WO2025076486A1

WO2025076486A1 - Biomarkers for repeat expansion diseases

Info

Publication number: WO2025076486A1
Application number: PCT/US2024/050140
Authority: WO
Inventors: Clarence Wang; Vinod Yadav; Baoguang HAN
Original assignee: Dyne Therapeutics Inc
Current assignee: Dyne Therapeutics Inc
Priority date: 2023-10-06
Filing date: 2024-10-04
Publication date: 2025-04-10
Anticipated expiration: 2026-04-06

Abstract

The present disclosure, in some aspects, provides one or more disease state-associated genes for repeat expansion diseases (e.g., a repeat expansion disease associated with spliceopathy, such as DM1). In some embodiments, each of the disease-state associated gene described herein is a disease-state associated gene for a repeat expansion disease (e.g., DM1). Methods of measuring the splicing activity of disease state- associated genes, e.g., for determining disease state and monitoring patient as well as treatment efficacy are also provided.

Description

BIOMARKERS FOR REPEAT EXPANSION DISEASES

FIELD OF THE INVENTION

[0001] The present application relates to genes associated with disease state of repeat expansion diseases (e.g., repeat expansion diseases associated with spliceopathy) and their uses. A non-limiting example of a such repeat expansion disease is myotonic dystrophy (DM).

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (D082470085WO00-SEQ- CBD.xml; Size: 55,045 bytes; and Date of Creation: September 25, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

[0003] Myotonic dystrophy (DM) is a dominantly inherited genetic disease that is characterized by myotonia, muscle loss or degeneration, diminished muscle function, insulin resistance, cardiac arrhythmia, smooth muscle dysfunction, and neurological abnormalities. DM is the most common form of adult-onset muscular dystrophy, with a worldwide incidence of about 1 in 8000 people worldwide. Two types of the disease, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), have been described. DM1, the more common form of the disease, results from a repeat expansion of a CTG trinucleotide repeat in the 3' non-coding region of DMPK on chromosome 19; DM2 results from a repeat expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9 on chromosome 3. In DM1 patients, the repeat expansion of a CTG trinucleotide repeat, which may comprise greater than about 50 to about 3,000 or more total repeats, leads to generation of toxic RNA repeats capable of forming hairpin structures that bind essential intracellular proteins, e.g., muscleblind-like proteins, with high affinity resulting in protein sequestration and the loss-of-function phenotypes that are characteristic of the disease. Apart from supportive care and treatments to address the symptoms of the disease, no effective therapeutic for DM1 is currently available.

SUMMARY

[0004] Myotonic dystrophy type I (DM1) is the most common form of adult muscular dystrophy. DM1 is a repeat expansion disease caused by expansion of a CTG triplet repeat in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase (DMPK) gene. The pathological CTG repeats result in protein trapping by expanded transcripts, a decreased DMPK translation and the disruption of the chromatin structure, affecting neighboring genes expression. The muscleblind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) are two families of tissue-specific regulators of developmentally programmed alternative splicing that act as antagonist regulators of various pre-mRNA targets, including, e.g., troponin 2 (TNNT2), insulin receptor (INSR), chloride channel 1 (CLCN1) and MBNL2. Sequestration of MBNL proteins and up-regulation of CELF1 are key to DM1 pathology, inducing a spliceopathy that leads to a developmental remodeling of the transcriptome due to an adult-to- fetal splicing switch, which results in the loss of cell function and viability.

[0005] Alternative splicing is a regulatory mechanism of gene expression that allows the generation of more than one unique mRNA species from a single gene. Alternative splicing occurs in a range of tissues from adult organs such as brain, heart and skeletal muscle, to embryonic stem and precursor cells, particularly during differentiation or reprogramming of various cell lineages as well as epithelial-mesenchymal transitions. Alternative splicing contributes to cell differentiation and lineage determination, tissue identity acquisition and maintenance and organ development.

[0006] Skeletal muscle is one of the tissues with the highest number of differentially expressed exons. Its splicing program is complex may differ even from that of cardiac muscle. In skeletal muscle, the muscleblind-like (MBNL) family, CUG-BP and ETR-3-like factors (CELF) family and the RNA binding Fox (RBFOX) are the most important splicing regulators.

[0007] Without wishing to be bound by any scientific theory, the expanded CUG repeats in mutant DMPK transcripts form imperfect stable hairpin structures that accumulate in the cell nucleus in small ribonuclear complexes or microscopically visible inclusions, which impair the physiological function of proteins implicated in transcription, splicing or RNA export. These aggregations lead to the deregulation of the alternative splicing of different transcripts due to the alteration of the splicing machinery, specifically MBNL1 and CELF1. Hundreds of splicing events are misregulated due to MBNL1 sequestration and CELF1 upregulation. These alterations in turn cause loss of cell function and viability, and some of them directly correlate with common symptoms of the disease, explaining its extensive multisystemic affection.

[0008] The misregulation of the alternative splicing is a well-characterized effect in DM1 cells. To date, more than thirty transcripts missplicing have been characterized in different tissues in DM1 patients (e.g., as described in Lopez-Martinez et al., Genes (Basel). 2020 Sep; 11(9): 1109, incorporated herein by reference). Splicing biomarkers and their use in determining disease severity in myotonic dystrophy have also been described (e.g., in Nakamori et al., Ann Neurol. 2013 Dec; 74(6): 862-872, incorporated herein by reference). Prior reports used composite measures of splicing events of a list of RNAs whose splicing are known to be affected in DM1, however without taking into consideration the relative timing of when dysregulation of splicing starts to occur for each RNA tested on a continuum of disease state. In other words, these reports failed to recognize that the splicing events of any given RNA in the list contributes to the composite splicing activity of the entire list of RNAs to a different extent, depending on disease severity and how much MBNL1 is sequestered. As a result, the composite measures of splicing activities as described in prior reports are less accurate and less sensitive as an indicator of disease severity, progression of disease, or treatment efficacy.

[0009] The present disclosure, in some aspects, recognizes and demonstrates that, as on a continuum of disease state of the repeat expansion disease (e.g., DM1), e.g., from mild to moderate to severe, and as more splicing regulators such as MBNL1 become sequestered, alternative splicing of certain genes are affected earlier than other genes. The present disclosure further identifies different sets of genes as sets of disease state-associated genes whose alternative splicing begin to be affected, collectively, at different disease state, e.g., when the disease is on a continuum of disease state from mild to moderate to severe. Composite measures of splicing activities of one or more such defined sets of disease state- associated genes can be used, e.g., as a more accurate and more sensitive indicator of disease severity and/or treatment efficacy.

[00010] Accordingly, the present disclosure, in some aspects, provides one or more genes and sets of genes that are associated with a disease state of a repeat expansion disease (e.g., a repeat expansion disease associated with spliceopathy, such as DM1). The splicing activity of the different sets of disease state-associated genes described herein can be used to indicate the disease state of a repeat expansion disease (e.g., mild, moderate, or severe). [00011] Further provided herein are compositions and methods (e.g., assays) for detecting the splicing events of the one or more disease state associated genes, determining the splicing activity (e.g., an alternative splicing index) of a given disease state associated gene, determining the composite measure of splicing activity (e.g., a composite alternative splicing index) of a set of disease state associated genes. The present disclosure further provides methods of using the splicing activity and/or composite splicing activity of the different sets of disease state associated genes for determining the disease state, monitoring the disease progression, and/or evaluating treatment efficacy of a subject having or suspected of having a repeat expansion disease (e.g., a repeat expansion disease associated with spliceopathy such as DM1).

[00012] The present disclosure, in other aspects, further provides methods of identifying additional disease state-associated genes suitable for use in any one of the compositions and methods described herein.

[00013] Some aspects of the present disclosure provide methods comprising determining a composite measure of splicing activity for a set of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state-associated genes in a nucleic acid sample obtained from a subject. In some embodiments, the composite measure of splicing activity is based on an inclusion or an exclusion of an RNA segment in the one or more RNA transcripts of each gene of the set of disease state-associated genes in the nucleic acid sample. In some embodiments, the RNA segment is an exonic region of the one or more RNA transcripts of each gene of the set of disease state-associated genes. In some embodiments, the composite measure of splicing activity is based on alternative splicing indices.

[00014] Some aspects of the present disclosure provide methods comprising:

(a) determining an alternative splicing index (ASI) for each gene of the set of disease state-associated genes based on the detected splicing events of one or more RNA transcripts of the gene detected in a nucleic acid sample obtained from a subject, wherein the splicing events are detected using nucleic acid hybridization assays; and

(b) determining a composite splicing index (CASI) for the set of disease state- associated genes based on the alternative splicing index determined in step (a) for each gene of the set of disease state-associated genes; wherein the subject has or is suspected of having a repeat expansion disease.

[00015] In some embodiments, the method further comprises obtaining the nucleic acid sample from the subject; subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events; and obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events.

[00016] In some embodiments, wherein the CASI determined in step (b) for the set of disease state-associated genes is above a first threshold CASI value. In some embodiments, each gene of the set of disease state-associated genes comprises a sequence exhibiting a lower binding affinity to MBNL1, relative to a set of genes with a CASI of below the first threshold CASI value. In some embodiments, the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, OPA1, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1. In some embodiments, the subject has a repeat expansion disease that is at a mild to moderate state. [00017] In some embodiments, the CASI determined in step (b) for the set of disease state-associated genes is below a second threshold CASI value. In some embodiments, each gene of the set of disease state-associated genes comprises a sequence exhibiting a higher binding affinity to MBNL1, relative to a set of genes with a CASI of above the second threshold CASI value. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, BINI, RYR1, MBNL2, NFIX, CLASP 1, VPS39, BEST3, SOS1, CLCN1, and MBNL1. In some embodiments, the subject has a repeat expansion disease that is at a moderate to severe state.

[00018] In some embodiments, the CASI determined in step (b) for the set of disease state-associated genes is above a third threshold CASI value. In some embodiments, each gene of the set of disease state-associated genes comprises a sequence exhibiting a higher binding affinity to MBNL1, relative to a set of genes with a CASI of above the second threshold CASI value. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, the subject has a repeat expansion disease that is at a severe state.

[00019] In some embodiments, the nucleic acid sample is obtained from a tissue sample obtained from the subject. In some embodiments, the tissue sample is a muscle biopsy sample. In some embodiments, the nucleic acid sample is obtained from a blood sample obtained from the subject. In some embodiments, the repeat expansion disease is a muscle disease or disorder. In some embodiments, the muscle disease or disorder is myotonic dystrophy. In some embodiments, the myotonic dystrophy is myotonic dystrophy type 1 (DM1). In some embodiments, the subject is undergoing treatment or has been treated for the repeat expansion disease. In some embodiments, the subject will be treated for the repeat expansion disease. [00020] In some embodiments, the nucleic acid hybridization assays comprise RT-PCR and/or sequencing. [00021] In some embodiments, the CASI determined in step (b) is between a value of 0 and 1. In some embodiments, the ASI determined in step (a) is between a value of 0 and 1. In some embodiments, the repeat expansion disease is associated with spliceopathy.

[00022] Other aspects of the present disclosure provide methods comprising determining composite measures of splicing activity for at least two sets of disease state- associated genes based on splicing events detected for one or more RNA transcripts of each gene of the sets of disease state-associated genes in a nucleic acid sample obtained from a subject; and evaluating the disease state of the subject based on the composite measures of splicing activity.

[00023] Other aspects of the present disclosure provide methods of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease, the method comprising:

(a) determining an alternative splicing index (ASI) for each gene of a first set of disease state-associated genes and for each gene of a second set of disease state- associated genes based on detected splicing events for one or more RNA transcripts of the respective genes detected in a nucleic acid sample obtained from a subject, wherein the splicing events are detected using nucleic acid hybridization assays; and

(b) determining a first composite splicing index (CASI) for the first set of disease state-associated genes based on the alternative splicing index determined in step (a) for each gene of the first set of disease state-associated genes;

(c) determining a second CASI for the second set of disease state- associated genes based on the alternative splicing index determined in step (a) for each gene of the second set of disease state-associated genes; and

(d) evaluating the disease state of the subject based on the first and/or the second CASI determined in step (b) and step (c), respectively.

[00024] Other aspects of the present disclosure provide methods comprising determining composite measures of splicing activity for at least two sets of disease state- associated genes based on splicing events detected for one or more RNA transcripts of each gene of the sets of disease state-associated genes in a nucleic acid sample obtained from a subject; and evaluating the disease state of the subject based on the composite measures of splicing activity. In some embodiments, the method further comprises obtaining the nucleic acid sample from the subject; subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events; and obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events.

[00025] In some embodiments, the first set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, the second set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, the first CASI determined in step (b) for the first set of disease state-associated genes is below a first threshold CASI value. In some embodiments, the subject is determined to have a repeat expansion disease that is at a mild state.

[00026] In some embodiments, the first CASI determined in step (b) for the first set of disease state-associated genes is above a second threshold CASI value. In some embodiments, the second CASI determined in step (c) for the second set of disease state-associated genes is below the second threshold CASI value. In some embodiments, the first CASI determined in step (b) for the first set of disease state- associated genes is larger than the second CASI determined in step (c) for the second set of disease state-associated genes. In some embodiments, the subject is determined to have a repeat expansion disease that is at a moderate state.

[00027] In some embodiments, the second CASI determined in step (c) for the second set of disease state-associated genes is above a third threshold CASI value. In some embodiments, wherein the subject is determined to have a repeat expansion disease that is at a severe state.

[00028] In some embodiments, the nucleic acid sample is obtained from a tissue sample obtained from the subject. In some embodiments, the tissue sample is a muscle biopsy sample. In some embodiments, the nucleic acid sample is obtained from a blood sample obtained from the subject. In some embodiments, the repeat expansion disease is a muscle disease or disorder. In some embodiments, the muscle disease or disorder is myotonic dystrophy. In some embodiments, the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

[00029] In some embodiments, the method further comprises treating the subject for the repeat expansion disease if the subject is determined to be at a targeted disease state. In some embodiments, the repeat expansion disease is DM1 and the treating comprises administering to the subject a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

[00030] In some embodiments, the nucleic acid hybridization assays comprise RT-PCR and/or sequencing. In some embodiments, step (b) and step (c) are performed in any order or in parallel. In some embodiments, the first CASI determined in step (b) and/or second CASI determined in step (c) is between a value of 0 and 1. In some embodiments, the ASI determined in step (a) is between a value of 0 and 1. In some embodiments, the repeat expansion disease is associated with spliceopathy.

[00031] Other aspects of the present disclosure provide methods of monitoring a subject undergoing a treatment for a repeat expansion disease, the method comprising determining, during a period of time, at least two composite measures of splicing activity for a set of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state-associated genes in a nucleic acid sample obtained from the subject; and determining the disease state of the subject based on the at least two composite measures of splicing activity to thereby monitor the subject.

[00032] Other aspects of the present disclosure provide methods of monitoring a subject undergoing a treatment for a repeat expansion disease, the method comprising, over a period of time:

(a) determining a first composite splicing index (CASI) for a set of disease state- associated genes;

(b) determining a second composite splicing index (CASI) for the set of disease state- associated genes; and

(c) comparing the first CASI and the second CASI to thereby monitor the subject. [00033] Other aspects of the present disclosure provide methods of evaluating effectiveness of a treatment for a repeat expansion disease in a subject undergoing the treatment for the repeat expansion disease, the method comprising, over a period of time:

(c) comparing the first CASI and the second CASI to thereby determine the effectiveness of the treatment.

[00034] In some embodiments, a period of time described herein is 1-24 (e.g., 1-24, 2- 24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 1-18, 1-12, 1-8, 1-6, 1-5, 1-4, 1-3, 1-2, 2-18, 2-12, 2-8, 2-6, 2-4, 4-18, 4-12, 4-8, 4-6, 6-18, 6-12, 6-8, 8-18, 8-12, or 12-18) months. In some embodiments, a period of time described herein is 1-20 (e.g., 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11- 20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, 19-20, 2-18, 2-12, 2-10, 2-5, 4-18, 4-12, 4-8, 6-18, 6-12, or 6-8) years. In some embodiments, a period of time described herein is the remainder of the subject’s life. In some embodiments, a period of time described herein occurs during treatment for a repeat expansion disease. In some embodiments, a period of time described herein occurs prior to treatment for a repeat expansion disease. In some embodiments, a period of time described herein occurs after treatment for a repeat expansion disease.

[00035] In some embodiments, the first CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI for each gene is determined based on splicing events detected for one or more RNA transcripts for the gene in a first nucleic acid sample obtained from the subject. In some embodiments, the second CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI is determined based on splicing events detected for one or more RNA transcripts of the gene in a second nucleic acid sample obtained from the subject.

[00036] In some embodiments, the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained from the subject after treatment. In some embodiments, the first nucleic acid sample is obtained from the subject earlier in time than the second nucleic acid sample during a period in which the subject is undergoing the treatment. In some embodiments, if the first CASI is larger than the second CASI, the treatment is effective. In some embodiments, the repeat expansion disease is DM1. In some embodiments, the treatment comprises administering to the subject an agent that treats the repeat-expansion disease. In some embodiments, the subject is undergoing treatment with an agent that treats DM1. In some embodiments, the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA. In some embodiments, In some embodiments, the repeat expansion disease is DM1 and the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

[00037] In some embodiments, the set of disease state- associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and MBNL1. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. [00038] In some embodiments, the first nucleic acid sample and/or the second nucleic acid sample is obtained from a tissue sample obtained from the subject. In some embodiments, the tissue sample is a muscle biopsy sample. In some embodiments, the first nucleic acid sample and/or the second nucleic acid sample is obtained from a blood sample obtained from the subject. In some embodiments, the splicing events are detected via subjecting the first nucleic acid sample and/or the second nucleic acid sample to nucleic acid hybridization assays. In some embodiments, the nucleic acid hybridization assays comprise RT-PCR and/or sequencing.

[00039] In some embodiments, the first CASI determined in step (a) and/or the second CASI determined in step (b) is between a value of 0 and 1. In some embodiments, the repeat expansion disease is associated with spliceopathy.

[00040] Other aspects of the present disclosure provide computer-implemented method of processing genomic data, the method comprising: for each gene in a selected set of genes of a reference genome, obtaining measures of splicing activity based on splicing events detected across a population of individuals having the repeat expansion disease at differing disease states; and assigning genes of the selected set into different groups based on the measures of splicing activity across the population of individuals having the repeat expansion disease, wherein the different groups are associated with the differing disease states.

[00041] In some embodiments, the method further comprises determining scores between each gene pair in the selected set based on the measures of splicing activity across the population.

[00042] Other aspects of the present disclosure provide computer-implemented method of processing genomic data, the method comprising: for each gene in a selected set of genes of a reference genome, obtaining measures of splicing activity based on splicing events detected across a population of individuals having the repeat expansion disease at differing disease states; determining similarity scores between each gene pair in the selected set based on in the measures of splicing activity across the population; and assigning genes of the selected set into different groups, wherein the genes within each of the different groups are determined, based on the similarity scores, to have statistically similar variances in the measures of splicing activity across the population of individuals having the repeat expansion disease.

[00043] Other aspects of the present disclosure provide computer-implemented methods of processing genomic data, the method comprising: obtaining sequence data of a reference genome from a database, the sequence data comprising RNA transcript characteristics associated with genes of the reference genome; selecting a set of genes of the reference genome having desired RNA transcript characteristics; for each gene in the selected set, obtaining alternative splicing indices across a population of individuals having the repeat expansion disease at differing disease states; determining similarity scores between each gene pair in the selected set based on the alternative splicing indices across the population; and assigning genes of the selected set into different groups, wherein the genes within each of the different groups are determined, based on the similarity scores, to have statistically similar variances in alternative splicing indices across the population of individuals having the repeat expansion disease.

[00044] In some embodiments, the method further comprises associating each of the different groups with an indicator of a corresponding disease state of the repeat expansion disease.

[00045] In some embodiments, the method further comprises determining a composite alternative splicing index based on alternative splicing indices of genes from two or more different groups; and associating each of the different groups with an indicator of a corresponding disease state of the repeat expansion disease, wherein the association is based on the relative contribution of alternative splicing indices of genes of the different groups to the composite alternative splicing index in relation to the disease state of the individuals from which the alternative splicing indices are obtained.

[00046] In some embodiments, the disease state is a degree of severity of the repeat expansion disease or a degree of progression of the repeat expansion disease.

[00047] In some embodiments, the method further comprises determining, based on the variance in measured alternative splicing indices, a value, n, reflecting the number of different groups.

[00048] In some embodiments, the method further comprises assigning genes into the n different groups using an iterative approach that minimizes the within-group variance in measured alternative splicing indices.

[00049] In some embodiments, the method further comprises assigning the RNA transcript characteristics to genes of the reference genome, and optionally storing the RNA transcript characteristics in the database. [00050] In some embodiments, the RNA transcript characteristics comprise relative splicing factor binding affinities of RNAs encoded by the genes and/or of the number of splicing factor binding elements present in RNAs encoded by the genes. In some embodiments, the RNA transcript characteristics comprise alternative splicing isoform information.

