancreatic islet repair or regeneration therapy, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; and administering therapy to reduce PAC P expression in the subj ect; wherein the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; wherein the biological sample is a serum sample; and wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1, 0.2, 0.3, 0.4.0.

5. 0.6, 0.7, 0.8, 0.

9. 1.0, 1.1, 1.2, 1.

3. 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.

0. 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg RNA.

49. The method of claim 48, wherein the therapy to reduce PAC P expression comprises administering a siRNA to the subject.

50. The method of claim 49, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30.

51. The method of claim 49, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.

52. The method of claim 49, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36 and any one of the antisense strands consisting of SEQ ID NO: 25-30.

53. A method for delaying or preventing the onset of Type 1 diabetes (T1D), comprising: selecting a subject suspected to be pre-symptomatic for T1D; assessing PAC P expression in a biological sample obtained from the subject; identifying the subject as pre-symptomatic for T1D, when the PAC P expression is assessed at levels in excess of a cut-off value in the biological sample; administering therapy to reduce PAC P expression in the subject; wherein the therapy to reduce PAC P expression comprises gene silencing methods comprising RNAi, siRNA, CRISPR, or any combination thereof; wherein PAC P expression is assessed by a RT-PCR assay; wherein the RT-PCR assay comprises at least one forward primer comprising or consisting of any one of SEQ ID NOs: 1-7 and 15-19, and at least one reverse primer comprising or consisting of any one of SEQ ID NO: 8-14 and 20-24; wherein the biological sample is a serum sample; and wherein the amount of RNA from the biological sample used for cDNA synthesis in the RT-PCR assay is, is at least, or is not more than 0.1 , 0.2, 0.3, 0.4.0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.

0. 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.7, 2.8, 2.9, 3.

0. 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.

1. 4.2, 4.3, 4.4, 4.

5. 4.6, 4.7, 4.8, 4.

9. or 5.0 pg RNA.

54. The method of claim 53, wherein the therapy to reduce PAC P expression comprises administering a siRNA to the subject.

55. The method of claim 54, wherein the siRNA comprises any one of the antisense strands consisting of SEQ ID NO: 25-30.

56. The method of claim 54, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36.

57. The method of claim 54, wherein the siRNA comprises any one of the sense strands consisting of SEQ ID NO: 31-36 and any one of the antisense strands consisting of SEQ ID NO: 25-30.

58. The method, kit, assay, or device of any one of the preceding claims, wherein the primers comprise the forward and reverse primers comprising any one of the primer sets consisting of: m) SEQ ID NO: 1 and SEQ ID NO: 8; n) SEQ ID NO: 2 and SEQ ID NO: 9; o) SEQ ID NO: 3 and SEQ ID NO: 10; p) SEQ ID NO: 4 and SEQ ID NO: 11 ; q) SEQ ID NO: 5 and SEQ ID NO: 12; r) SEQ ID NO: 6 and SEQ ID NO: 13; s) SEQ ID NO: 7 and SEQ ID NO: 14; t) SEQ ID NO: 15 and SEQ ID NO: 20; u) SEQ ID NO: 16 and SEQ ID NO: 21; v) SEQ ID NO: 17 and SEQ ID NO: 22; w) SEQ ID NO: 18 and SEQ ID NO: 23; and x) SEQ ID NO: 19 and SEQ ID NO: 24.

59. A double-stranded short interfering ribonucleic acid (siRNA) molecule for suppressing expression of pancreatic alpha cell protein, comprising: an antisense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 25-30; and a sense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 31 -36; which is complementary to a portion of the preproglucagon gene.

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