[00051] In some embodiments, the method further comprises, for at least each gene in the selected set, assigning one or more characteristics indicative of the alternative splicing indices across the population, the one or more characteristics comprising a variance in alternative splicing indices across the population. In some embodiments, the repeat expansion disease is myotonic muscular dystrophy type 1 (DM1), myotonic muscular dystrophy type 2 (DM2), Fuchs endothelial corneal dystrophy (FEDC), or spinocerebellar ataxia type 8(SCA8). [00052] In some embodiments, the repeat expansion disease is associated with spliceopathy. In some embodiments, the repeat expansion disease is myotonic dystrophy type 1 (DM1). In some embodiments, the alternative splicing indices are based on normalized levels of inclusion splice isoforms and/or exclusion splice isoforms present in nucleic acid samples obtained from individuals of the population.

[00053] Other aspects of the present disclosure provide methods of determining a disease state of a subject who has or is suspected of having a repeat expansion disease, the method comprising: (al) determining a first composite splicing index (CASI) for a first set of disease state-associated genes, (a2) determining a second composite splicing index (CASI) for the first set of disease state-associated genes, (a3) determining a first comparator between the first CASI of step (al) and the second CASI of step (a2), wherein the first set of disease state- associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1; (bl) determining a first CASI for a second set of disease state-associated genes, (b2) determining a second CASI for the second set of disease state-associated genes, (b3) determining a second comparator between the second CASI of step (bl) and the second CASI of step (b2), wherein the second set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP 1, VPS39, BEST3, SOS1, CLCN1, and MBNL1; (cl) determining a first CASI for a third set of disease state-associated genes, (c2) determining a second CASI for the third set of disease state-associated genes, (c3) determining a third comparator between the first CASI of step (cl) and the second CASI of step (c2), wherein the third set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI; and (d) determining the disease state of the subject based on the first comparator, second comparator, and/or third comparator. BRIEF DESCRIPTION OF THE DRAWINGS

[00054] FIGs. 1A-1B shows the identification of different sets of genes that start to contribute to the composite alternative splicing index (CASI) at different disease states by unsupervised clustering. The optimal number of clusters (FIG. 1A) was determined, followed by K-means clustering based on distribution of per-gene alternative splicing index (ASI) values across patients (FIG. IB).

[00055] FIG. 2 shows the grouping of individual splice events according to the timing of origin of splicing contribution (early-stage (e.g., mild disease state), intermediate-stage (e.g., moderate disease state), or late-stage (e.g., severe disease state) on a continuum of disease state from no disease to very severe disease) to CASI.

[00056] FIG. 3 shows a model of MBNL binding affinities to genes whose splicing activity is affected relatively earlier (lower MBNL binding affinity) and to genes whose splicing activity is affected relatively later (higher MBNL binding affinity) on a continuum of disease state from no disease to very severe disease.

[00057] FIGs. 4A-4B show that genes with different MBNL binding affinity are suitable for indicating therapeutic efficacy at different disease states. FIG. 4A shows that genes with higher MBNL binding affinity are better indicators for therapeutic efficacy when disease is at a severe state. FIG. 4B shows that genes with lower MBNL binding affinity are better indicators for therapeutic efficacy when disease is at a mild state.

[00058] FIG. 5 is a block diagram of an illustrative environment 500 in which some embodiments described herein may be implemented.

[00059] FIG. 6 is a flowchart of an illustrative process for evaluating or determining the disease state of a subject based on composite measures of splicing activity, in accordance with some embodiments described herein.

[00060] FIG. 7 is a further flowchart of an illustrative process for evaluating or determining the disease state of a subject based on composite measures of splicing activity, in accordance with some embodiments described herein.

[00061] FIG. 8 is a flowchart of an illustrative process for assigning genes of a selected set into different groups based on scores such that each group is associated with a disease state, in accordance with some embodiments described herein.

[00062] FIG. 9 shows overall CASI for all 22 genes determined from baseline (BL) and 3-month (3M) longitudinal samples from 34 patients with DM1, as well as CASI based on the specific subsets of genes within each severity stratum. “Mild” refers to mild-disease state, “Moderate” refers to moderate disease state, and “Severe” refers to severe disease state. “BL” refers to Baseline (BL) and (3M) refers to 3-months. CASE Mild = 0 - 0.25, Moderate = 0.25 - 0.65 and Severe = 0.65 - 1.0.

DETAILED DESCRIPTION

[00063] Some aspects of the present disclosure provide one or more genes and sets of genes associated with disease states of a repeat expansion disease (e.g., a repeat expansion disease associated with spliceopathy, such as DM1). In some embodiments, different sets of disease state-associated genes are associated with different relative disease states of a repeat expansion disease (e.g., relatively mild, moderate, or severe) on a continuum of disease state from no disease to very severe disease. In some embodiments, a disease state of the repeat expansion disease can be as determined by functional parameters (e.g., those determined clinically).

[00064] In some embodiments, each set of disease state-associated genes described herein comprise genes that are similar in structural features (e.g., comprising sequences that confer similar binding affinity and/or binding profile of splicing regulators such as MBNL1) and/or functional features (e.g., similar tissue (e.g., muscle) specificity, similar expression profile across a population of subjects having a repeat expansion disease at a similar disease state; similarly affected alternative splicing at a given disease state, etc.).

[00065] Further provided herein are compositions and methods (e.g., assays) for detecting the splicing events of the one or more disease state-associated genes, determining the splicing activity (e.g., an alternative splicing index) of a given disease state-associated gene, determining the composite measure of splicing activities (e.g., a composite splicing index) of a set of disease state-associated genes associated with a certain disease state. The present disclosure further provides methods of using the splicing activity and/or composite splicing activities of the different sets of disease state-associated genes for determining the disease state, monitoring the disease progression, and/or evaluating treatment efficacy of a subject having or suspected of having a repeat expansion disease (e.g., a repeat expansion disease associated with spliceopathy such as DM1). In some embodiments, in methods of evaluating treatment efficacy described herein, the subject is treated with an agent for treating the repeat expansion disease (e.g., DM1). In some embodiments, the agent comprises a muscle-targeting complex described herein, e.g., a muscle-targeting complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide targeting a DMPK RNA. Nonlimiting examples of such muscle targeting complexes are also provided herein. [00066] The present disclosure, in other aspects, further provides methods of identifying additional disease state-associated genes suitable for use in any one of the compositions and methods described herein.

[00067] Further aspects of the disclosure, including a description of defined terms, are provided below.

I. Definitions

[00068] Administering: As used herein, the terms “administering” or “administration” means to provide a complex to a subject in a manner that is physiologically and/or (e.g., and) pharmacologically useful e.g., to treat a condition in the subject).

[00069] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[00070] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen. In some embodiments, an antibody is a full-length antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence. In another embodiment, an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or (e.g., and) a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (e), gamma (y) or mu (p) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (e), gamma (y) or mu ( ) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CHI, CH2, and/or (e.g., and) CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or (e.g., and) methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N- acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still further, an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

[00071] CDR: As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and/or (e.g., and) the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000);

Lefranc, M.-P, Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P, Nucleic Acids Res., 31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs. As used herein, a CDR may refer to the CDR defined by any method known in the art. Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.

[00072] There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Sub-portions of CDRs may be designated as LI, L2 and L3 or Hl, H2 and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are provided below.

¹ IMGT®, the international ImMunoGeneTics information system®, imgt.org, Lefranc, M.-P. et

al., Nucleic Acids

Res., 27:209-212 (1999)

² Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242

³ Chothia et al., J. Mol. Biol. 196:901-917 (1987))

[00073] CDR-grafted antibody: The term "CDR-grafted antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or (e.g., and) VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.

[00074] Chimeric antibody: The term "chimeric antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

[00075] Complementary: As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleosides or two sets of nucleosides. In particular, complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleosides or two sets of nucleosides. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of a target nucleic acid (e.g., an mRNA), then the bases are considered to be complementary to each other at that position. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil- type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.

[00076] Conservative amino acid substitution: As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[00077] Covalently linked: As used herein, the term “covalently linked” refers to a characteristic of two or more molecules being linked together via at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, e.g., a disulfide bond or disulfide bridge, that serves as a linker between the molecules. However, in some embodiments, two or more molecules can be covalently linked together via a molecule that serves as a linker that joins the two or more molecules together through multiple covalent bonds. In some embodiments, a linker may be a cleavable linker. However, in some embodiments, a linker may be a non-cleavable linker.

[00078] Cross-reactive: As used herein and in the context of a targeting agent (e.g., antibody), the term “cross-reactive,” refers to a property of the agent being capable of specifically binding to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity. For example, in some embodiments, an antibody that is cross-reactive against human and non-human primate antigens of a similar type or class (e.g., a human transferrin receptor and non-human primate transferrin receptor) is capable of binding to the human antigen and non-human primate antigens with a similar affinity or avidity. In some embodiments, an antibody is cross- reactive against a human antigen and a rodent antigen of a similar type or class. In some embodiments, an antibody is cross -reactive against a rodent antigen and a non-human primate antigen of a similar type or class. In some embodiments, an antibody is cross-reactive against a human antigen, a non-human primate antigen, and a rodent antigen of a similar type or class. [00079] Disease-associated-repeat: As used herein, the term “disease-associated- repeat” refers to a repeated nucleotide sequence at a genomic location for which the number of units of the repeated nucleotide sequence is correlated with and/or (e.g., and) directly or indirectly contributes to, or causes, genetic disease such as DM1. Each repeating unit of a disease associated repeat may be 2, 3, 4, 5 or more nucleotides in length. For example, in some embodiments, a disease associated repeat is a dinucleotide repeat. In some embodiments, a disease associated repeat is a trinucleotide repeat. In some embodiments, a disease associated repeat is a tetranucleotide repeat. In some embodiments, a disease associated repeat is a pentanucleotide repeat. In some embodiments, embodiments, the disease-associated-repeat comprises CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTG repeats, or a nucleotide complement of any thereof. In some embodiments, a disease- associated-repeat is in a non-coding portion of a gene. However, in some embodiments, a disease-associated-repeat is in a coding region of a gene. In some embodiments, a disease- associated-repeat is expanded from a normal state to a length that directly or indirectly contributes to, or causes, genetic disease. In some embodiments, a disease-associated-repeat is in RNA (e.g., an RNA transcript). In some embodiments, a disease-associated-repeat is in DNA (e.g., a chromosome, a plasmid). In some embodiments, a disease-associated-repeat is expanded in a chromosome of a germline cell. In some embodiments, a disease-associated- repeat is expanded in a chromosome of a somatic cell. In some embodiments, a disease- associated-repeat is expanded to a number of repeating units that is associated with congenital onset of disease. In some embodiments, a disease-associated-repeat is expanded to a number of repeating units that is associated with childhood onset of disease. In some embodiments, a disease-associated-repeat is expanded to a number of repeating units that is associated with adult onset of disease. In DM1, a trinucleotide repeat region of CTG units in the 3' untranslated region (3’-UTR) of DMPK is disease-associated. A normal DMPK allele comprises about 5 to about 37 CTG repeat units, whereas in patients with DM1, the length of the CTG repeat region is significantly increased, up to hundreds or thousands of trinucleotide repeats.

[00080] DMPK: As used herein, the term “DMPK” refers to a gene that encodes myotonin-protein kinase (also known as myotonic dystrophy protein kinase or dystrophia myotonica protein kinase), a serine/threonine protein kinase. Substrates for this enzyme may include myogenin, the beta-subunit of the L-type calcium channels, and phospholemman. In some embodiments, DMPK may be a human (Gene ID: 1760), non-human primate (e.g., Gene ID: 456139, Gene ID: 715328), or rodent gene (e.g., Gene ID: 13400). In humans, a CTG repeat expansion in the 3' non-coding, untranslated region of DMPK is associated with myotonic dystrophy type I (DM1). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001081563.2, NM_004409.4, NM_001081560.2, NM_001081562.2, NM_001288764.1, NM_001288765.1, and NM_001288766.1) have been characterized that encode different protein isoforms.

[00081] DMPK allele: As used herein, the term “DMPK allele” refers to any one of alternative forms (e.g., wild-type or mutant forms) of a DMPK gene. In some embodiments, a DMPK allele may encode for wild-type myotonin-protein kinase that retains its normal and typical functions. In some embodiments, a DMPK allele may comprise one or more disease- associated-repeat expansions. In some embodiments, normal subjects have two DMPK alleles comprising in the range of 5 to 37 repeat units. In some embodiments, the number of CTG repeat units in subjects having DM1 is in the range of about 50 to about 3,000 or more with higher numbers of repeats leading to an increased severity of disease. In some embodiments, mildly affected DM1 subjects have at least one DMPK allele having in the range of 50 to 150 repeat units. In some embodiments, subjects with classic DM1 have at least one DMPK allele having in the range of 100 to 1,000 or more repeat units. In some embodiments, subjects having DM1 with congenital onset may have at least one DMPK allele comprising more than 2,000 repeat units.

[00082] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region. Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art may be used in the antibodies disclosed herein.

[00083] Human antibody: The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[00084] Humanized antibody: The term "humanized antibody" refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or (e.g., and) VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR- grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfRl antibodies and antigen binding portions are provided. Such antibodies may be generated by obtaining murine anti- TfRl monoclonal antibodies using traditional hybridoma technology followed by humanization using in vitro genetic engineering, such as those disclosed in Kasaian et al PCT publication No. WO 2005/123126 A2.

[00085] Internalizing cell surface receptor: As used herein, the term, “internalizing cell surface receptor” refers to a cell surface receptor that is internalized by cells, e.g., upon external stimulation, e.g., ligand binding to the receptor. In some embodiments, an internalizing cell surface receptor is internalized by endocytosis. In some embodiments, an internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, an internalizing cell surface receptor is internalized by a clathrin- independent pathway, such as, for example, phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake or constitutive clathrin-independent endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and/or (e.g., and) an extracellular domain, which may optionally further comprise a ligand-binding domain. In some embodiments, a cell surface receptor becomes internalized by a cell after ligand binding. In some embodiments, a ligand may be a muscle-targeting agent or a muscle-targeting antibody. In some embodiments, an internalizing cell surface receptor is a transferrin receptor.

[00086] Isolated antibody: An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities e.g., an isolated antibody that specifically binds transferrin receptor is substantially free of antibodies that specifically bind antigens other than transferrin receptor). An isolated antibody that specifically binds transferrin receptor complex may, however, have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or (e.g., and) chemicals.

[00087] Kabat numbering: The terms "Kabat numbering", "Kabat definitions and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[00088] Molecular payload: As used herein, the term “molecular payload” refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, a molecular payload is linked to, or otherwise associated with a muscle-targeting agent. In some embodiments, the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a target gene. [00089] Muscle-targeting agent: As used herein, the term, “muscle-targeting agent,” refers to a molecule that specifically binds to an antigen expressed on muscle cells. The antigen in or on muscle cells may be a membrane protein, for example an integral membrane protein or a peripheral membrane protein. Typically, a muscle-targeting agent specifically binds to an antigen on muscle cells that facilitates internalization of the muscle-targeting agent (and any associated molecular pay load) into the muscle cells. In some embodiments, a muscle-targeting agent specifically binds to an internalizing, cell surface receptor on muscles and is capable of being internalized into muscle cells through receptor mediated internalization. In some embodiments, the muscle-targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle-targeting agent is linked to a molecular payload.

[00090] Muscle-targeting antibody: As used herein, the term, “muscle-targeting antibody,” refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, a muscle-targeting antibody specifically binds to an antigen on muscle cells that facilitates internalization of the muscletargeting antibody (and any associated molecular payment) into the muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing, cell surface receptor present on muscle cells. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a transferrin receptor.

[00091] Myotonic dystrophy (DM): As used herein, the term “Myotonic dystrophy (DM)” refers to a genetic disease caused by mutations in the DMPK gene or CNBP (ZNF9) gene that is characterized by muscle loss, muscle weakening, and muscle function. Two types of the disease, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), have been described. DM1 is associated with an expansion of a CTG trinucleotide repeat in the 3' non-coding region of DMPK. DM2 is associated with an expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9. In both DM1 and DM2, the nucleotide expansions lead to toxic RNA repeats capable of forming hairpin structures that bind critical intracellular proteins, e.g., muscleblind-like proteins, with high affinity. Myotonic dystrophy, the genetic basis for the disease, and related symptoms are described in the art (see, e.g., Thornton, C.A., “Myotonic Dystrophy” Neurol Clin. (2014), 32(3): 705-719.; and Konieczny et al. “Myotonic dystrophy: candidate small molecule therapeutics” Drug Discovery Today (2017), 22:11.) In some embodiments, subjects are bom with a variation of DM1 called congenital myotonic dystrophy. Symptoms of congenital myotonic dystrophy are present from birth and include weakness of all muscles, breathing problems, clubfeet, developmental delays and intellectual disabilities. DM1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 160900. DM2 is associated with OMIM Entry # 602668.

[00092] Oligonucleotide: As used herein, the term “oligonucleotide” refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNAs), etc. Oligonucleotides may be single- stranded or double-stranded. In some embodiments, an oligonucleotide may comprise one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, an oligonucleotide may comprise one or more modified intemucleoside linkages. In some embodiments, an oligonucleotide may comprise one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.

[00093] Recombinant antibody: The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more details in this disclosure), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425- 445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. One embodiment of the disclosure provides fully human antibodies capable of binding human transferrin receptor which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries such as those disclosed in Jermutus et al., PCT publication No. WO 2005/007699.

[00094] Region of complementarity: As used herein, the term “region of complementarity” refers to a nucleotide sequence, e.g., of an oligonucleotide, that is sufficiently complementary to a cognate nucleotide sequence, e.g., of a target nucleic acid, such that the two nucleotide sequences are capable of annealing to one another under physiological conditions (e.g., in a cell). In some embodiments, a region of complementarity is fully complementary to a cognate nucleotide sequence of target nucleic acid. However, in some embodiments, a region of complementarity is partially complementary to a cognate nucleotide sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, or 4 mismatches compared with a cognate nucleotide sequence of a target nucleic acid.

[00095] Specifically binds: As used herein, the term “specifically binds” refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to an antibody, the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, e.g., to an extent that permits preferential targeting to certain cells, e.g., muscle cells, through binding to the antigen, as described herein. In some embodiments, an antibody specifically binds to a target if the antibody has a KD for binding the target of at least about 10'⁴ M, 10'⁵ M, 10'⁶ M, IO’⁷ M, 10'⁸ M, 10'⁹ M, 10⁴⁰ M, 10⁴¹ M, 10 ² M, 10 ³ M, or less. In some embodiments, an antibody specifically binds to the transferrin receptor, e.g., an epitope of the apical domain of transferrin receptor.

[00096] Subject: As used herein, the term “subject” refers to a mammal. In some embodiments, a subject is non-human primate, or rodent. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has or is suspected of having a disease resulting from a disease-associated-repeat expansion, e.g., in a DMPK allele. [00097] Transferrin receptor: As used herein, the term, “transferrin receptor” (also known as TFRC, CD71, p90, or TFR1) refers to an internalizing cell surface receptor that binds transferrin to facilitate iron uptake by endocytosis. In some embodiments, a transferrin receptor may be of human (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin. In addition, multiple human transcript variants have been characterized that encoded different isoforms of the receptor (e.g., as annotated under GenBank RefSeq Accession Numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[00098] 2’-modified nucleoside: As used herein, the terms “2’ -modified nucleoside” and “2’ -modified ribonucleoside” are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2’ position. In some embodiments, the 2’ -modified nucleoside is a 2’-4’ bicyclic nucleoside, where the 2’ and 4’ positions of the sugar are bridged (e.g., via a methylene, an ethylene, or a (S)-constrained ethyl bridge). In some embodiments, the 2’- modified nucleoside is a non-bicyclic 2’-modified nucleoside, e.g., where the 2’ position of the sugar moiety is substituted. Non-limiting examples of 2’ -modified nucleosides include: 2’-deoxy, 2’-fluoro (2’-F), 2’-O-methyl (2’-0-Me), 2’-O-methoxyethyl (2’-M0E), 2’-O- aminopropyl (2’-O-AP), 2’-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O- dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O-N-methylacetamido (2’-0-NMA), locked nucleic acid (LNA, methylene-bridged nucleic acid), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt). In some embodiments, the 2’ -modified nucleosides described herein are high-affinity modified nucleosides and oligonucleotides comprising the 2’ -modified nucleosides have increased affinity to a target sequences, relative to an unmodified oligonucleotide. Examples of structures of 2’-modified nucleosides are provided below:

2'-O-methoxyethyl '

locked nucleic acid ethylene-bridged (S)-constrained

These examples are shown with phosphate groups, but any internucleoside linkages are contemplated between 2’ -modified nucleosides.

[00099] Ranges: All ranges provided in the present disclosure are inclusive of the end points.

[000100] Disease state-associated gene: As used herein, the term “disease state- associated gene” refers to a gene whose expression (e.g., expression of RNA, protein, and/or relative levels of different isoforms) and/or activity differs in the presence or absence of a disease such as a repeat expansion disease described herein or known in the art, and/or is affected by the progression and/or severity of the disease. In some embodiments, the disease state-associated gene encodes RNAs whose splicing and/or alternative splicing activities differ in the presence or absence of a disease such as a repeat expansion disease described herein or known in the art, and/or is affected by the progression and/or severity of the disease. For example, a disease state-associated gene as described herein may have different expression profiles and/or encode RNAs with different splicing and/or alternative splicing profiles at different disease states including, without limitation: no disease (i.e., in healthy subject), mild disease state, moderate disease state, and severe disease state. It is to be understood that disease states referred to herein are intended to describe relative severity of a disease on a continuum as the disease becomes more severe, i.e., from no disease to very several disease, rather than any discretely characterized disease states. For example, a mild disease state as referred to herein means the disease has progressed in its severity relative to no disease (i.e., in a healthy subject); a moderate disease as referred to herein means the disease is in its severity relative to a mild disease state; and a severe disease as referred to herein means the disease is in its severity relative to a moderate disease state. In some embodiments, a disease state described herein can correspond to clinically established disease states (e.g., as indicated by functional measures determined clinically) for a disease such as a repeat expansion disease described herein or known in the art.

[000101] In some embodiments, a disease state- associated gene as described herein is deemed to be associated with a mild disease state when it has different (e.g., detectable difference and/or statistically significant difference) expression profiles and/or encode RNAs with different splicing and/or alternative splicing profiles in a subject at a mild disease state relative to a healthy subject, i.e., a gene that is “earlier” affected by the disease on the continuum of disease state from no disease to very severe disease. In some embodiments, further progression of disease severity further affects the expression profiles of the gene and/or splicing and/or alternative splicing profiles of RNAs encoded by the gene. In some embodiments, the effect of disease severity on genes associated with a mild disease state may saturate as disease progresses in severity, and in such situations, further progression of disease severity may not further affect the expression profiles of the gene and or splicing and/or alternative splicing profiles of RNAs encoded by the gene. Nonlimiting examples of genes associated with a mild disease state include: CCPG1, KIF13A, INSR, CAMK2B, and OPA1. [000102] In some embodiments, a disease state- associated gene as described herein is deemed to be associated with a severe disease state when it continues to have similar expression profiles and/or encode RNAs with similar splicing and/or alternative splicing profiles in a subject at a mild or moderate disease state relative to a healthy subject, i.e., a gene that is “later” affected by the disease on the continuum of disease state from no disease to very severe disease. In other words, a gene associated with a severe disease state may not exhibit changes (e.g., detectable changes and/or statistically significant changes) in its expression profile and/or in the splicing and/or alternative splicing profiles of the RNAs it encodes when the disease is mild or even moderate, but start to exhibit changes (e.g., detectable changes and/or statistically significant changes) when the disease is towards the severe end of the continuum. Nonlimiting examples of genes associated with a severe disease state include: CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. [000103] In some embodiments, a disease state- associated gene as described herein is deemed to be associated with a moderate disease state when the expression profiles of the gene and/or the splicing and/or alternative splicing profiles of the RNAs it encodes start to exhibit changes (e.g., detectable changes and/or statistically significant changes) at a point on the continuum between where a gene associated with a mild disease state start to exhibit change (e.g., detectable changes and/or statistically significant changes) and where a gene associated with a severe disease state start to exhibit change (e.g., detectable changes and/or statistically significant changes). Nonlimiting examples of genes associated with a moderate disease state include: RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1.

[000104] In any one of the disease states described herein, the disease is a repeat expansion disease. In any one of the disease states described herein, the disease is a repeat expansion disease associated with spliceopathy. In any one of the disease states described herein, the disease is myotonic dystrophy, type 1 (DM1). In the context of DM1, in some embodiments, the disease states described herein correspond to disease states indicated by functional measures (e.g., measures of muscle integrity and/or strength). Tests used clinically for determining the severity of DM1 include, without limitation: handgrip strength test, hand grip relaxation time, video hand opening time (vHOT), Quantitative Myometry Testing (QMT), 10-meter walk/run test (10-MWRT), stair-ascend/descend test, 5 times sit to stand (5xSTS), 9-hole peg test (9-HPT), ankle dorsiflexion test; 30 foot-go test, 4-step climb test, and test of myotonia.

[000105] Set of disease state-associated genes: As used herein, a “set of disease state- associated genes” refers to a set of genes that is associated with a similar disease state, e.g., mild, moderate, or severe on the continuum of disease severity.

[000106] In some embodiments, genes in any set of disease state-associated genes described herein may have similarities, e.g., structural and/or functional similarities. For example, in the context of DM1, MBNL1 is known as the splicing regulator of many genes encoding RNAs whose splicing and/or alternative splicing is affected by DM1. MBNL1 binds and is sequestered by the mutant DMPK RNA that contain expanded CUG repeats, leading to deregulation of splicing and/or alternative splicing of genes normally regulated by MBNL1. As such, in some embodiments, genes in any sets of DM1 disease state-associated genes described herein may encode RNAs that comprise similar number of MBNL1 binding sites and/or exhibit similar binding affinity to MBNL1.

[000107] In some embodiments, a set of disease state-associated genes as described herein are deemed to be associated with a mild disease state when the genes collectively have different (e.g., detectable difference and/or statistically significant difference) expression profiles and/or encode RNAs with different splicing and/or alternative splicing profiles in a subject at a mild disease state relative to a healthy subject, i.e., a set of genes that collectively are “earlier” affected by the disease on the continuum of disease state from no disease to very severe disease. In some embodiments, further progression of disease severity further affects the collective expression profiles of the gene and or collective splicing and/or alternative splicing profiles of RNAs encoded by the genes. In some embodiments, the effect of disease severity on the set of genes associated with a mild disease state may saturate as disease progresses in severity, and in such situations, further progression of disease severity may not further affect the collective expression profiles of the gene and or collective splicing and/or alternative splicing profiles of RNAs encoded by the gene. A nonlimiting example of a set of genes associated with a mild disease state comprises two or more of CCPG1, KIF13A, INSR, CAMK2B, and 0PA1. In some embodiments, a set of genes associated with a mild disease state comprises CCPG1, KIF13A, INSR, CAMK2B, and 0PA1. In some embodiments, each gene of a set of genes associated with a mild disease state (e.g., mild DM1 disease state) encodes RNAs that exhibit similar binding affinity to a splicing regulator (e.g., MBNL1), which binding affinity is lower than a binding affinity of RNAs encoded by a gene of a set of genes associated with moderate disease state (e.g., moderate DM1 disease state) and/or RNAs encoded by a gene of a set of genes associated with severe disease state (e.g., severe DM1 disease state).

[000108] In some embodiments, a set of disease state-associated gene as described herein is deemed to be associated with a severe disease state when the genes collectively continue to have similar expression profiles and/or encode RNAs with similar splicing and/or alternative splicing profiles in a subject at a mild or moderate disease state relative to a healthy subject but collectively start to exhibit changes (e.g., detectable changes and/or statistically significant changes) when disease is at a severe state, i.e., a set of genes that collectively are “later” affected by the disease on the continuum of disease state from no disease to very severe disease. In other words, a set of genes associated with a severe disease state may collectively not exhibit any changes (e.g., detectable changes and/or statistically significant changes) in expression profiles and/or in the splicing and/or alternative splicing profiles of the RNAs they encode when the disease is mild or even moderate, but start to exhibit changes when the disease is towards the severe end of the continuum. A nonlimiting example of a set of genes associated with a severe disease state comprises two or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, a set of genes associated with a severe disease state comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, each gene of a set of genes associated with a severe disease state (e.g., severe DM1 disease state) encodes RNAs that exhibit similar binding affinity to a splicing regulator (e.g., MBNL1), which binding affinity is higher than a binding affinity of RNAs encoded by a gene of a set of genes associated with mild disease state (e.g., mild DM1 disease state), and which binding affinity is higher than a binding affinity of RNAs encoded by a gene of a set of genes associated with moderate disease state (e.g., moderate DM1 disease state). In some embodiments, a set of genes associated with severe disease state exhibits higher degree of muscle specificity than a set of genes associated with mild disease state.

[000109] In some embodiments, a set of disease state-associated genes as described herein is deemed to be associated with a moderate disease state when, collectively, the expression profiles of the set of genes and/or the splicing and/or alternative splicing profiles of the RNAs they encode start to exhibit change (e.g., detectable changes and/or statistically significant changes) at a point on the continuum between where a set of genes associated with a mild disease state collectively start to exhibit change (e.g., detectable changes and/or statistically significant changes) and where a set of genes associated with a severe disease state collectively start to exhibit change (e.g., detectable changes and/or statistically significant changes). A nonlimiting example of a set of genes associated with a moderate disease state comprises two or more of RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1. In some embodiments, a set of genes associated with a moderate disease state comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1. In some embodiments, each gene of a set of genes associated with a moderate disease state (e.g., severe DM1 disease state) encodes RNAs that exhibit similar binding affinity to a splicing regulator (e.g., MBNL1), which binding affinity is higher than a binding affinity of RNAs encoded by a gene of a set of genes associated with mild disease state (e.g., moderate DM1 disease state), and which binding affinity is lower than a binding affinity of RNAs encoded by a gene of a set of genes associated with severe disease state (e.g., severe DM1 disease state).

[000110] Splicing: As used herein, the term “splicing” refers to the RNA splicing process, which is a process by which a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). The splicing process removes all the introns (non-coding regions of RNA) and joins together exons (coding regions). For nuclear-encoded genes, splicing occurs in the nucleus either during or immediately after transcription. For eukaryotic genes that contain introns, splicing is usually needed to create an mRNA molecule that can be translated into protein. In some embodiments, splicing regulates gene expression in a cell. In some embodiments, splicing regulates protein levels in a cell. In some embodiments, splicing is catalyzed by small nucleal ribonucleoproteins (snRNPs). In some embodiments, splicing occurs in spliceosomes, which is a complex of small nuclear ribonucleoproteins (snRNPs). For many eukaryotic introns, splicing occurs in a series of reactions which are catalyzed by the spliceosome. In some embodiments, an RNA molecule can splice itself (e.g., self-splicing), e.g., via self-splicing introns, that is, ribozymes that can catalyze their own excision from their parent RNA molecule.

[000111] Spliceosomal splicing and self-splicing involve two sequential transesterification reactions. First, the 2'OH of a specific branchpoint nucleotide within the intron, defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site, forming the lariat intermediate. Second, the 3'OH of the released 5' exon then performs a nucleophilic attack at the first nucleotide following the last nucleotide of the intron at the 3' splice site, thus joining the exons and releasing the intron lariat. tRNA splicing, however, does not involve a transesterification reaction.

[000112] Disease state: The term “disease state” refers to the relative severity of a repeat expansion disease (e.g., DM1). For example, a disease state referred to herein may be mild, moderate, or severe on a continuum of disease severity. In some embodiments, a disease state referred to herein relates to DM1 disease state. In some embodiments, DM1 disease state can be determined clinically. Tests used clinically for determining the severity of DM1 include, without limitation: handgrip strength test, hand grip relaxation time, video hand opening time (vHOT), Quantitative Myometry Testing (QMT), 10-meter walk/run test (10-MWRT), stair-ascend/descend test, 5 times sit to stand (5xSTS), 9-hole peg test (9-HPT), ankle dorsiflexion test; 30 foot-go test, 4-step climb test, and test of myotonia.

[000113] For DM1, classifications on the disease state based on the age of onset of symptoms have been described, e.g., in Thornton et al., (Neurol Clin. 2014 August ; 32(3): 705-719), Bird et al., Myotonic Dystrophy Type 1; GeneReviews, 1999; Johnson et al., Neurology. 2021 Feb 16; 96(7): el045-el053; Jean et al., Orphanet Journal of Rare Diseases volume 9, Article number: 186 (2014); and Johnson et al., Neurology: Clinical Practice October 2019 vol. 9 no. 5 443-454, each of which is incorporated herein by reference in its entirety. As described in prior reports, DM1 disease state which span a continuum from mild to severe have been categorized into three overlapping phenotypes: mild, classic, and congenital. Mild DM1 is characterized by cataract and mild myotonia (sustained muscle contraction), and patient life span is normal. Classic DM1 is characterized by muscle weakness and wasting, myotonia, cataract, and often cardiac conduction abnormalities; adults may become physically disabled and may have a shortened life span. Congenital DM1 is characterized by hypotonia and severe generalized weakness at birth, often with respiratory insufficiency and early death; and subjects with congenital DM1 commonly has intellectual disability.

[000114] In some embodiments, a subject is considered as having the mild phenotype of DM1 when presenting at least two of the following three criteria: (1) Less than 200 CTG repeats; (2) Muscular Impairment Rating Scale (MIRS) grade 1 or 2; (3) age at onset over 40 years old.

[000115] Exon: As used herein, the term “exon” (also known as an “exonic region”) refers to a part of a gene that will form a part of the final mature RNA (e.g., mRNA) produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature RNA (e.g., mRNA).

[000116] Intron: As used herein, the term “intron” refers to nucleotide sequences within a gene that is not included, expressed and/or operative in a mature RNA (e.g., mRNA) product. The term intron refers to both the DNA sequence within a gene and the corresponding RNA sequence in RNA transcripts. Intron sequences are included in primary RNA transcripts upon transcription, but are removed during RNA splicing. During RNA splicing, non-intron sequences (exons) are joined to form mature RNA (e.g., mRNA) products.

[000117] Alternative splicing: As used herein the term “alternative splicing” refers to splicing processes (e.g., occurring on a pre-mRNA transcript) that result in a range of RNA (e.g., mRNA) transcripts from the same gene that contain varying exon compositions, in turn leading to a range of unique proteins encoded by the RNA (e.g., mRNA) transcripts.

Alternative splicing can occur in many ways. Exons can be extended or skipped, or introns can be retained. It is estimated that 95% of transcripts from multiexon genes undergo alternative splicing, some instances of which occur in a tissue- specific manner and/or under specific cellular conditions. Development of high throughput mRNA sequencing technology can help quantify the expression levels of mRNA transcripts resulting from alternative splicing. Differential expression levels across tissues and cell lineages allowed computational approaches to be developed to predict the functions of these isoforms. In many instances, alternative splicing of pre-mRNA transcripts is regulated by a system of trans-acting proteins (activators and repressors) that bind to cis-acting sites or "elements" (enhancers and silencers) on the pre-mRNA transcript itself. These proteins and their respective binding elements promote or reduce the usage of a particular splice site. The binding specificity is associated with the sequence and structure of the cis-elements. For example, in accordance with the present disclosure, MBNL1 is a splicing regulator that regulates the alternative splicing of many genes whose splicing or alternative splicing activity is affected by a repeat expansion disease (e.g., DM1).

[000118] Splicing activity: As used herein, the term “splicing activity” refers to splicing events occurring to a RNA, e.g., a pre-mRNA transcript, including alternative splicing activities. When referring to the “splicing activity” of a gene (e.g., a disease state-associated gene as described herein), or a set of genes (e.g., a set of disease state-associated genes as described herein), it is to be understood that the term encompasses any one or more of the splicing events occurring to RNAs (e.g., all RNAs or a subset of RNAs) encoded by the gene or set of genes detected (e.g., using a set of primers that detect inclusion or exclusion of one or more specific exons) or detectable (e.g., any activity at any one or more of the exons that is above detection threshold) in a nucleic acid hybridization assay (e.g., any one or more of the nucleic acid hybridization assays described herein).

[000119] Percent spliced in (PSI): As used herein, the term “percent spliced in (PSI)” is used as a measure of an individual splicing event (e.g., the splicing or alternative splicing of a specific exon of a gene). For example, PSI may refer to the fractional inclusion (e.g., inclusion in a mature RNA (e.g., mRNA) after splicing) of an exon or an alternative exon of a gene that is detected (e.g., using a set of primers that detect inclusion or exclusion of the exon or alternative exon) in a sample. In some embodiments, the sample may be a blood sample or a tissue sample (e.g., muscle tissue sample). “Fractional inclusion” means the fraction of the exon included in a mature RNA (e.g., mRNA) after splicing relative to the total detected exon in the sample. PSI values can be determined using samples obtained from subjects including control subjects (e.g., healthy subjects or subjects that do not have a repeat expansion disease such as DM1), or subjects that have a repeat expansion disease such as DM1. In some embodiments, PSI values are determined for an exon (e.g., an exon whose inclusion or exclusion via alternative splicing that is affected by a repeat expansion disease) in a diseasestate associated gene (e.g., a disease-state associated gene that is described herein) in different subjects. As such, in some embodiments, a change in PSI values for an exon in a disease stage-associated gene as described herein is observed in a subject having a repeat expansion disease (e.g., DM1), relative to a control subject (e.g., healthy subjects or subjects that do not have a repeat expansion disease such as DM1). In some embodiments, a PSI for an individual splicing event is calculated using the equation below (Equation A): 100

[000120] The “included reads” and “excluded reads” in Equation A maybe obtained using any suitable techniques, e.g., nucleic acid hybridization assays known in the art and/or described herein, which include, without limitation, PCR based methods such as RT-PCR,

RT-qPCR, and/or microarrays, and sequencing based methods such as Multiplex Alternative Splice sequencing (MASseq), and/or RNAseq.

[000121] Alternative splicing index (ASI): As used herein, the term “alternative splicing index (ASI)” is used to indicate a normalized change of an individual splicing event (e.g., the splicing or alternative splicing of a specific exon of a gene) detected in a sample (e.g., a sample obtained from a subject or multiple subjects having a repeat expansion disease such as DM1), relative to a corresponding splicing event in a control sample (e.g., a sample obtained from one or multiple healthy subjects or one or multiple subjects who do not have a repeat expansion disease such as DM1). Methods for determining alternative splicing index have been described in the art, e.g., in Tanner et al. (Nucleic Acids Research, 2021, Vol. 49, No. 4), the entire contents of which are incorporated herein by reference.

[000122] In some embodiments, an ASI for an individual splicing event z (e.g., the splicing or alternative splicing of a specific exon of a disease state-associated gene as described herein) is determined using the equation below (Equation B):

in which, “PSIi” is the PSI determined experimentally for the splicing event z, “PSI(control, P50)” is the median PSI for the splicing event z across the control subjects (e.g., one or multiple healthy subjects or one or multiple subjects who do not have a repeat expansion disease such as DM1); “PSI(disease, P95)” is the 95th percentile PSIs for most severely affected splicing events in subjects having the repeat expansion disease such as DM1. For example, in some embodiments, PSI(disease,P95) is determined from multiple subjects of a disease model (e.g., mouse model) for the repeat expansion disease such as DM1. In some embodiments, any one of the ASI values provided in the present disclosure may be determined using Equation B. In some embodiments, any one of the PSI values in Equation B is determined using Equation A.

[000123] In some embodiments, an ASI value determined in accordance with the present disclosure (e.g., using Equation B) is between the value of 0 and 1 (inclusive). In some embodiments, an ASI value determined in accordance with the present disclosure (e.g., using Equation B) is above 1. [000124] Composite alternative splicing index (CASI): As used herein, the term “composite alternative splicing index (CASI)” is used to indicate a normalized collective change of a number of splicing events (e.g., splicing events of one or more exons of one or more genes) detected in a sample (e.g., a sample obtained from a subject or multiple subjects having a repeat expansion disease such as DM1), relative to corresponding splicing events, collectively, in a control sample (e.g., a sample obtained from one or multiple healthy subjects or one or multiple subjects who do not have a repeat expansion disease such as DM1). In some embodiments, a CASI is determined for a set of disease state-associated genes known and/or described herein. In some embodiments, a CASI for a set of disease state-associated genes (e.g., those known and/or described herein) is determined using the equation below (Equation C):

in which, n is the number of genes in a set of disease state-associated genes, ASIi is determined as described herein (e.g., using Equation B). In some embodiments, any one of the CASI values provided in the present disclosure may be determined using Equation C. [000125] In some embodiments, a CASI value determined in accordance with the present disclosure (e.g., using Equation C) is between the value of 0 and 1 (inclusive). In some embodiments, an CASI value determined in accordance with the present disclosure (e.g., using Equation B) is above 1.

[000126] First threshold composite alternative splicing index (CASI) value: As used herein, the term “first threshold composite alternative splicing index (CASI) value” refers to a control CASI value determined for a set of splicing events (e.g., on RNA transcripts encoded by one or more genes) that are known to be affected when the repeat expansion disease (e.g., DM1) is mild on the continuum of disease state from no disease to very severe disease. In the context of DM1, in some embodiments, the mild disease state corresponds to mild disease states indicated by functional measures (e.g., measures of muscle integrity and/or strength). Tests used clinically for determining the severity of DM1 include, without limitation: handgrip strength test, hand grip relaxation time, video hand opening time (vHOT), Quantitative Myometry Testing (QMT), 10-meter walk/run test (10-MWRT), stair- ascend/descend test, 5 times sit to stand (5xSTS), 9-hole peg test (9-HPT), ankle dorsiflexion test; 30 foot-go test, 4- step climb test, and test of myotonia.

[000127] Second threshold composite alternative splicing index (CASI) value: As used herein, the term “second threshold composite alternative splicing index (CASI) value” refers to a control CASI value determined for a set of splicing events (e.g., on RNA transcripts encoded by one or more genes) that are known to be affected when the repeat expansion disease (e.g., DM1) has advanced from mild to moderate on the continuum of disease state from no disease to very severe disease. In the context of DM1, in some embodiments, the moderate disease state corresponds to moderate disease states indicated by functional measures (e.g., measures of muscle integrity and/or strength). Tests used clinically for determining the severity of DM1 include, without limitation: handgrip strength test, hand grip relaxation time, video hand opening time (vHOT), Quantitative Myometry Testing (QMT), 10-meter walk/run test (10-MWRT), stair-ascend/descend test, 5 times sit to stand (5xSTS), 9- hole peg test (9-HPT), ankle dorsiflexion test; 30 foot-go test, 4-step climb test, and test of myotonia.

[000128] Third threshold composite alternative splicing index (CASI) value: As used herein, the term “third threshold composite alternative splicing index (CASI) value” refers to a control CASI value determined for a set of splicing events (e.g., on RNA transcripts encoded by one or more genes) that are known to be affected when the repeat expansion disease (e.g., DM1) has advanced from moderate to severe on the continuum of disease state from no disease to very severe disease. In the context of DM1, in some embodiments, the severe disease state corresponds to severe disease states indicated by functional measures (e.g., measures of muscle integrity and/or strength). Tests used clinically for determining the severity of DM1 include, without limitation: handgrip strength test, hand grip relaxation time, video hand opening time (vHOT), Quantitative Myometry Testing (QMT), 10-meter walk/run test (10-MWRT), stair-ascend/descend test, 5 times sit to stand (5xSTS), 9-hole peg test (9- HPT), ankle dorsiflexion test; 30 foot-go test, 4-step climb test, and test of myotonia.

[000129] Comparator: As used herein, the term “comparator” refers to a quantity or representation useful for discriminating between conditions, states, values, or events. A comparator, for example, may be a numerical representation indicative of a difference between two values (e.g., two CASI values), such as a subtraction results, ratio, or other similar parameter. In some embodiments, a comparator may be a scalar quantity or a vector quantity.

[000130] Nucleic acid hybridization assay: As used herein, the term “nucleic acid hybridization assay” refers to any assay that involves one or more steps of nucleic acid hybridization. The hybridization may be the hybridization of a primer specifically to a target sequence to be detected, or the hybridization of a common primer to a common sequence in a range of different genes or RNAs, e.g., for the purposes of amplification and subsequent processing (e.g., sequencing). Nucleic acid hybridization assays suitable for use in accordance with the present disclosure are described in, e.g., Nakamori et al. (Ann Neurol. 2013 December ; 74(6)), Wagner et al., (PLoS Genet 12, el006316), Tanner et al. (Nucleic Acids Research, 2021, Vol. 49, No. 4), Wang et al. (RNA Gain-of-function Mechanism and Biomarker Development for Myotonic Dystrophy Type 1 ; Dissertation, University of Rochester, 2017), the entire contents of each of which are incorporated herein by reference. Nonlimiting examples of nucleic acid hybridization assays suitable for use in accordance with the present disclosure include PCR based methods such as RT-PCR, RT-qPCR, and/or microarrays, and sequencing based methods such as Multiplex Alternative Splice sequencing (MASseq), and/or RNAseq.

[000131] Repeat expansion disease: As used herein, the term “repeat expansion disease” (also known as repeat expansion disorder) refers to a class of genetic diseases that are caused by expansions in DNA repeats. Throughout the human genome there are repeating sequences or tandem repeats called microsatellites (1-9 bp repeats), minisatellites (10-99 bp repeats), or satellites (greater than 100 bp repeats). Repeat expansion diseases arise from normal polymorphic repeats expanding (e.g., increasing number of repeat sequences), with the symptomatic threshold of repeat expansion variable depending on the disease. In some embodiments, repeat expansion affects a coding region of a gene. In some embodiments, repeat expansion affects a non-coding region of a gene. In some embodiments, a repeat expansion disease is a trinucleotide repeat disease, trinucleotide repeat expansion, or trinucleotide repeat disorder. In some embodiments, a repeat expansion disease has trinucleotide repeats (e.g., DM1), tetranucleotides repeats (e.g., DM2), pentanucleotides repeats (e.g., SCAIO, SCA31), hexanucleotides repeats (e.g., C9ORF72 FTD/ALS, SCA36), or dodecanucleotides repeats (e.g., EPM1). Repeat expansion is the cause a variety of diseases such as myotonic dystrophy (DM1 and DM2), myotrophic lateral sclerosis /frontotemporal dementia, Huntington disease, Friedreich ataxia, oculopharyngeal muscular dystrophy, and myoclonic epilepsy.

[000132] Spliceopathy: As used herein, the term “spliceopathy” refers to splicing deregulation, abnormal splicing of genes, or misregulated splicing. In some embodiments, spliceopathy is the general alteration of the mRNA processing pathways. In some embodiments, spliceopathy occurs when different transcripts from different tissues are incorrectly spliced. For example, in some embodiments, spliceopathy causes myotonia and insulin resistance in DM1. In some embodiments, patients with DM1 have spliceopathy of the INSR, MBNL1, MBNL2, ADD3, CRTC2 genes. In some embodiments, spliceopathy causes the accumulation of toxic RNAs, wherein there is an accumulation in the nucleus of splicing and cleavage factors (e.g., heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear ribonucleoproteins (snRNPs)).

[000133] CCPG1: As used herein, the term “CCPG1” (also known as cell cycle progression 1 and CPR8) refers to a gene that is involved in positive regulation of cell cycle and cell population proliferation. CCPG1 encodes a protein that is an effector of endoplasmic reticulum stress response, which drives endoplasmic reticulum autophagy and endoplasmic reticulum remodeling. In some embodiments, CCPG1 may be a human (Gene ID: 9236), nonhuman primate (e.g., Gene ID: 467688, Gene ID: 698918), or rodent gene (e.g., Gene ID: 72278, Gene ID: 363098). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001204450.2, NM_001204451.2, NM_004748.6, and NM_020739.5) have been characterized that encode different protein isoforms.

[000134] KIF13A: As used herein, the term “KIF13A” (also known as RBKIN, bA500Cl 1.2, and kinesin family member 13 A) encodes a member of the kinesin family of microtubule-based motor proteins that have a role in the positioning of endosomes. KIF13A mediates the transport of mannose-6-phosphate receptor (M6PR) to the plasma membrane, via interaction with the AP-1 complex. In some embodiments, KIF13A may be a human (Gene ID: 63971), non-human primate (e.g., Gene ID: 471862, Gene ID: 704126), or rodent gene (e.g., Gene ID: 16553, Gene ID: 308173). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001105566.3, NM_001105567.3, NM_001105568.4, NM_001243423.2, and NM_022113.6) have been characterized that encode different protein isoforms.

[000135] INSR: As used herein, the term “INSR” (also known as insulin receptor, HHF5, and CD2220) encodes an insulin receptor, a receptor of the tyrosine kinase family of proteins. During transcription alternate splicing of exon 11 results in either an insulin receptor- A (IR-A) or insulin receptor-B (IR-B) isoform. Translation of IR-A results in exon 11 exclusion, while translation of IR-B results in the inclusion of exon 11. Muscleblind-like 1 (MBNL1), which is upregulated by insulin, promotes ISNR exon 11 inclusion. Posttranslation these isoforms result in either heterodimerization or homodimerization producing an insulin receptor. The insulin receptor plays a key role in glucose homeostasis and fat metabolism. Activation of the insulin receptor by a ligand (e.g., insulin) activates the insulin signaling pathway, which regulates glucose update and release, as well as synthesis and storage of carbohydrates, lipids, and proteins. In some embodiments, INSR may be a human (Gene ID: 3643), non-human primate (e.g., Gene ID: 455649, Gene ID: 706009), or rodent gene (e.g., Gene ID: 16337, Gene ID: 24954). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_000208.4 and NM_001079817.3) have been characterized that encode different protein isoforms.

[000136] CAMK2B: As used herein, the term “CAMK2B” (also known as calcium/calmodulin dependent protein kinase II beta, CAM2, CAMK2, CAMKB, MRD54, and CaMKIip) encodes an enzyme that belongs to the serine/threonine protein kinase family, and to the calcium/calmodulin-dependent protein kinase subfamily. The CAMK2B gene encodes the beta subunit of the enzyme, which comprises an alpha, beta, gamma, and delta subunit. Multiple transcript variants of the gene are produced by alterative splicing. In some embodiments, CAMK2B may be a human (Gene ID: 816), non-human primate (e.g., Gene ID: 738167, Gene ID: 706977), or rodent gene (e.g., Gene ID: 12323, Gene ID: 24245). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001220.5, NM_001293170.2, NM_001293170.2, NM_172079.3, NM_172080.3, NM_172081.3, NM_172082.3, NM_172083.3, and NM_172084.3) have been characterized that encode different protein isoforms.

[000137] OPA1: As used herein, the term “OPA1” (also known as OPA1 mitochondrial dynamin like GTPase, mitochondrial dynamin like GTPase, NPG, NTG, MGM1, BERHS, largeG, MTDPS14, Optic atrophy 1, and BERHS) encodes a nuclear-encoded mitochondrial protein with similarity to dynamin-related GTPases. OPA1 is necessary for normal mitochondrial morphology and energy output, regulating mitochondrial fusion and cristae structure. In some embodiments, OPA1 may be a human (Gene ID: 4976), non-human primate (e.g., Gene ID: 471042, Gene ID: 698540), or rodent gene (e.g., Gene ID: 171116, Gene ID: 74143). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001354663.2, NM_001354664.2, NM_015560.3, NM_130831.3, NM_130832.3, NM_130833.3, NM_130834.3, NM_130835.3, NM_130836.3, and NM_130837.3) have been characterized that encode different protein isoforms.

[000138] RYR1: As used herein, the term “RYR1” (also known as CCO, MHS, MHS1, PPP1R137, RYDR, RYR, RYR-1, SKRR, ryanodine receptor 1, KDS, CMYP1A, CMYP1B, and PPP1R137) encodes a skeletal muscle receptor. The protein functions as a channel that releases calcium in the sarcoplasmic reticulum and as a connection between the sarcoplasmic reticulum and transverse tubule. RYR1 has been shown to interact with calmodulin, FKBP1A, HOMER1, HOMER2, HOMER3, and TRDN. In some embodiments, RYR1 may be a human (Gene ID: 6261), non-human primate (e.g., Gene ID: 100612063, Gene ID: 693612), or rodent gene (e.g., Gene ID: 20190, Gene ID: 114207). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_000540.3 and NM_001042723.2) have been characterized that encode different protein isoforms.

[000139] MBNL2: As used herein, the term “MBNL2” (also known as MBLL, MBLL39, PRO2032, and muscleblind like splicing regulator 2) encodes a CSH-type zinc finger protein. MBLN2 modulates alternative splicing of pre-mRNAs, such as inhibiting cardiac troponin-T (TNNT2) pre-mRNA exon inclusion and induction of insulin receptor pre- mRNA exon inclusion in muscle. In some embodiments, MBNL2 may be a human (Gene ID: 10150), non-human primate (e.g., Gene ID: 467304, Gene ID: 697097), or rodent gene (e.g., Gene ID: 105559, Gene ID: 680445). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001306070.2,

NM_001382649.1, NM_001382650.1, NM_001382651.1, NM_001382652.1,

NM_001382653.1, NM_001382654.1, NM_001382656.1, NM_001382660.1, NM_001382661.1, NM_001382663.1, NM_001382666.1, NM_001382667.1, NM_001382668.1, NM_001382669.1, NM_001382670.1, NM_001382671.1, NM_001382672.1, NM_001382673.1, NM_001382674.1, NM_001382675.1, NM_001382676.1, NM_001382677.1, NM_001382678.1, NM_001382679.1, NM_001382680.1, NM_001382681.1, NM_001382682.1, NM_001382683.1, NM_001382684.1, NM_001382685.1, NM_001382686.1, NM_001382687.1, NM_001382688.1, NM_001382689.1, NM_001382690.1, NM_001382691.1, NM_001382692.1, NM_001382693.1, NM_001382694.1, NM_001382695.1, NM_001382696.1, NM_001382697.1, NM_144778.4, and NM_207304.3) have been characterized that encode different protein isoforms.

[000140] NFIX: As used herein, the term “NFIX” (also known as MRSHSS, NF1A, SOTOS2, nuclear factor I X, NF1A, NF1-X, NF-I/X, CTF, and MALNS) encodes a transcript factor that, in some embodiments, binds the palindromic sequence 5'- TTGGCNNNNNGCCAA-3 (SEQ ID NO: 26) found in viral and cellular promoters. NFIX also interacts with myostatin and regulates the progression of muscle regeneration over time. In some embodiments, NFIX is regulated by MBNL1. In some embodiments, NFIX may be a human (Gene ID: 4784), non-human primate (e.g., Gene ID: 468742, Gene ID: 717108), or rodent gene (e.g., Gene ID: 18032, Gene ID: 81524). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001271043.2, NM-001271044.3, NM_001365902.3, NM_001365982.2, NM_001365983.2, NM_001365984.2, NM_001365985.2, NM_001378404.1, NM_OO 1378405.1, and NM_002501.4) have been characterized that encode different protein isoforms.

[000141] CLASP1: As used herein, the term “CLASP1” (also known as MAST1 and cytoplasmic linker associated protein 1) encodes nonmotor microtubule-associated proteins that interact with CAP-GLY domain containing linker proteins (CLIPs). CLASP 1 is ubiquitously expressed and promotes stabilization of dynamic microtubules. In some embodiments, exon inclusion of the CLASP 1 exon 19 is promoted by MBNL1. In some embodiments, CLASP1 may be a human (Gene ID: 23332), non-human primate (e.g., Gene ID: 459583, Gene ID: 696719), or rodent gene (e.g., Gene ID: 76707, Gene ID: 304740). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001142273.2, NM_001142274.2, NM_001207051.2,

NM_001378003.1, NM_001378004.1, NM_001378005.1, NM_001395891.1, and NM_015282.3) have been characterized that encode different protein isoforms.

[000142] VPS39: As used herein, the term “VPS39” (also known as TLP, VAM6, hVam6p, HOPS complex subunit, vacuolar protein sorting 39, and VPS39 subunit of HOPS complex) encodes a protein that promotes late endosome and lysosome clustering and fusion with autophagosomes. Additionally, VPS39 can modulate transforming growth factor-beta response by acting as an adaptor protein. In some embodiments, VPS39 may be a human (Gene ID: 23339), non-human primate (e.g., Gene ID: 453358, Gene ID: 707791), or rodent gene (e.g., Gene ID: 269338, Gene ID: 362199). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001301138.3 and NM_015289.5) have been characterized that encode different protein isoforms.

[000143] BEST3: As used herein, the term “BEST3” (also known as bestrophin 3, Vitelliform Macular Dystrophy 2-Like Protein 3, Vitelliform Macular Dystrophy 2-Like 3, VMD2L3, MGC40411, and MGC13168) encodes transmembrane protein that belongs to the bestrophin family of anion channels. The BEST3 anion channel is a calcium sensitive channel. Alternative splicing of BEST3 mRNA can cause the BEST3 protein to lose the ability to act as an ion channel, while another splice variant of BEST3 controls intracellular calcium release. Additionally, BEST3 has been found to have anti- apop to tic functions, the ability to reduce TNF induced inflammation, and be protective after endoplasmic reticulum stress. In some embodiments, BEST3 may be a human (Gene ID: 144453), non-human primate (e.g., Gene ID: 467066, Gene ID: 718450), or rodent gene (e.g., Gene ID: 382427, Gene ID: 314847). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001282613.2, NM_001282614.2, NM_001282615.2, NM_001282616.2, NM_032735.3, and NM_152439.4) have been characterized that encode different protein isoforms.

[000144] SOS1: As used herein, the term “SOS1” (also known as GF1, GGF1, GINGF, HGF, NS4, SOS Ras/Rac guanine nucleotide exchange factor 1, SOS-1, and Son of Sevenless Homolog 1) encodes a guanine nucleotide exchange factor (GEF) which regulates the activation of the RAS/MAPK signaling pathway. This pathway regulates cell growth, cell proliferation, cell differentiation, cell migration, and apoptosis. SOS1 protein interacts with RAS proteins phosphorylating GDP into GTP, activating cell proliferation. In some embodiments, SOS1 may be a human (Gene ID: 6654), non-human primate (e.g., Gene ID: 459171, Gene ID: 713777), or rodent gene (e.g., Gene ID: 20662, Gene ID: 313845). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001382394.1, NM_001382395.1, and NM_005633.4) have been characterized that encode different protein isoforms.

[000145] CLCN1: As used herein, the term “CLCN1” (also known as CLC1, and chloride voltage-gated channel 1) encodes a voltage-dependent chloride channel. The CLCN family comprises members; CNCL1-7, Ka, and Kb. Normal function of skeletal muscle cells relies on CLCN1, as the CLCN1 ion channel functions to stabilize the electrical charge of the cell, allowing normal muscle contractions. Alternative splicing of CLCN1 is regulated in part by MBNL1 and CELF. Aberrant splicing of CLCN1 can result in myotonia in myotonic dystrophy (DM). In some embodiments, CLCN1 may be a human (Gene ID: 1180), non- human primate (e.g., Gene ID: 703944, Gene ID: 472560), or rodent gene (e.g., Gene ID: 12723, Gene ID: 25688). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_000083.3 and NR_046453.2) have been characterized that encode different protein isoforms.

[000146] MBNL1: As used herein, the term “MBNL1” (also known as EXP, EXP35, EXP40, EXP42, MBNL, and muscleblind like splicing regulator 1) encodes a member of the muscleblind protein family. MBNL1 regulates RNA alternative splicing, localization, and integrity. MBNL1 promotes the maturation of skeletal and cardiac muscle controlling the alternative splicing of key exons. Additionally, MBNL1 regulates the fetal to adult transition of the heart, muscle, and brain. The main pathogenesis of myotonic dystrophy type 1 (DM1) is caused by the sequestration of MBNL1, which is bound to expanded poly-CUG repeats in DMKP mRNA. In some embodiments, MBNL1 may be a human (Gene ID: 4154), non- human primate (e.g., Gene ID: 460787, Gene ID: 708735), or rodent gene (e.g., Gene ID: 56758, Gene ID: 282635). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001314057.2, NM_001363870.1, NM_001376818.1, NM_001376819.1, NM_001376820.1, NM_001376821.1, NM_001376822.1, NM_001376823.1, NM_001376824.1, NM_001376825.1, NM_001376826.1, NM_001376827.1, NM_001376828.1, NM_001376829.1, NM_001376830.1, NM_001376831.1, NM_001376832.1, NM_001376833.1, NM_001376834.1, NM_001376835.1, NM_001376836.1, NM_001376837.1, NM_001376838.1, NM_001376839.1, NM_001376840.1, NM_001376841.1, NM_001376842.1, NM_001376843.1, NM_001376844.1, NM_001376845.1, NM_001376846.1, NM_001376847.1, NM_001376848.1, NM_001376849.1, NM_001376851.1, NM_001376853.1, NM_001387781.1, NM_001387782.1, NM_001387783.1, NM_001387784.1, NM_001387785.1, NM_001387786.1, NM_001387787.1, NM_001387788.1, NM_001387789.1, NM_001387790.1, NM_001387791.1, NM_001387792.1, NM_001387793.1, NM_001387794.1, NM_001387795.1, NM_001387796.1, NM_001387797.1, NM_001387798.1, NM_001387799.1, NM_001387800.1, NM_001387801.1, NM_001387802.1, NM_001387803.1, NM_001387804.1, NM_001387805.1, NM_001387806.1, NM_001387807.1, NM_001387808.1, NM_001387809.1, NM_001387810.1, NM_001387811.1, NM_001387812.1, NM_001387813.1, NM_001387814.1, NM_001387815.1, NM_001387816.1, NM_001387817.1, NM_001387818.1, NM_001387819.1, NM_001387820.1, NM_001387821.1, NM_001387822.1, NM_001387823.1, NM_001387824.1, NM_001387825.1, NM_001387826.1, NM_001387827.1, NM_001387828.1_i NM_001387829.1, NM_001387830.1, NM_001387831.1, NM_001387832.1, NM_001387833.1, NM_001387834.1, NM_021038.5, NM_207292.3, NM_207293.2, NM_207294.2, NM_207295.2, NM_207296.2, and NM_207297.2) have been characterized that encode different protein isoforms.

[000147] CACNA1S: As used herein, the term “CACNA1S” (also known as Cavl.l, ACNL1A3, CCHL1A3HOKPP, HOKPP1, MHS5, TTPP1, hypoPP, calcium voltage-gated channel subunit alpha 1 S, dihydropyridine receptor, DHPR, DHPRM, CMYP18, and CACNL1A3) encodes a subunit of a slowly inactivating L-type voltage dependent calcium channel. This channel is located in skeletal muscle cells, and have a role in excitationcontraction coupling, in which electrical signals trigger muscle contraction or tensing.

CACNA1S protein interact with ryanodine receptor 1 (RYR1), activating the RYR1 channel, releasing calcium ions from the cell. In some embodiments, alternate splicing of CACNA1S is controlled by MBNL1. Skipping of CACNA1S exon 29 has been linked facioscapulohumeral muscular dystrophy (FSHD) and DM1. Symptoms of CACNA1S exon 29 skipping can be muscle contraction impairment and muscle weakness. In some embodiments, CACNA1S may be a human (Gene ID: 779), non-human primate (e.g., Gene ID: 469635, Gene ID: 708706), or rodent gene (e.g., Gene ID: 12292, Gene ID: 682930). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_000069.3 and XM_005245478.4) have been characterized that encode different protein isoforms.

[000148] CAPZB: As used herein, the term “CAPZB” (also known as CAPB, CapZp, CAPPB, CAPZ, capping actin protein of muscle Z-line beta subunit, F-actin-capping protein subunit beta, and capping actin protein of muscle Z-line subunit beta) encodes the beta subunit of the barbed-end actin binding protein, a member of the F-actin capping protein family. CAPZB regulates actin filament growth, blocking actin filament assembly and disassembly at the barbed end. CAPZB further functions in regulating and stabilizing actin filaments in cells. In some embodiments, CAPNZB may be a human (Gene ID: 832), non-human primate (e.g., Gene ID: 100612860, Gene ID: 100429776), or rodent gene (e.g., Gene ID: 12345, Gene ID: 298584). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001206540.3, NM_001206541.3, NM_001282162.2, NM_001313932.2, and NM_004930.5) have been characterized that encode different protein isoforms.

[000149] GOLGA4: As used herein, the term “GOLGA4” (also known as CRPF46, GCP2, GOLG, MU-RMS-40.18, golgin A4, Trans-Golgil p230, Golgin 245, p230, Golgin subfamily A member 4, and golgin A4) encodes a one of the six Golgi matrix proteins. GOLGA4 is a member of the golgin subfamily A, which plays a role in Rab6-regulated membrane tethering in the Golgi apparatus. Multiple isoforms of this gene have been identified, encoded by alternatively spliced transcript variants. In some embodiments, GOLGA4 may be a human (Gene ID: 2803), non-human primate (e.g., Gene ID: 460264, Gene ID: 697455), or rodent gene (e.g., Gene ID: 54214, Gene ID: 501069). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001172713.2, NM_001410721.1, and NM_002078.5) have been characterized that encode different protein isoforms.

[000150] GFPT1: As used herein, the term “GFPT1” (also known as CMSTA1, GFA, GFAT, GFAT 1, GFAT1, GFATlm, GFPT, GFPT1L, MSLG, CMS12, and glutamine- fructose-6-phosphate transaminase 1) encodes an enzyme that controls the flow of glucose into the hexosamine pathway. GFPT further catalyzes the formation of glucosamine 6- phosphate. In the hexosamine pathway the GFPT1 enzyme is the first rate limiting enzyme. GFPT1 is a homodimeric cytoplasmic enzyme. In DM1 patients GFPT1 is incorrectly spliced into skeletal muscle. In some embodiments, GFPT1 may be a human (Gene ID: 2673), nonhuman primate (e.g., Gene ID: 450203, Gene ID: 574376), or rodent gene (e.g., Gene ID: 14583, Gene ID: 297417). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001244710.2 and NM_002056.4) have been characterized that encode different protein isoforms.

[000151] ATP2A1: As used herein, the term “ATP2A1” (also known as ATP2A, SERCA1, and ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1) encodes a sarco(endo)plasmic reticulum calcium- ATPase 1, an intracellular pump found in the sarcoplasmic or endoplasmic reticula of muscle cells. ATP2A1 helps control the level of positively charged calcium ions inside a cell, which plays a role in muscle contraction and relaxation. When calcium ions are transported into the sarcoplasmic reticulum muscles relax, and when calcium ions are released from the sarcoplasmic reticulum, muscles contract. There are three transcript variants of ATP2A1, the result of alternative splicing. Mis-spliced ATP2A1 has been associated with DM1, and downregulation of ATP2A1 intracellular pumps have been found in amyotrophic lateral sclerosis (ALS). In some embodiments, MBNL1 has been found to regulate alternative splicing of ATP2A1. In some embodiments, ATP2A1 may be a human (Gene ID: 487), non-human primate (e.g., Gene ID: 454019, Gene ID: 708058), or rodent gene (e.g., Gene ID: 11937, Gene ID: 116601). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001286075.2, NM_004320.6, and NM_173201.5) have been characterized that encode different protein isoforms.

[000152] ANK2: As used herein, the term “ANK2” (also known as ANK-2, LQT4, brank-2, ankyrin 2, neuronal, ankyrin 2, AP87, and CFAP87) encodes a member of the ankyrin family of proteins, Ankyrin-B (also known as Ankyrin 2). ANK2 protein, in some embodiments, comprises four domains; a membrane binding domain, a spectrin binding domain, a death domain, and a C-terminal domain. ANK2 plays a role in the localization and membrane stabilization of ion channels on the cell, to allow for cells to properly control the flow of ions. ANK2 variants that have a loss-of-function are associates with different heart diseases, such as long QT syndrome, type 4. In some embodiments, ANK2 may be a human (Gene ID: 287), non-human primate (e.g., Gene ID: 461444, Gene ID: 702180), or rodent gene (e.g., Gene ID: 109676, Gene ID: 362036). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001127493.3, NM_001148.6, NM_001354225.2, NM_001354228.2, NM_001354230.2, NM_001354231.2, NM_001354232.2, NM_001354235.2, NM_001354236.2, NM_001354237.2, NM_001354239.2, NM_001354240.2, NM_001354241.2, NM_001354242.2, NM_001354243.2, NM_001354244.2, NM_001354245.2, NM_001354246.2, NM_001354249.2, NM_001354252.2, NM_001354253.2, NM_001354254.2, NM_001354255.2, NM_001354256.2, NM_001354257.2, NM_001354258.2, NM_001354260.2, NM_001354261.2, NM_001354262.2, NM_001354264.2, NM_001354265.2, NM_001354266.2, NM_001354267.2, NM_001354268.2, NM_001354269.3, NM_001354270.2, NM_001354271.2, NM_001354272.2, NM_001354273.2, NM_001354274.2, NM_001354275.2, NM_001354276.2, NM_001354277.2, NM_001354278.2, NM_001354279.2, NM_001354280.2, NM_001354281.2, NM_001354282.2, NM_001386142.1, NM_001386143.1, NM_001386143.1, NM_001386146.1, NM_001386147.1, NM_001386148.2, NM_001386149.1, NM_001386150.1, NM_001386151.1, NM_001386152.1, NM_001386153.1, NM_001386154.1, NM_001386156.1, NM_001386157.1, NM_001386158.1, NM_001386160.1, NM_001386161.1, NM_001386162.1, NM_001386166.1, NM_001386167.1, NM_001386174.1, NM_001386175.1, NM_001386186.2, NM_001386187.2, and NM_020977.5) have been characterized that encode different protein isoforms.

[000153] DMD: As used herein, the term “DMD” (also known as BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85, and dystrophin) encodes dystrophin a rod-shaped cytoplasmic protein. DMD is the largest known human gene, spanning approximately 0.1% of the entire genome, containing 79 exons. The dystrophin protein is found primarily in muscle cells, such as skeletal and cardiac muscle cells. Dystrophin connects the cytoskeleton of the cell to the surrounding extracellular matrix, strengthening muscle fibers, aiding in movement, and helping prevent muscle fiber injury. Mutations in the DMD gene can cause different types of muscular dystrophy, the most common being Duchenne muscular dystrophy. In some embodiments, DMD may be a human (Gene ID: 1756), non-human primate (e.g., Gene ID: 465559, Gene ID: 708073), or rodent gene (e.g., Gene ID: 13405, Gene ID: 24907). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_000109.4, NM_004006.3, NM_004009.3, NM_004010.3, NM_004011.4, NM_004012.4, NM_004013.3, NM_004014.3, NM_004015.3, NM_004016.3, NM_004017.3, NM_004018.3, NM_004019.3, NM_004020.4, NM_004021.3, NM_004022.3, and NM_004023.3) have been characterized that encode different protein isoforms.

[000154] BINI: As used herein, the term “BINI” (also known as AMPH2, AMPHL, SH3P9, bridging integrator 1, Myc box-dependent-interacting protein 1, Amphiphy sin-2, and CNM2) encodes multiple isoforms of a nucleocytoplasmic adaptor protein. BINI was originally discovered as a MYC-interacting protein and tumor suppressor but is now known to be a key regulator of endocytosis, membrane recycling, cytoskeleton regulation, DNA repair, cell cycle progression and apoptosis. Ubiquitously expressed isoforms and isoforms expressed in muscle cells activate a caspase-independent apoptotic process and localize in both the cytoplasm and nucleus. Isoforms expressed in the muscle are further involved in the formation of transverse tubules (T-tubules). In some embodiments, isoforms expressed in the central nervous system (CNS) interact with clatherin, dynamin, amphiphysin 1, synaptojanin, and endophilin. In some embodiments, BINI in the CNS also regulates synaptic vesicle endocytosis and cytoskeletal dynamics, functioning as an adapter protein. Mutations in BINI have been associated with skeletal myopathies causing muscle weakness, myotonic dystrophy, myotonia, cataracts, heat condition defects, and Alzheimer’s disease. In some embodiments, BINI may be a human (Gene ID: 274), non-human primate (e.g., Gene ID: 735972, Gene ID: 713587), or rodent gene (e.g., Gene ID: 30948, Gene ID: 117028). In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001320632.2, NM_001320633.2, NM_001320634.1, NM_001320640.2,

NM_001320641.2, NM_001320642.1, NM_004305.4, NM_139343.3, NM_139344.3, NM_139345.3, NM_139346.3, NM_139347.3, NM_139348.3, NM_139349.3, NM_139350.3, and NM_139351.3) have been characterized that encode different protein isoforms.

[000155] Program or Software: As used herein, the terms “program” or “software” refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of the present disclosure as discussed above. Additionally, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present disclosure.

[000156] Database: As used herein, the term “database” generally refers to a collection of data arranged for ease and speed of search and retrieval. Further, a database typically comprises logical and physical data structures. Those skilled in the art will recognize the methods described herein may be used with any type of database including a relational database, an object-relational database and an XML-based database, where XML stands for "eXtensible-Markup-Language". For example, gene or splicing activity information or patient information or other information may be stored in and retrieved from a database. In some cases, gene or splicing information may be stored in or indexed in a manner that relates the gene or splicing information with a variety of other relevant information (e.g., information relevant for creating a report or document that aids a physician in establishing treatment protocols and/or making diagnostic determinations, or information that aids in tracking patient samples). Such relevant information may include, for example, patient identification information, ordering physician identification information, information regarding an ordering physician’s office (e.g., address, telephone number), information regarding the origin of a biological sample (e.g., tissue type, date of sampling), biological sample processing information, sample quality control information, biological sample storage information, gene annotation information, disease state information, splicing activity information, payment information, order date information, etc.

II. Disease State-associated Genes and Methods of Use

[000157] The present disclosure, in some aspects, recognizes and demonstrates that, the repeat expansion disease (e.g., DM1) state spans, e.g., from mild to moderate to severe, and as more splicing regulators such as MBNL1 become sequestered, alternative splicing of certain genes are affected earlier than other genes. The present disclosure further identifies different sets of genes as sets of disease state-associated genes whose alternative splicing begin to be affected, collectively, at different disease state, e.g., when the disease is on a continuum from mild to moderate to severe. Composite measures of splicing activities of one or more such defined sets of disease state-associated genes can be used, e.g., as a more accurate and more sensitive indicator of disease severity and/or treatment efficacy.

[000158] Accordingly, the present disclosure, in some aspects, provides one or more genes and sets of genes that are associated with a disease state of a repeat expansion disease (e.g., a repeat expansion disease associated with spliceopathy, such as DM1). The splicing activity of the different sets of disease state-associated genes described herein can be used to indicate the relative disease state of a repeat expansion disease (e.g., mild, moderate, or severe) on a continuum of disease states. [000159] The present disclosure provides non-limiting examples of the different sets of disease state-associated genes that are associated with the disease state of a repeat expansion disease (e.g., DM1).

[000160] A nonlimiting example of a set of genes associated with a mild disease state comprises two or more of CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, a set of genes associated with a mild disease state comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1.

[000161] A nonlimiting example of a set of genes associated with a moderate disease state comprises two or more of RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1. In some embodiments, a set of genes associated with a moderate disease state comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1.

[000162] A nonlimiting example of a set of genes associated with a severe disease state comprises two or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, a set of genes associated with a severe disease state comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

[000163] Further provided herein are compositions and methods (e.g., assays) for detecting the splicing events of the one or more disease state associated genes, determining the splicing activity (e.g., an alternative splicing index) of a given disease state associated gene, determining the composite measure of splicing activity (e.g., a composite alternative splicing index) of a set of disease state associated genes.

[000164] In some embodiments, splicing events (e.g., individual splicing events) for any one of the disease state-associated genes can be detected for certain exons known to be affected in these genes. As a non-limiting example, Table 1 provides the genomic regions (corresponding to exon(s)) that may be used for detecting splicing activity of disease state associated genes. In some embodiments, when referring to measuring splicing activity of a disease state-associated gene in the present disclosure, the splicing activity is based on the detected splicing events in Table 1.

Table 1. Genomic regions for detecting splicing events

[000165] The present disclosure further provides methods of using the splicing activity and/or composite splicing activity of the different sets of disease state associated genes for determining the disease state, monitoring disease progression, and/or evaluating treatment efficacy in a subject having or suspected of having a repeat expansion disease (e.g., a repeat expansion disease associated with spliceopathy such as DM1).

Assays

[000166] In some aspects, the present disclosure provides methods (e.g., assay methods) comprising determining the splicing activity of one or more disease state-associated genes. In some aspects, the present disclosure provides methods (e.g., assay methods) comprising determining the composite splicing activity of a set of disease state-associated genes. In some embodiments, a composite measure of splicing activity for a set of disease state-associated genes can be based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state-associated genes. In some embodiments, the one or more RNA transcripts are in a nucleic acid sample obtained from a subject (e.g., a subject having or suspected of having a repeat expansion disease such as DM1, or a control subject). In some embodiments, the composite measure of splicing activity is based on an inclusion or an exclusion of an RNA segment (e.g., an exonic region or an exon) in the one or more RNA transcripts of each gene of the set of disease state-associated genes in the nucleic acid sample. In some embodiments, the composite measure of splicing activity is based on alternative splicing indices (ASIs), e.g., ASIs for individual splicing events of each gene of the set of disease state-associated genes. In some embodiments, the composite measure of splicing activity is a composite alternative splicing index (CASI).

[000167] In some embodiments, a method (e.g., assay method) described herein comprises: a. determining an alternative splicing index (ASI) for each gene of the set of disease state-associated genes based on the detected splicing events of one or more RNA transcripts of the gene detected in a nucleic acid sample obtained from a subject, wherein the splicing events are detected using nucleic acid hybridization assays; and b. determining a composite splicing index (CASI) for the set of disease state- associated genes based on the alternative splicing index determined in step (a) for each gene of the set of disease state-associated genes; wherein the subject has or is suspected of having a repeat expansion disease. [000168] In some embodiments, a method (e.g., assay method) described herein further comprises obtaining the nucleic acid sample from the subject. In some embodiments, a method (e.g., assay method) described herein further comprises subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events. In some embodiments, a method (e.g., assay method) described herein further comprises obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events. [000169] In some embodiments, a method (e.g., assay method) described herein comprises: a. obtaining the nucleic acid sample from a subject has or is suspected of having a repeat expansion disease; b. subjecting the nucleic acid sample to nucleic acid hybridization assays to detect splicing events of one or more RNA transcripts of each gene of the a of disease state-associated genes; c. determining an alternative splicing index (ASI) for each gene of the set of disease state-associated genes based on the detected splicing events of one or more RNA transcripts of the gene detected in a nucleic acid sample obtained from a subject; and d. determining a composite splicing index (CASI) for the set of disease state- associated genes based on the alternative splicing index determined in step (c) for each gene of the set of disease state-associated genes.

[000170] In some embodiments, in a method (e.g., assay method) described herein, the nucleic acid sample is obtained from a sample (e.g., a tissue sample or a blood sample) obtained from the subject. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a muscle biopsy sample.

[000171] In some embodiments, in a method (e.g., assay method) described herein, the repeat expansion disease is a muscle disease or disorder. In some embodiments, in a method (e.g., assay method) described herein, wherein the repeat expansion disease is associated with spliceopathy. In some embodiments, in a method (e.g., assay method) described herein, the muscle disease or disorder is myotonic dystrophy. In some embodiments, in a method (e.g., assay method) described herein, the myotonic dystrophy is myotonic dystrophy type 1 (DM1). [000172] In some embodiments, in a method (e.g., assay method) described herein, the subject is undergoing treatment or has been treated for the repeat expansion disease (e.g., DM1). In some embodiments, in a method (e.g., assay method) described herein, the subject will be treated for the repeat expansion disease (e.g., DM1).

[000173] In some embodiments, in a method (e.g., assay method) described herein, the nucleic acid hybridization assays used to detect splicing events can be any suitable methods known in the art or described herein, e.g., RT-PCR and/or sequencing. For example, the nucleic acid hybridization assays may comprise one or more of PCR based methods such as RT-PCR, RT-qPCR, and microarrays, and sequencing based methods such as Multiplex Alternative Splice sequencing (MASseq), and RNAseq.

[000174] In some embodiments, in a method (e.g., assay method) described herein, any of the ASI and CASI values may be determined as described herein, e.g., using Equation B and Equation C, respectively. In some embodiments, in a method (e.g., assay method) described herein, any CASI value determined is between a value of 0 and 1. In some embodiments, in a method (e.g., assay method) described herein, any ASI value determined is between a value of 0 and 1. In some embodiments, in a method (e.g., assay method) described herein, any CASI value determined is more than 1. In some embodiments, in a method (e.g., assay method) described herein, any ASI value determined is more than 1. [000175] In some embodiments, in a method (e.g., assay method) described herein, the subject has a repeat expansion disease that is at a mild to moderate state on the continuum of disease state from no disease to very severe disease. In some embodiments, the CASI value determined for a set of disease state-associated genes is above a first threshold CASI value. In some embodiments, each gene of the set of disease state-associated genes comprises a sequence exhibiting a lower binding affinity to MBNL1, relative to a set of genes with an CASI of below the first threshold CASI value. In some embodiments, the set of disease state- associated genes comprises any one or more of CCPG1, KIF13A, INSR, CAMK2B, and/or OPA1. In some embodiments, the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, the set of disease state- associated genes comprises any one or more of CCPG1, KIF13A, INSR, CAMK2B, OPA1, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and/or MBNL1. In some embodiments, the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, OPA1, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and MBNL1. In some embodiments, the set of disease state-associated genes comprises any one or more of RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and/or MBNL1. In some embodiments, the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and MBNL1.

[000176] In some embodiments, in a method (e.g., assay method) described herein, the subject has a repeat expansion disease that is at a moderate to severe state on the continuum of disease state from no disease to very severe disease. In some embodiments, the CASI determined for a set of disease state-associated genes is below a second threshold CASI value. In some embodiments, in in a method (e.g., assay method) described herein, each gene of the set of disease state-associated genes comprises a sequence exhibiting a higher binding affinity to MBNL1, relative to a gene with a CASI of above the second threshold CASI value. In some embodiments, the set of disease state-associated genes comprises any one or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, the set of disease state-associated genes comprises any one or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, BINI, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and/or MBNL1. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, BINI, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and MBNL1. In some embodiments, the set of disease state-associated genes comprises any one or more of RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and/or MBNL1. In some embodiments, the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1.

[000177] In some embodiments, in a method (e.g., assay method) described herein, the subject has a repeat expansion disease that is at a severe state on the continuum of disease state from no disease to very severe disease. In some embodiments, the CASI determined for a set of disease state-associated genes is above a third threshold CASI value. In some embodiments, in a method (e.g., assay method) described herein, each gene of the set of disease state-associated genes comprises a sequence exhibiting a higher binding affinity to MBNL1, relative to a gene with a CASI value of below the third threshold CASI value. In some embodiments, the set of disease state-associated genes comprises any one or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI. In some embodiments, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

[000178] In some embodiments, in a method (e.g., assay method) described herein, the subject has DM1, and the disease states described herein correspond to disease states indicated by functional measures (e.g., measures of muscle integrity and/or strength). Tests used clinically for determining the severity of DM1 include, without limitation: handgrip strength test, hand grip relaxation time, video hand opening time (vHOT), Quantitative Myometry Testing (QMT), 10-meter walk/run test (10-MWRT), stair-ascend/descend test, 5 times sit to stand (5xSTS), 9-hole peg test (9-HPT), ankle dorsiflexion test; 30 foot-go test, 4- step climb test, and test of myotonia.

Method of Evaluating Disease State or Patient Selection

[000179] The present disclosure, in some aspects, provides that the splicing activity of one or more sets of disease state-associated genes described herein can be used to evaluate the disease state of a subject (e.g., a subject having or suspected of having a repeat expansion disease such as DM1). Accordingly, further provided herein are methods (e.g., methods of evaluating a disease state of a subject) comprising determining composite measures of splicing activity for at least two (e.g., two, three, or more) sets of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the sets of disease state-associated genes in a nucleic acid sample obtained from a subject; and evaluating the disease state of the subject based on the composite measures of splicing activity. In some embodiments, a method described herein (e.g., method of evaluating or determining a disease state of a subject who has or is suspected of having a repeat expansion disease) is performed in vitro. In some embodiments, a method described herein (e.g., method of evaluating or determining a disease state of a subject who has or is suspected of having a repeat expansion disease) is performed ex vivo.

[000180] In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) comprises: a. determining an alternative splicing index (ASI) for each gene of the first set of disease state-associated genes and for each gene of the second set of disease state- associated genes based on the detected splicing events for one or more RNA transcripts of the gene detected in a nucleic acid sample obtained from a subject, wherein the splicing events are detected using nucleic acid hybridization assays; and b. determining a first composite splicing index (CASI) for the first set of disease state-associated genes based on the alternative splicing index determined in step (a) for each gene of the first set of disease state-associated genes; c. determining a second CASI for the second set of disease state- associated genes based on the alternative splicing index determined in step (a) for each gene of the second set of disease state-associated genes and d. evaluating the disease state of the subject based on the first and/or the second CASI determined in step (b) and step (c), respectively.

[000181] In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, step (b) and step (c) can be performed in either order (e.g., step (b) before step (c), or step (c) before step (b)) or in parallel.

[000182] In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises obtaining the nucleic acid sample from the subject. In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events. In some embodiments, a method (e.g., methods of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events. [000183] In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein comprises treating the subject for the repeat expansion disease if the subject is determined to be at a targeted disease state (e.g., any disease state of interest on the continuum of disease state from no disease to very severe disease).

[000184] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the nucleic acid sample is obtained from a sample (e.g., a tissue sample or a blood sample) obtained from the subject. In some embodiments, the tissue sample is a muscle biopsy sample. [000185] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the repeat expansion disease is a muscle disease or disorder. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease), the repeat expansion disease is associated with spliceopathy. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease), the muscle disease or disorder is myotonic dystrophy. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease), the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

[000186] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the nucleic acid hybridization assays used to detect splicing events can be any suitable methods known in the art or described herein, e.g., RT-PCR and/or sequencing. For example, the nucleic acid hybridization assays may comprise one or more of PCR based methods such as RT-PCR, RT-qPCR, and microarrays, and sequencing based methods such as Multiplex Alternative Splice sequencing (MASseq), and RNAseq.

[000187] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any of the ASI and CASI values may be determined as described herein, e.g., using Equation B and Equation C, respectively. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any CASI value determined is between a value of 0 and 1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any ASI value determined is between a value of 0 and 1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any CASI value determined is more than 1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any ASI value determined is more than 1.

[000188] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the first set of disease state-associated genes of step (b) comprises any one or more of CCPG1, KIF13A, INSR, CAMK2B, and/or OPA1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the second set of disease state-associated genes of step (c) comprises any one or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI.

[000189] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the first set of disease state-associated genes of step (b) comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the second set of disease state-associated genes of step (c) comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

[000190] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the first CASI determined in step (b) for the first set of disease state-associated genes is below a first threshold CASI value (e.g., a first threshold CASI value as described herein). As such, the subject can be determined to have a repeat expansion disease (e.g., DM1) that is at a relatively mild state on the continuum of disease state from no disease to very severe disease. In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises treating the subject that is determined to have a repeat expansion disease (e.g., DM1) that is at a relatively mild state on the continuum of disease state from no disease to very severe disease.

[000191] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the first CASI determined in step (b) for the first set of disease state-associated genes is above a second threshold CASI value. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject) described herein, the second CASI determined in step (c) for the second set of disease state-associated genes is below the second threshold CASI value (e.g., a second threshold CASI value as described herein). In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the first CASI determined in step (b) for the first set of disease state-associated genes is above a second threshold CASI value, and the second CASI determined in step (c) for the second set of disease state-associated genes is below the second threshold CASI value. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the first CASI determined in step (b) for the first set of disease state-associated genes is larger than the second CASI determined in step (c) for the second set of disease state-associated genes. As such, the subject can be determined to have a repeat expansion disease (e.g., DM1) that is at a moderate state (i.e., the disease has advanced from a relatively mild state to a relatively moderate state on the continuum of disease state from no disease to very severe disease). In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises treating the subject that is determined to have a repeat expansion disease (e.g., DM1) that is at a relatively moderate state on the continuum of disease state from no disease to very severe disease.

[000192] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the second CASI determined in step (c) for the second set of disease state-associated genes is above a third threshold CASI value (e.g., a third threshold CASI value as described herein). As such, the subject can be determined to have a repeat expansion disease (e.g., DM1) that is at a severe state (i.e., the disease has advanced from a relatively moderate state to a relatively severe state on the continuum of disease state from no disease to very severe disease). In some embodiments, a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises treating the subject that is determined to have a repeat expansion disease (e.g., DM1) that is at a relatively severe state on the continuum of disease state from no disease to very severe disease. [000193] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the subject is undergoing treatment or has been treated for the repeat expansion disease (e.g., DM1) prior to the evaluation. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the subject will be treated for the repeat expansion disease (e.g., DM1) after the evaluation.

[000194] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the repeat expansion disease is DM1 and the subject is treated after being evaluated for disease state. In some embodiments, the treatment comprises administering to the subject an agent that treats DM1. In some embodiments, the agent comprises a complex comprising a muscle targeting agent (e.g., an anti-TfRl antibody) covalently linked to a molecular payload (e.g., an oligonucleotide that targets a DMPK RNA).

[000195] In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the repeat expansion disease is DM1. In some embodiments, the subject undergoes treatment with an agent that treats DM1 after being evaluated for disease state. In some embodiments, the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

Monitoring Subject and Treatment Efficacy

[000196] The present disclosure, in some aspects, provides that changes during a period of time (e.g., a period of time when a subject having a repeat expansion disease is undergoing treatment for a repeat expansion disease) in the splicing activity of a set of disease state- associated genes described herein can be used to evaluate the changes in disease state of a subject and/or the efficacy of any treatment a subject is receiving for treating the repeat expansion disease. Accordingly, further provided herein are methods (e.g., methods of monitoring a subject undergoing treatment for a repeat expansion disease) comprising determining, at least two (e.g., 2, 3, 4, 5, or more) times during a period of time, composite measures of splicing activity for a set of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the sets of disease state- associated genes in a nucleic acid sample obtained from a subject; and evaluating the disease state of the subject based on the composite measures of splicing activity. In some embodiments, a period of time described herein is 1-24 (e.g., 1-24, 2-24, 3-24, 4-24, 5-24, 6- 24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 1-18, 1- 12, 1-8, 1-6, 1-5, 1-4, 1-3, 1-2, 2-18, 2-12, 2-8, 2-6, 2-4, 4-18, 4-12, 4-8, 4-6, 6-18, 6-12, 6-8, 8-18, 8-12, or 12-18) months. In some embodiments, a period of time described herein is 1-20 (e.g., 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, 19-20, 2-18, 2-12, 2-10, 2-5, 4-18, 4-12, 4-8, 6-18, 6-12, or 6-8) years. In some embodiments, a period of time described herein is the remainder of the subject’s life. In some embodiments, a period of time described herein occurs during treatment for a repeat expansion disease. In some embodiments, a period of time described herein occurs prior to treatment for a repeat expansion disease. In some embodiments, a period of time described herein occurs after treatment for a repeat expansion disease.

[000197] In some embodiments, a method (e.g., method of monitoring a subject undergoing a treatment for a repeat expansion disease) comprises determining, during a period of time, at least two composite measures of splicing activity for a set of disease state- associated genes based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state-associated genes in a nucleic acid sample obtained from the subject; and determining the disease state of the subject based on the at least two (e.g., 2, 3, 4, 5, or more) composite measures of splicing activity to thereby monitor the subject.

[000198] In some embodiments, a method (e.g., method of monitoring a subject undergoing a treatment for a repeat expansion disease) comprises, over a period of time: a. determining a first composite splicing index (CASI) for a set of disease state- associated genes; b. determining a second composite splicing index (CASI) for the set of disease state- associated genes; and c. comparing the first CASI and the second CASI to thereby monitor the subject. [000199] In some embodiments, a method (e.g., method of evaluating effectiveness of a treatment for a repeat expansion disease in a subject undergoing the treatment for the repeat expansion disease) comprises, over a period of time: a. determining a first composite splicing index (CASI) for a set of disease state- associated genes; b. determining a second composite splicing index (CASI) for the set of disease state- associated genes; and c. comparing the first CASI and the second CASI to thereby determine the effectiveness of the treatment. [000200] In some embodiments, a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein further comprises obtaining the nucleic acid sample from the subject. In some embodiments, a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein further comprises subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events. In some embodiments, a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein further comprises obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events.

[000201] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the first and/or second nucleic acid sample is obtained from a sample (e.g., a tissue sample or a blood sample) obtained from the subject. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a muscle biopsy sample.

[000202] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the repeat expansion disease is a muscle disease or disorder. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the repeat expansion disease is associated with spliceopathy. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the muscle disease or disorder is myotonic dystrophy. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

[000203] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the nucleic acid hybridization assays used to detect splicing events can be any suitable methods known in the art or described herein, e.g., RT-PCR and/or sequencing. For example, the nucleic acid hybridization assays may comprise one or more of PCR based methods such as RT-PCR, RT-qPCR, and microarrays, and sequencing based methods such as Multiplex Alternative Splice sequencing (MASseq), and RNAseq.

[000204] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, any of the ASI and CASI values may be determined as described herein, e.g., using Equation B and Equation C, respectively. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, any CASI value determined is between a value of 0 and 1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any ASI value determined is between a value of 0 and 1. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, any CASI value determined is more than 1. In some embodiments, in a method (e.g., method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, any ASI value determined is more than 1.

[000205] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, to determine the genes used to determine the first CASI and the second CASI, a CASI value for a panel of 22 genes (e.g., CCPG1, KIF13A, INSR, CAMK2B, OPA1, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SCSI, CLCN1, MBNL1, CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI) is determined, which informs the relative disease state the subject is at and which set(s) of disease state associated genes is (are) to be used in a method described herein (e.g., method of monitoring a subject or evaluating effectiveness of a treatment).

[000206] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the first CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI for each gene is determined based on splicing events detected for one or more RNA transcripts for the gene in a first nucleic acid sample obtained from the subject. [000207] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the second CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state- associated genes, wherein the ASI is determined based on splicing events detected for one or more RNA transcripts of the gene in a second nucleic acid sample obtained from the subject.

[000208] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained from the subject after treatment.

[000209] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the first nucleic acid sample is obtained from the subject earlier in time than the second nucleic acid sample during a period (e.g., a period in which the subject is undergoing the treatment). [000210] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the second nucleic acid sample is obtained from the subject after the first nucleic acid sample is obtained from the subject (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks, 76 weeks, 80 weeks, 84 weeks, 88 weeks, 92 weeks, 96 weeks, 100 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months after the first nucleic acid sample is obtained from the subject).

[000211] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the set of disease state-associated genes comprises any one or more of CCPG1, KIF13A, INSR, CAMK2B, and/or OPA1. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, wherein the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, a set of disease state-associated genes comprising any one or more of CCPG1, KIF13A, INSR, CAMK2B, and/or OPA1 is suitable (e.g., more sensitive and more accurate for indicating efficacy) for a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein when the subject has a repeat expansion disease (e.g., DM1) at a relatively mild state on the continuum of disease state from no disease to very severe disease. [000212] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the set of disease state-associated genes comprises any one or more of RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and/or MBNL1. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and MBNL1. In some embodiments, a set of disease state-associated genes comprising any one or more of RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and/or MBNL1 is suitable (e.g., more sensitive and more accurate for indicating efficacy) for a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein when the subject has a repeat expansion disease (e.g., DM1) at a relatively moderate state on the continuum of disease state from no disease to very severe disease.

[000213] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the set of disease state-associated genes comprises any one or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI. In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the set of disease state- associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI. In some embodiments, a set of disease state-associated genes comprising any one or more of CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI is suitable (e.g., more sensitive and more accurate for indicating efficacy) for a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein when the subject has a repeat expansion disease (e.g., DM1) at a relatively severe state on the continuum of disease state from no disease to very severe disease.

[000214] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, if the first CASI is larger than the second CASI, the treatment is effective. In some embodiments, when treatment is determined to be effective, the subject continues to receive treatments (e.g., same treatments). In some embodiments, when treatment is determined to be ineffective, the subject may continue to receive the same treatment, or may be revaluated and/or be treated differently.

[000215] In some embodiments, in a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein, the subject is undergoing treatment for the repeat expansion disease (e.g., DM1). In some embodiments, the subject is undergoing treatment with an agent that treats the repeat-expansion disease. In some embodiments, the repeat expansion disease is DM1. In some embodiments, the treatment comprises administering to the subject an agent that treats DM1. In some embodiments, the agent comprises a complex comprising a muscle targeting agent (e.g., an anti-TfRl antibody) covalently linked to a molecular payload (e.g., an oligonucleotide that targets a DMPK RNA). [000216] Further provided herein are methods of determining a disease state of a subject, comprising determining at least one comparator between splicing events detected for one or more RNA transcripts of each gene of one or more sets of disease state-associated genes in a nucleic acid sample obtained from the subject and determining the disease state of the subject based on the at least one comparator. [000217] Further provided herein are methods of monitoring a subject, comprising determining the disease state of the subject based on at least one comparator between splicing events detected for one or more RNA transcripts of each gene of two or more sets of disease state-associated genes in a nucleic acid sample obtained from the subject; and monitoring the subject based on splicing events detected for one or more RNA transcripts of each gene of a set of disease state-associated genes selected based on the determined disease state.

[000218] Further provided herein are methods of determining a disease state of a subject, the method comprising determining at least one comparator for at least two composite splicing indices (CASIs) for one or more sets of disease state-associated genes, and determining the disease state of the subject based on the at least one comparator.

[000219] Further provided herein are methods of monitoring a subject, comprising determining the disease state of the subject based on at least one comparator for composite splicing indices (CASIs) for two or more sets of disease state-associated genes; and monitoring the subject based on a CASI for a set of disease state-associated genes selected based on the determined disease state.

[000220] Further provided herein are methods (e.g., method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease) comprising:

(al) determining a first composite splicing index (CASI) for a first set of disease state- associated genes,

(a2) determining a second composite splicing index (CASI) for the first set of disease state-associated genes,

(a3) determining a first comparator between the first CASI of step (al) and the second CASI of step (a2),

(bl) determining a first CASI for a second set of disease state-associated genes,

(b2) determining a second CASI for the second set of disease state- associated genes, (b3) determining a second comparator between the second CASI of step (bl) and the second CASI of step (b2),

(cl) determining a first CASI for a third set of disease state-associated genes,

(c2) determining a second CASI for the third set of disease state-associated genes,

(c3) determining a third comparator between the first CASI of step (cl) and the second CASI of step (c2), and

(d) determining the disease state of the subject based on the first comparator, second comparator, and/or third comparator. [000221] In some embodiments, the first set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and/or (e.g., and) OPA1. In some embodiments, the second set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP 1, VPS39, BEST3, S0S1, CLCN1, and/or (e.g., and) MBNL1. In some embodiments, the third set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and/or (e.g., and) BINI. In some embodiments, the disease state of the subject is selected from a mild disease state, a moderate disease state, or a severe disease state. In some embodiments, the disease state of the subject is a mild disease state corresponding to a reference CASI for a combination of the first, second, and third sets of disease state-associated genes having a value in the range of 0 and 0.25. In some embodiments, the disease state of the subject is a moderate disease state corresponding to a reference CASI for a combination of the first, second, and third sets of disease state- associated genes having a value in the range of 0.25 - 0.65. In some embodiments, the disease state of the subject is a severe disease state corresponding to a reference CASI for a combination of the first, second, and third sets of disease state-associated genes having a value in the range of 0.65 - 1.0. In some embodiments, the reference CASI is determined from a sample obtained from a reference subject at a time corresponding to a time at or before which a sample was obtained for determining the first CASIs in steps (al), (bl), and (cl). In some embodiments, the first CASI in (al) and the second CASI (a2) are determined based on a first alternative splicing index (ASI) for each gene of the first set of disease state-associated genes. In some embodiments, the first CASI in (bl) and the second CASI in (b2) are determined based on a second alternative splicing index (ASI) for each gene of the second set of disease state-associated genes. In some embodiments, the first CASI in (cl) and the second CASI in (c2) are determined based on a third alternative splicing index (ASI) for each gene of the third set of disease state-associated genes. In some embodiments, the ASI for each gene in each set of disease state-associated genes in (al), (bl), and (cl) are determined based on detected splicing events for one or more RNA transcripts of the respective genes detected in a first nucleic acid sample obtained from the subject. In some embodiments, the ASI for each gene in each set of disease state-associated genes in (a2), (b2), and (c2) are determined based on detected splicing events for one or more RNA transcripts of the respective genes detected in a second nucleic acid sample obtained from the subject. In some embodiments, the splicing events are detected using nucleic acid hybridization assays. In some embodiments, the nucleic acid hybridization assays comprise RT-PCR and/or sequencing. In some embodiments, the first CASI determined in step (al) and/or second CASI determined in step (a2) is between a value of 0 and 1. In some embodiments, the first CASI determined in step (bl) and/or second CASI determined in step (b2) is between a value of 0 and 1. In some embodiments, the first CASI determined in step (cl) and/or second CASI determined in step (c2) is between a value of 0 and 1. In some embodiments, the first, second, and/or third ASI is between a value of 0 and 1.

[000222] In some embodiments, a method (e.g., method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises obtaining the first nucleic acid sample and second nucleic acid sample from the subject; subjecting the first nucleic acid sample and second nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events; and obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events. The first nucleic acid sample and second nucleic acid sample may independently be obtained from the subject before treatment, during a period in which the subject is undergoing treatment or after treatment. In some embodiments, the second nucleic acid sample is obtained from the subject after the first nucleic acid sample is obtained from the subject (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks, 76 weeks, 80 weeks, 84 weeks, 88 weeks, 92 weeks, 96 weeks, 100 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months after the first nucleic acid sample is obtained from the subject). In some embodiments, the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained during a period in which the subject is undergoing the treatment. In some embodiments, the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained from the subject after treatment. In some embodiments, the first nucleic acid sample is obtained from the subject earlier in time than the second nucleic acid sample during a period in which the subject is undergoing the treatment.

[000223] In some embodiments, in a method (e.g., method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the disease state of the subject is determined to be a mild disease state based on the first comparator, second comparator, and/or third comparator. In some embodiments, the disease state of the subject is determined to be a moderate disease state based on the first comparator, second comparator, and/or third comparator. In some embodiments, the disease state of the subject is determined to be a severe disease state based on the first comparator, second comparator, and/or third comparator.

[000224] In some embodiments, a method (e.g., method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein further comprises treating the subject for the repeat expansion disease if the subject is determined to be at a targeted disease state. In some embodiments, the repeat expansion disease is DM1 and the treating comprises administering to the subject a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA. In some embodiments, the method further comprises monitoring the subject undergoing the treatment according to a method (e.g., method of monitoring a subject or evaluating effectiveness of a treatment) described herein. In some embodiments, if the subject is determined to have a repeat expansion disease that is at a mild state, the set of disease state- associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. In some embodiments, if the subject is determined to have a repeat expansion disease that is at a moderate state, the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, S0S1, CLCN1, and MBNL1. In some embodiments, if the subject is determined to have a repeat expansion disease that is at a severe state, the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

[000225] In some embodiments, in a method (e.g., method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease) described herein, the nucleic acid sample is obtained from a tissue sample obtained from the subject. In some embodiments, the tissue sample is a muscle biopsy sample. In some embodiments, the nucleic acid sample is obtained from a blood sample obtained from the subject. In some embodiments, the repeat expansion disease is a muscle disease or disorder. In some embodiments, the muscle disease or disorder is myotonic dystrophy. In some embodiments, the myotonic dystrophy is myotonic dystrophy type 1 (DM1). In some embodiments, the repeat expansion disease is associated with spliceopathy.

Computer Implemented. Methods

[000226] Aspects of the disclosure provide computer implemented methods for determining, using alternative splicing data, the disease state of a subject having a repeat- associated disease, such as DM1 and others. Methods disclosed herein may be implemented in any of numerous ways. For example, certain embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. Such processors may be implemented as integrated circuits, with one or more processors in an integrated circuit component. Though, a processor may be implemented using circuitry in any suitable format. [000227] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a smart phone or any other suitable portable or fixed electronic device.

[000228] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible format.

[000229] Such computers may be interconnected by one or more networks in any suitable form, including as a local area network or a wide area network, such as an enterprise network or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

[000230] Also, the various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

[000231] In this respect, aspects of the disclosure may be embodied as a computer readable medium (or multiple computer readable media) (e.g., a computer memory, one or more floppy discs, compact discs (CD), optical discs, digital video disks (DVD), magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory, tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the disclosure discussed above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various embodiments of the disclosure.

[000232] In some embodiments, a software program may provide a user with a visual representation presenting information related to a subject or patient’s alternative splicing data (e.g., alternative splicing index, composite alternative splicing index), and predicted or determined disease state using a graphical user interface (GUI). Such a software program may execute in any suitable computing environment including, but not limited to, a cloudcomputing environment, a device co-located with a user (e.g., the user’s laptop, desktop, smartphone, etc.), one or more devices remote from the user (e.g., one or more servers), etc. [000233] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[000234] For example, in some embodiments, the techniques described herein may be implemented in the illustrative environment 500 shown in FIG. 5A. As shown in FIG. 5A, within illustrative environment 500, one or more biological samples of a patient 502 may be provided to a laboratory 504. Laboratory 504 may process the biological sample(s) to obtain alternative splicing data (e.g., inclusion events, exclusion events, alternative splicing indices, etc.) and provide it, via network 508, to at least one database 506 that stores information about patient 502 in association with the alternative splicing data.

[000235] Network 508 may be a wide area network (e.g., the Internet), a local area network (e.g., a corporate Intranet), and/or any other suitable type of network. Any of the devices shown in FIG. 5A may connect to the network 508 using one or more wired links, one or more wireless links, and/or any suitable combination thereof.

[000236] In the illustrated embodiment of FIG. 5A, the at least one patient information database 506 may store alternative splicing data for the patient 502, medical history data for the patient 502 (e.g., muscle function, disease repeat status, etc.), test result data for the patient 502, and/or any other suitable information about the patient 502, as well as similar data for other subjects or patients. Examples of stored test result data for the patient include muscle biopsy test results, muscle function test results (e.g., results of hand grip relaxation time, myotonia as measured by video hand opening time (vHOT), quantitative myometry testing (QMT), lO-meter walk/run test (10-MWRT), stair-ascend/descend test, 5 times sit to stand (5xSTS), and 9-hole peg test (9-HPT) results,), and ambulatory status. The information stored in at least one database 506 may be stored in any suitable format and/or using any suitable data structure(s), as aspects of the technology described herein are not limited in this respect. The at least one database 506 may store data in any suitable way (e.g., one or more databases, one or more files). The at least one database 506 may be a single database or multiple databases.

[000237] As shown in FIG. 5, illustrative environment 500 includes one or more external databases 503, which may store information for subjects or patients other than patient 502.

For example, external databases 503 may store alternative splicing data (of any suitable type) for one or more patients or subjects (e.g., a population of subjects), medical history data for one or more patients or subjects, test result data (e.g., muscle function test results, biopsy results, blood test results) for one or more subjects, demographic and/or biographic information for one or more subjects, and/or any other suitable type of information. In some embodiments, external database(s) 503 may store information available in one or more publicly accessible databases such as the Entrez Molecular Sequence Database, Single Nucleotide Polymorphism Database, or UCSC Genome Browser, one or more databases of clinical trial information, and/or one or more databases maintained by commercial nucleic acid sequencing providers. The external database(s) 503 may store such information in any suitable way using any suitable hardware, as aspects of the technology described herein are not limited in this respect.

[000238] In some embodiments, the at least one database 506 and the external database(s) 503 may be the same database, may be part of the same database system, or may be physically co-located, or physically positioned in separate geographic locations.

[000239] In some embodiments, information stored in patient information database 506 and/or in external database(s) 503 may be used to perform any of the techniques described herein related to determining a patient or subject’s disease status. For example, the information stored in the database(s) 503 and/or 506 may be accessed, via network 508, by software executing on server(s) 507 to perform any one or more of the techniques described herein in connection with FIGs. 6, 7, and 8.

[000240] For example, in some embodiments, server(s) 507 may access information stored in database(s) 503 and/or 506 and use this information to perform process 509, described with reference to FIG. 6, for evaluating or determining the disease state of the subject based on composite measures of splicing activity. In some embodiments, the server(s) 507 may use information stored in database(s) 503 and/or 506 as inputs for a computational or predictive model for evaluating or determining the disease state of the subject based on the composite measures of splicing activity.

[000241] As another example, server(s) 507 may access information stored in database(s) 503 and/or 506 and use this information to perform process 512, described with reference to FIG. 7, for evaluating or determining the disease state of the subject based on a first and/or a second composite alternative splicing index. For example, in some embodiments, the server(s) 507 may use information stored in database(s) 503 and/or 506 as inputs for a computational or predictive model for evaluating or determining the disease state of the subject based on the composite measures of splicing activity.

[000242] As yet another example, server(s) 507 may access information stored in database(s) 503 and/or 506 and use this information to perform process 517, described with reference to FIG. 8, for assigning genes of a selected set of genes of a reference genome (e.g., a human genome) into different groups based on scores such that each group is associated with a disease state.

[000243] In some embodiments, server(s) 507 may include one or multiple computing devices. When server(s) 507 include multiple computing devices, the device(s) may be physically co-located (e.g., in a single room) or distributed across multiple physical locations. In some embodiments, server(s) 507 may be part of a cloud computing infrastructure. In some embodiments, one or more server(s) 507 may be co-located in a facility operated by an entity (e.g., a hospital, research institution) with which an end user (e.g., doctor, researcher) 501 is affiliated. In some embodiments, server(s) 507 have access to private medical data for the patient 502.

[000244] As shown in FIG. 5, in some embodiments, the results of the analysis performed by server(s) 507 may be provided to end user 502 through a computing device 505 (which may be a portable computing device, such as a laptop or smartphone, or a fixed computing device such as a desktop computer). The results may be provided in a written report, an e-mail, a graphical user interface, and/or any other suitable way. It should be appreciated that although in the embodiment of FIG. 5A, the results are typically provided to a doctor, in other embodiments, the results of the analysis may be provided to patient 502 or a caretaker of patient 502, a healthcare provider such as a nurse, or a person or entity involved with a clinical trial or other end user. [000245] In some embodiments, the results may be part of a graphical user interface (GUI) presented to the end user 502 via the computing device 505. In some embodiments, the GUI may be presented to the user as part of a webpage displayed by a web browser executing on the computing device 505. In some embodiments, the GUI may be presented to the user using an application program (different from a web-browser) executing on the computing device 505. For example, in some embodiments, the computing device 505 may be a mobile device (e.g., a smartphone) and the GUI may be presented to the user via an application program (e.g., “an app”) executing on the mobile device.

[000246] FIG. 6 is a flowchart of an illustrative computer-implemented process 509 for evaluating or determining the disease state of a subject based on the composite measures of splicing activity, in accordance with some embodiments of the disclosure. Process 509 may be performed by any suitable computing device(s). For example, it may be performed by a laptop computer, a desktop computer, one or more servers, in a cloud computing environment, or in any other suitable way.

[000247] Process 509 begins at act 510, where composite measures of splicing activity are determined for at least two sets of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of sets of disease state-associated genes in a nucleic acid sample obtained from a subject. Examples of measures of splicing activity include counts of exon inclusion and exclusion events, e.g., one or more splicing locations in an RNA transcript as well as alternative splicing indices as described herein. In some embodiments, the splicing events are determined using a nucleic acid hybridization assay performed on an RNA sample obtained from a subject. In some embodiments, the RNA sample may be obtained from a muscle biopsy sample obtained from a subject.

[000248] Next, process 509 proceeds to act 511, where the disease state of the subject is evaluated or determined based on the composite measures of splicing activity according to methods provided herein. In some embodiments, the disease state is determined to be mild, moderate, or severe disease (e.g., in which the disease is DM1). In some embodiments the disease state is determined to be mild DM1, classic DM1, or congenital DM1.

[000249] FIG. 7 is a flowchart of an illustrative computer-implemented process 512 for evaluating or determining the disease state of a subject. Here the disease state of the subject is evaluated or determined based on one or more composite alternative splicing indices. Process 512 may be performed by any suitable computing device(s). For example, it may be performed by a laptop computer, a desktop computer, one or more servers, in a cloud computing environment, or in any other suitable way. [000250] Process 512 begins at act 513, where an alternative splicing index (ASI) is determined for each gene of a first set of disease state-associated genes and for each gene of a second set of disease state-associated genes based on detected splicing events for one or more RNA transcripts detected in a nucleic acid sample obtained from a subject. Next, in act 514, a first composite splicing index (CASI) is determined for the first set of disease state- associated genes based on the alternative splicing index determined in step 513 for each gene of the first set of disease state-associated genes. Then, in act 515, a second CASI is determined for the second set of disease state-associated genes based on the alternative splicing index determined in 514 for each gene of the second set of disease state-associated genes. Finally, the disease state of the subject is evaluated or determined based on the first and/or the second CASI determined in act 513 and act 514, respectively. In some embodiments, one or more further CASIs are determined for one or more further sets of disease state-associated genes to inform the evaluation or determination of disease state.

[000251] FIG. 8 is a flowchart of an illustrative computer-implemented process 517 for assigning genes of a selected set (e.g., of a reference genome) into different groups based on alternative splicing indices and/or related scores such that each group is associated with a disease state. Process 517 may be performed by any suitable computing device(s). For example, it may be performed by a laptop computer, a desktop computer, one or more servers, in a cloud computing environment, or in any other suitable way.

[000252] Process 517 begins at act 518, where sequence data is obtained of a reference genome, e.g., a human genome. The sequence data may include, among other information, gene sequence information, gene location in the reference genome, transcript information including intron-exon boundaries, transcription start and end sites and other annotation information as well as sequences of transcripts of a gene. Next, in act 519, a set of genes of the reference genome having desired RNA transcript characteristics are selected based on the obtained sequence data. In act 520, alternative splicing indices are obtained across a population of subjects having a repeat expansion disease, in which subjects in the population have different disease states (e.g., different degrees of disease severity or different degrees of clinical severity, e.g., as determined based on clinical and/or genetic presentations) such that a range of disease states are reflected in the population.

[000253] In some embodiments, certain subjects of the population have mild disease. In some embodiments, certain subjects of the population have moderate disease. In some embodiments, certain subjects of the population have severe disease. In some embodiments, certain subjects of the population have mild DM1. In some embodiments, mild DM1 is characterized by or associated with cataracts and mild myotonia. In some embodiments, certain subjects of the population have classic DM1. In some embodiments, classic DM1 is characterized by or associated with muscle weakness and/or wasting, myotonia, cataract, and/or cardiac conduction abnormalities. In some embodiments, a subject’s disease state is associated with a certain grade of a muscular impairment rating scale (MIRS). For example, in some embodiments, a MIRS of grade 1 reflects no muscular impairment. In some embodiments, a MIRS of grade 2 reflects minimal detectable muscular impairment. In some embodiments, a MIRS of grade 3 reflects distal muscular weakness. In some embodiments, a MIRS of grade 4 reflects mild to moderate proximal muscular weakness. In some embodiments, a MIRS of grade 5 reflects severe proximal muscular weakness. Such grading as assigned to a subject can be used to understand disease state.

[000254] Next, in act 521, scores are determined pairwise between each gene in the selected set based on the alternative splicing indices across the population. In some embodiments, the scores are similarity scores. In some embodiments, the scores are k-means scores reflecting the within-cluster (or within-set) sum of squares of alternative splicing indices of genes in the selected set. In act 522, genes of the selected set are assigned into different groups based on scores determined in act 521 such that each group is associated with a disease state. In some embodiments, genes of the selected set are assigned into k clusters or groups (e.g., 2, 3, 4, 5 or more clusters or groups) using k-means clustering based on alternative splicing indices. In some embodiments, each gene is assigned a feature matrix or vector reflecting alternative splicing across the population. In some embodiments, the data is normalized and/or centered. In some embodiments, an appropriate number of clusters, k, are determined, for example, using a data-driven approach such as the elbow or scree-plot method. In some embodiments, an algorithm initializes k cluster centroids and iteratively assigns genes to the cluster with the closest centroid, updating centroids accordingly. In some embodiments, once convergence is reached, the clusters are analyzed to uncover shared splicing patterns and/or disease states, which can provide insights into RNA splicing in relation to disease states. In some embodiments, visualization and statistical tests further elucidate cluster or group characteristics, and validation measures assess the quality of the clustering. In some embodiments, adjustments are made such as exploring different k values or distance metrics (e.g., Euclidean distance, Manhattan distance, cosine similarity, Mahalanobis distance) to establish appropriate groupings of genes associated with disease state. In other embodiments, genes are grouped based on splicing activity, e.g., alternative splicing indices, using other algorithms such as, for example, partial least squares, linear discriminant analysis, quadratic discriminant analysis, neural networks, naive Bayes, C4.5 decision tree, k-nearest neighbor, random forest, and support vector machine.

III. Agents and Methods for Treating Repeat Expansion Diseases

[000255] In some embodiments, a subject of the present disclosure that has a repeat expansion disease (e.g., DM1) is undergoing treatment for the repeat expansion disease (e.g., DM1) with any suitable agents. Nonlimiting examples of repeat expansion disease and treatments have been described, e.g., in Ellerby et al. (Neurotherapeutics. 2019 Oct; 16(4): 924-927), the entire contents of which are incorporated herein by reference.

[000256] In some embodiments, the repeat expansion disease is DM1 and the subject is undergoing a treatment for DM1 with complexes that comprise a targeting agent, e.g., an antibody, covalently linked to a molecular payload. In some embodiments, a complex comprises a muscle-targeting antibody covalently linked to an oligonucleotide. A complex may comprise an antibody that specifically binds a single antigenic site or that binds to at least two antigenic sites that may exist on the same or different antigens. In some embodiments, the molecular payload is an oligonucleotide that targets a coding or non-coding region of a DMPK transcript (e.g., a pre-mRNA or mRNA), such as a 3 ’-untranslated region, intronic region, or exonic region of a DMPK transcript (e.g., a pre-mRNA or mRNA) in cells (e.g., muscle cells or CNS cells).

[000257] In some embodiments, a complex for use in accordance with the present disclosure for treating DM1 comprises a muscle-targeting agent, e.g., an anti-TfRl antibody, covalently linked to a molecular payload, e.g., an antisense oligonucleotide that targets DMPK, such as a nucleic acid comprising a disease-associated repeat, e.g., a DMPK allele. In some embodiments, the oligonucleotide is an antisense oligonucleotide that targets a DMPK RNA to reduce expression or activity of DMPK (e.g., reduce the level of a mutant or wildtype DMPK RNA, or the activity of a DMPK gene product). In some embodiments, a complex comprises a muscle-targeting antibody (e.g., an anti-transferrin receptor 1 (TfRl) antibody) covalently linked to one or more oligonucleotides. In some embodiments, complexes described herein comprise a linker that covalently links an antibody (e.g., an anti- TfRl antibody) described herein to an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration). A linker comprises at least one covalent bond.

[000258] In some embodiments, non-limiting examples of complexes that may be used for treating DM1 in accordance with the present disclosure are provided in US20190298847, US11446387, US20210308272, US20210261680, US20230144436, US20230226212, US20230256112, US20230050911, WO2022026152, and WO2022147209.

[000259] In some embodiments, complexes for use in accordance with the present disclosure for treating DM1 comprise a structure of formula (I): [R^ni-R², in which each R¹ independently comprises a compound comprising an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration) and R² comprises an antibody (e.g., an anti-TfRl antibody), and wherein in each complex nl is independently an integer (e.g., one or greater) representing the number of instances of R¹ in each complex. In some embodiments, each R¹ independently comprises a group comprising an oligonucleotide. In some embodiments, each R¹ independently comprises a group that comprises additional elements in addition to an oligonucleotide. In some embodiments, R² comprises an antibody e.g., an anti-TfRl antibody) comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, each R¹ of a complex is independently covalently linked to a different amino acid residue (e.g., lysine or cysteine) of R².

[000260] In some embodiments, in each complex nl is independently an integer (e.g., one or greater). In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprises a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. In some embodiments, the antibody is a Fab fragment.

[000261] In some embodiments, the value of nl of each or any complex (e.g., any complex in any of the compositions or formulations disclosed herein) is an integer up to the number of amino acid residues in the antibody to which conjugation is desired or targeted (e.g., the number of lysine residues). In some embodiments, in each complex the value of nl is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, in each complex the value of nl is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26. In some embodiments, in each complex the value of nl is independently in the range of 1-27, 1-26, 1-10, 1-5, or 1-3. In some embodiments, the average value of nl of complexes of the composition is in the range of 1 to 5 (e.g., 1-5, 1-4, 1- 3, 3-5, or 1-2). In some embodiments, compositions described herein comprise complexes that comprise a structure of formula (I): [R^ni-R², wherein nl is 0. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1- 5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, 0.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2). In some embodiments, in each complex type nl is independently an integer of one or greater representing the number of instances of R¹ in each complex of the complex type, and in which the different complex types of the composition are characterized by having different nl values (e.g., nl values in the range of 1-27, 1-26, 1-25, 1-20, 1-15, 1- 10, 1-5, or 1-3).

[000262] In some embodiments, compositions are provided that comprise a plurality of different complexes for use in accordance with the present disclosure for treating DM1. In some embodiments, the plurality of different complexes comprise a common targeting agent (e.g., an antibody) and a common oligonucleotide (e.g., an oligonucleotide comprising a 5’-X- Y-Z-3’ configuration, such as a DMPK- targeting oligonucleotide). In such embodiments, different complex types are characterized by having different numbers of oligonucleotides covalently linked to an antibody. For example, in some embodiments, compositions are provided that comprise a plurality of complexes comprising a structure of formula (I): [ R ¹1 _n i - R², in which each R¹ independently comprises a compound comprising an oligonucleotide (e.g., a DMPK-targeting oligonucleotide) and R² comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer representing the number of instances of R¹ in a complex, and in which different complexes of the composition may have different nl values (e.g., nl values in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, in complexes of a composition nl is independently an integer. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1- 5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, 0.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9- 1.3, or 0.9- 1.2). In some embodiments, compositions described herein comprise complexes in which nl is 0.

[000263] In some embodiments, a composition described herein comprises antibody that is not conjugated to an oligonucleotide (e.g., in trace amounts) and antibody conjugated to one or more oligonucleotides. In some embodiments, antibody that is not conjugated to an oligonucleotide may be referred to as a compound comprising a structure of formula (I): [R^ni-R², for which nl is zero. Accordingly, in some embodiments, a composition for administration to a subject in the methods described herein comprises compounds (e.g., complexes) comprising a structure of formula (I): [ R ¹1 _n i -R², for which each R¹ independently comprises a group comprising an oligonucleotide, R² comprises an antibody and nl is independently an integer of zero or greater that reflects the number of instances of R¹ in each compound (e.g., complex). In some embodiments, the fraction of compounds comprising a structure of formula (I): [R^ni-R², in a composition, for which nl is zero, compared with all compounds of that structure in the composition for which nl is one or greater, is less than 10%, less than 5%, less than 1% less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01%. As such, in some embodiments, the average value of nl of complexes in a composition disclosed herein is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, 0.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2). [000264] In some embodiments, each instance of R¹ in a complex is covalently linked to a different amino acid residue of the antibody. In some embodiments, an amino acid to which R¹ is covalently linked comprises an 8-amino group (e.g., lysine, arginine). However, in some embodiments, an amino acid to which R¹ is covalently linked is a cysteine. In some embodiments, R¹ is directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R¹ is indirectly covalently linked to an amino acid of the antibody, e.g., covalently linked to a glycosylation site on the amino acid. In some embodiments, R¹ is not covalently linked to an amino acid residue residing in a CDR region of the antibody.

[000265] In some embodiments, complexes provided herein for use in accordance with the present disclosure for treating DM1 comprise a structure of formula (I): [R^ni-R², in which each instance of R¹ independently comprises a group of the formula (la):

(la), in which R³ comprises an oligonucleotide, e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration; and R¹ is covalently linked e.g., indirectly or directly linked, e.g., directly linked) to R² at attachment point A. In some embodiments, R² comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R² comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R² comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R² comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R² comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. In some embodiments, R³ comprises an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, R³ comprises an oligonucleotide comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an LNA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’ -MOE modified ribonucleoside, oC represents a 5- methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, and * represents a phosphorothioate intemucleoside linkage.

[000266] In some embodiments, complexes provided herein for use in accordance with the present disclosure for treating DM1 comprise a structure of formula (I): [R^ni-R², in which each R¹ comprises a group of the formula (lb):

(lb), wherein +N represents an LNA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’- 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), wherein nl is an integer (e.g., one or greater) representing the number of instances of R¹ in each complex, and each R¹ is covalently linked to R² at attachment point A. In some embodiments, R² comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R² comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R² comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R² comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R² comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.

[000267] In some embodiments, complexes provided herein for use in accordance with the present disclosure for treating DM1 comprise a structure of formula (I): [ R ¹ ] _n i - R², in which each R¹ comprises a group of the formula (Ic):

(Ic), wherein R¹ is covalently linked to R² at attachment point A. In some embodiments, R² comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R² comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R² comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R² comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R² comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.

[000268] In some embodiments, complexes provided herein for use in accordance with the present disclosure for treating DM1 comprise a structure of the formula (Id):

(Id), wherein +N represents an LNA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’- 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R² comprises an antibody comprising a sequence as set forth in Table 2; wherein nl is an integer (e.g., one or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is a lysine. In some embodiments, R² comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ

ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R² comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R² comprises an antibody comprising a heavy chain comprising an amino acid sequence at least

85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R² comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R² comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. [000269] In some embodiments, complexes described herein for use in accordance with the present disclosure for treating DM comprise a structure of formula (A):

(A), wherein y is 0-15 (e.g., 3) and z is 0-15 (e.g., 4). In some embodiments, the amide shown adjacent the antibody (e.g., anti-TfRl antibody) in the structure (A) results from a reaction with an amine of the antibody, such as a lysine epsilon amine. In some embodiments, a complex described herein comprises an anti-TfRl antibody (e.g., an anti-TfRl Fab) covalently linked via a lysine of the antibody to the 5’ end of an oligonucleotide e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration). In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprises a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full- length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.

[000270] In some embodiments, for treating DM1 in accordance with any one of the compositions or methods described herein, complexes provided herein are formulated in a manner suitable for pharmaceutical use. In some embodiments, complexes can be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and/or (e.g., and) uptake, or provides another beneficial property to complexes in the formulation. In some embodiments, provided herein are formulations (e.g., aqueous solutions, lyophilized forms, or frozen forms) comprising complexes together with tris(hydroxymethyl)aminomethane and/or sucrose. Such formulations can be suitably prepared such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient amount of the complexes enter target muscle cells.

[000271] In some embodiments, a formulation of complexes described herein for treating DM1 in any one of the compositions and methods described herein comprises at a concentration of between 1-50 mg of the complex per mL of the formulation. In some embodiments, a formulation described herein comprises complexes at a concentration of 10- 50 mg/ml or 20-35 mg/mL (e.g., 15-45 mg/mL, 20-40 mg/mL, 25-35 mg/mL, 25.5-34.5 mg/mL, 26-34 mg/mL, 27-33 mg/mL, 28-32 mg/mL, 29-31 mg/mL, 29.5-30.5 mg/mL, 10-40 mg/mL, 15-35 mg/mL, 20-30 mg/mL, 21-29 mg/mL, 21.2-28.8 mg/mL, 22-28 mg/mL, 23-27 mg/mL, 24-26 mg/mL, or 24.5-25.5 mg/mL). In some embodiments, a formulation described herein comprises complexes at a concentration of approximately 25 mg/mL (e.g., 25 mg/mL). In some embodiments, a formulation comprises complexes at a concentration of approximately 30 mg/mL (e.g., 30 mg/mL). In some embodiments, the concentration of complexes in a formulation may vary by up to 20% (e.g., +/- up to 20%, +/- up to 15%, +/- up to 10%, or +/- up to 5%) of a set value. For example, in some embodiments, the concentration of complexes in the formulation is 30 mg/mL +/- up to 15% (e.g., 30 +/- 4.5 mg/mL). In some embodiments, the concentration of complexes in the formula is 25 mg/mL +/- up to 15% (e.g., 25 +/- 3.8 mg/mL).

[000272] In some embodiments, any one or a plurality of the complexes described herein is formulated with tris(hydroxymethyl)aminomethane and sucrose in an aqueous solution. In some embodiments, the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration in the range of 5-50 mM (e.g., 5-40 mM, 5-35 mM, 5-30 mM, 10-50 mM, 15-45 mM, 10-40 mM, 20-40 mM, 20-35 mM, 20-30 mM, 21-29 mM, 22-28 mM, 23-27 mM, 24-26 mM). In some embodiments, the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of approximately 25 mM (e.g., 25 mM). In some embodiments, the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%), for example, 7-13 w/v%, 8-15% w/v%, 9-15% w/v%, 9-11% w/v%, 9.5-11% w/v%, or for example, in the range of 9-10 w/v%, 10-11 w/v%, 10-12 VI/N%, or 8-12 VI/N%. In some embodiments, the sucrose is present in the aqueous solution at a concentration in the range of 7-13 VI/N%, 8-12 VI/N%, or 9-11 VI/N%. In some embodiments, the sucrose is present in the aqueous solution at a concentration of approximately 10 VI/N% (e.g., 10 w/v%). In some embodiments, the aqueous solution has a pH in the range of 6.5 to 8.5, for example, 6.5-6.5, 6.7-6.9, 6.9-7.1, 7.1-7.3, 7.2-7.8, 7.3-7.5, 7.4- 7.5, 7.4-7.6, 7.5-7.6; for example, 7.0-8.0, or for example, in the pH range of 7.0-7.3, 7.2-7.8, 7.3-7.5, 7.4-7.6, 7.5-7.6, 7.5-7.7, 7.7-7.' 9, 7.9-8.0, 8.0-8.2, 8.2-8.4, 8.3-8.4, 8.4-8.5, 8.5-8.6, or

7.3-7.7. In some embodiments, the aqueous solution has a pH in the range of 7.0-8.0 (e.g., 7.0-7.8, 7.1-7.8, 7.2-7.8, 7.3-7.7, 7.3-7.5, 7.3-7.6, 7.4-7.6, or 7.4-7.8). In some embodiments, the aqueous solution has a pH of approximately 7.5 (e.g., 7.5). In some embodiments, the aqueous solution has a pH in the range of 7.4-7.7. In some embodiments, the aqueous solution has a pH in the range of 7.4-7.6 (e.g., 7.5, or about 7.5).

[000273] In some embodiments, any one of the formulations described herein is an aqueous solution, wherein tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of approximately 25 mM (e.g., 25 mM), wherein sucrose is present in the aqueous solution at a concentration of approximately 10 VI/N% (e.g., 10 w/v%), and wherein the aqueous solution is at a pH of approximately 7.5 (e.g., 7.5).

[000274] In some embodiments, any one of the formulations described herein is an aqueous solution, wherein tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of approximately 25 mM (e.g., 25 mM), wherein sucrose is present in the aqueous solution at a concentration of approximately 10 VI/N% (e.g., 10 w/v%), wherein complexes are present in the aqueous solution at a concentration of approximately 20-35 mg/ml (e.g., 25 mg/ml or 30 mg/ml) and wherein the aqueous solution is at a pH of approximately 7.5 (e.g., 7.5).

[000275] In some embodiments, any one or a plurality of the complexes described herein is formulated with tris(hydroxymethyl)aminomethane and sucrose in a lyophilized form (e.g., lyophilized powder). In some embodiments, the lyophilized form (e.g., lyophilized powder) is obtained by lyophilization of any one of the aqueous solutions described herein.

[000276] In some embodiments, a lyophilized form is a lyophilized cake. In some embodiments, a lyophilized cake comprises a plurality of complexes provided herein, tris(hydroxymethyl)aminomethane, and sucrose. In some embodiments, a lyophilized cake comprises IxlO'² mg-1.5 mg (e.g., IxlO'² mg-1.5 mg, 4.02xl0'² mg-1.21 mg, 6xl0'² mg-1 mg, 8xl0'² mg-1 mg, 0.1 mg-1 mg, 0.05 mg-0.2 mg, 0.05 mg-0.3 mg, 0.05 mg-0.4 mg, 0.1 mg-0.2 mg, 0.1 mg-0.3 mg, 0.1 mg-0.4 mg, 0.1 mg-0.5 mg, 0.2 mg-0.3 mg, 0.2 mg-0.4 mg, 0.2 mg- 0.4 mg, 0.3 mg-0.4 mg, or 0.3 mg-0.5 mg) tris(hydroxymethyl)aminomethane per g of cake.

In some embodiments, a lyophilized cake comprises 900 mg-1100 mg (e.g., 900 mg-1100 mg, 989 mg-999 mg, 950 mg- 1000 mg, 980 mg- 1000 mg, 980 mg-990 mg, 990 mg- 1000 mg, 995 mg- 1000 mg, 992 mg-998 mg, 994 mg-999 mg) sucrose per g of cake. In some embodiments, a lyophilized cake comprises 0.5 mg- 10 mg (e.g., 0.5 mg- 10 mg, 0.5 mg-1.5 mg, 1 mg-2 mg, 1.5 mg-3 mg, 1 mg-8 mg, 0.5 mg-6 mg, 0.5 mg-5 mg, 1.5 mg-3.5 mg, 1.5 mg-4 mg, 1.5 mg-5 mg, 2 mg-5 mg, 2.5 mg-5 mg, 2 mg-3mg, 3 mg-4 mg, 4 mg-5 mg) of complexes per g of cake. [000277] In some embodiments, a lyophilized cake comprises 0.303 mg tris(hydroxymethyl)aminomethane per g of cake, 998.6 mg sucrose per g of cake, and/or (e.g., and) 0.999 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 997.7 mg sucrose per g of cake, and/or (e.g., and) 1.995 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 997.2 mg sucrose per g of cake, and/or (e.g., and) 2.49 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 996.7 mg sucrose per g of cake, and/or (e.g., and) 2.99 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 995.7 mg sucrose per g of cake, and/or (e.g., and) 3.98 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.301 mg tris(hydroxymethyl)aminomethane per g of cake, 994.7 mg sucrose per g of cake, and/or (e.g., and) 4.97 mg complexes per g of cake.

[000278] In some embodiments, for administration to a subject to treat DM1, a lyophilized cake may be reconstituted, e.g., with water, reconstitution of a lyophilized cake (e.g., with water) results in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM, sucrose at a concentration of 5 VI/N% to

and/or (e.g., and) complexes at a concentration of 10 mg/mL to 50 mg/mL. In some embodiments, reconstitution of 10.053 g of a lyophilized cake with a solvent (e.g., water) to form 1 mL of reconstituted solution results in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM (optionally 25 mM), sucrose at a concentration of 5 VI/N% to 15 VI/N% (optionally 10 w/v%), and/or (e.g., and) complexes at a concentration of 10 mg/mL to 50 mg/mL. In some embodiments, reconstitution of about 10 g of a lyophilized cake with a solvent (e.g., water) to form 1 mL of reconstituted solution results in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM (optionally 25 mM), sucrose at a concentration of 5 VI/N% to 15 VI/N% (optionally 10 w/v%), and/or (e.g., and) complexes at a concentration of 10 mg/mL to 50 mg/mL.

Antibodies

[000279] In some embodiments, complexes described herein for use in accordance with the present disclosure for treating DM1 comprise an antibody that binds human transferrin receptor 1 (TfRl). An example human TfRl amino acid sequence, corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKAN VTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGE DFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVEN QFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAAT VTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQ TKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKL FGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYV VVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFG SVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHP VTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTY KELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRA DIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFL SPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQ GAANALSGDVWDIDNEF (SEQ ID NO: 23).

[000280] Table 2 provides examples of sequences of an anti-TfRl antibody useful in the complexes provided herein.

Table 2. Examples of anti-TfRl antibody sequences

[000281] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 1 (according to the IMGT definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 2 (according to the IMGT definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the IMGT definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 4 (according to the IMGT definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the IMGT definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the IMGT definition system).

[000282] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 7 (according to the Kabat definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 8 (according to the Kabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 9 (according to the Kabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 10 (according to the Kabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 11 (according to the Kabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the Kabat definition system).

[000283] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 12 (according to the Chothia definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 13 (according to the Chothia definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 14 (according to the Chothia definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 15 (according to the Chothia definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the Chothia definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 16 (according to the Chothia definition system).

[000284] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain variable region (VH) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or in addition (e.g., in addition), the anti-TfRl antibody of the present disclosure comprises a light chain variable region (VL) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL comprising the amino acid sequence of SEQ ID NO: 18.

[000285] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or in addition (e.g., in addition), in some embodiments, the anti-TfRl antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL comprising the amino acid sequence of SEQ ID NO: 18.

[000286] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or in addition (e.g., in addition), in some embodiments, the anti-TfRl antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 18.

[000287] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 19.

Alternatively or in addition e.g., in addition), the anti-TfRl antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfRl antibody of the present disclosure is a Fab that comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 19. Alternatively or in addition (e.g., in addition), the anti-TfRl antibody of the present disclosure is a Fab that comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 20.

[000288] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19.

Alternatively or in addition (e.g., in addition), the anti-TfRl antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfRl antibody of the present disclosure is a Fab that comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 19. Alternatively or in addition (e.g., in addition), the anti-TfRl antibody of the present disclosure is a Fab that comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[000289] In some embodiments, the anti-TfRl antibody provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also called pyroglutamate formation (pyro-Glu), may occur in the antibody at N-terminal Glutamate (Glu) and/or Glutamine (Gin) residues during production. As such, it should be appreciated that an antibody specified as having a sequence comprising an N-terminal glutamate or glutamine residue encompasses antibodies that have undergone pyroglutamate formation resulting from a post-translational modification. In some embodiments, pyroglutamate formation occurs in a heavy chain sequence. In some embodiments, pyroglutamate formation occurs in a light chain sequence.

[000290] Other non-limiting examples of anti-TfRl antibodies that may be used as the muscle-targeting agent in the complexes for treating DM1 disclosed herein are provided in, e.g., US20230113823, US20230103793, US20230256112, US20190240346 A, US10913800, US11246941, US9708406, US10508151, each of which is incorporated herein by reference in its entirety.

[000291] Any other appropriate anti-TfRl antibodies known in the art may be used as the muscle-targeting agent in the complexes for treating DM1 disclosed herein. Examples of known anti-transferrin receptor antibodies, including associated references and binding epitopes, are listed in Table 2. In some embodiments, the anti-transferrin receptor antibody comprises the complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrin receptor antibodies provided herein, e.g., anti-transferrin receptor antibodies listed in Table 3. All references cited in Table 2 are hereby incorporated by reference in their entirety. [000292] Table 3 - List of anti-transferrin receptor antibody clones, including associated references and binding epitope information.

Oligonucleotides

[000293] In some embodiments, an oligonucleotide of the complexes described herein is a single stranded oligonucleotide. In some embodiments, the oligonucleotide is useful for targeting DMPK (e.g., for reducing expression or activity of a DMPK RNA, such as the level of a mutant or wild-type DMPK RNA). In some embodiments, an oligonucleotide that is useful for targeting DMPK RNAs. (e.g., for reducing expression or activity of a DMPK RNA, such as the level of a mutant or wild-type DMPK RNA). In some embodiments, the oligonucleotide comprises a region of complementarity to a DMPK RNA. In some embodiments, the oligonucleotide is useful for reducing levels of toxic DMPK having disease- associated repeat expansions, e.g., in a subject having or suspected of having myotonic dystrophy. In some embodiments, the oligonucleotide is designed to direct RNAse H mediated degradation of the target DMPK RNA residing in the nucleus of cells, e.g., muscle cells (e.g., myotubes) or cells of the nervous system (e.g., central nervous system (CNS) cells). In some embodiments, the oligonucleotide is designed to have desirable bioavailability and/or serum-stability properties. In some embodiments, the oligonucleotide is designed to have desirable binding affinity properties. In some embodiments, the oligonucleotide is designed to have desirable toxicity profiles. In some embodiments, the oligonucleotide is designed to have low-complement activation and/or cytokine induction properties.

[000294] In some embodiments, DMPK-targeting oligonucleotides described herein are designed to caused RNase H mediated degradation of DMPK mRNA. It should be appreciated that, in some embodiments, oligonucleotides in one format (e.g., antisense oligonucleotides) may be suitably adapted to another format (e.g., siRNA oligonucleotides) by incorporating functional sequences (e.g., antisense strand sequences) from one format to the other format.

[000295] Examples of oligonucleotides useful for targeting DMPK are provided in US Patent Application Publication US20230144436, published on May 11, 2023, entitled “Muscle targeting complexes and uses thereof for treating myotonic dystrophy '' US Patent Application Publication 20190298847A1, published on October 3, 2019, entitled “Compositions and methods of treating muscle atrophy and myotonic dystrophy'." US Patent Application Publication 20100016215A1, published on January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy, US Patent Application Publication 20130237585A1, published July 19, 2010, Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression', US Patent Application Publication 20150064181A1, published on March 5, 2015, entitled “Antisense Conjugates For Decreasing Expression Of Dmpk"', US Patent Application Publication 20150238627A1, published on August 27, 2015, entitled “Peptide-Einked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy"', US Patent Application Publication 20160304877A1, published on October 20, 2016, entitled “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression," and International Application Publication WO2022147209, published July 7, 2022, entitled “Muscle targeting complexes and uses thereof for treating myotonic dystrophy," the contents of each of which are incorporated herein in their entireties.

[000296] In some embodiments, oligonucleotides may comprise a region of complementarity to a sequence set forth as follows, which is an example human DMPK gene sequence (Gene ID 1760; NM_001081560.2): AGGGGGGCTGGACCAAGGGGTGGGGAGAAGGGGAGGAGGCCTCGGCCGGCCGC AGAGAGAAGTGGCCAGAGAGGCCCAGGGGACAGCCAGGGACAGGCAGACATGC AGCCAGGGCTCCAGGGCCTGGACAGGGGCTGCCAGGCCCTGTGACAGGAGGACC CCGAGCCCCCGGCCCGGGGAGGGGCCATGGTGCTGCCTGTCCAACATGTCAGCCG AGGTGCGGCTGAGGCGGCTCCAGCAGCTGGTGTTGGACCCGGGCTTCCTGGGGCT GGAGCCCCTGCTCGACCTTCTCCTGGGCGTCCACCAGGAGCTGGGCGCCTCCGAA CTGGCCCAGGACAAGTACGTGGCCGACTTCTTGCAGTGGGCGGAGCCCATCGTGG TGAGGCTTAAGGAGGTCCGACTGCAGAGGGACGACTTCGAGATTCTGAAGGTGA TCGGACGCGGGGCGTTCAGCGAGGTAGCGGTAGTGAAGATGAAGCAGACGGGCC AGGTGTATGCCATGAAGATCATGAACAAGTGGGACATGCTGAAGAGGGGCGAGG TGTCGTGCTTCCGTGAGGAGAGGGACGTGTTGGTGAATGGGGACCGGCGGTGGAT CACGCAGCTGCACTTCGCCTTCCAGGATGAGAACTACCTGTACCTGGTCATGGAG TATTACGTGGGCGGGGACCTGCTGACACTGCTGAGCAAGTTTGGGGAGCGGATTC

CGGCCGAGATGGCGCGCTTCTACCTGGCGGAGATTGTCATGGCCATAGACTCGGT

GCACCGGCTTGGCTACGTGCACAGGGACATCAAACCCGACAACATCCTGCTGGAC

CGCTGTGGCCACATCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTGCGGGCAG

ATGGAACGGTGCGGTCGCTGGTGGCTGTGGGCACCCCAGACTACCTGTCCCCCGA

GATCCTGCAGGCTGTGGGCGGTGGGCCTGGGACAGGCAGCTACGGGCCCGAGTG

TGACTGGTGGGCGCTGGGTGTATTCGCCTATGAAATGTTCTATGGGCAGACGCCC

TTCTACGCGGATTCCACGGCGGAGACCTATGGCAAGATCGTCCACTACAAGGAGC

ACCTCTCTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGGAGGCTCGAGACTTCAT

TCAGCGGTTGCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTGGAGCAGGCGA

CTTCCGGACACATCCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGGACAGCG

TGCCCCCCTTTACACCGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACTT

GGTGGAGGACGGGCTCACTGCCATGGAGACACTGTCGGACATTCGGGAAGGTGC

GCCGCTAGGGGTCCACCTGCCTTTTGTGGGCTACTCCTACTCCTGCATGGCCCTCA

GGGACAGTGAGGTCCCAGGCCCCACACCCATGGAACTGGAGGCCGAGCAGCTGC

TTGAGCCACACGTGCAAGCGCCCAGCCTGGAGCCCTCGGTGTCCCCACAGGATGA

AACAGCTGAAGTGGCAGTTCCAGCGGCTGTCCCTGCGGCAGAGGCTGAGGCCGA

GGTGACGCTGCGGGAGCTCCAGGAAGCCCTGGAGGAGGAGGTGCTCACCCGGCA

GAGCCTGAGCCGGGAGATGGAGGCCATCCGCACGGACAACCAGAACTTCGCCAG

TCAACTACGCGAGGCAGAGGCTCGGAACCGGGACCTAGAGGCACACGTCCGGCA

GTTGCAGGAGCGGATGGAGTTGCTGCAGGCAGAGGGAGCCACAGCTGTCACGGG

GGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATCTAGATGGCCCCCCGGCC

GTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGCCACC

TGCTGCTCCCTGCCAGGGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTC

CTGTTCGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGC

CCACGCCGGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCGCTCCC

TGAACCCTAGAACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGG

GGCACGGCACAGAAGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGT

GGGTCTCCGCCCAGCTCCAGTCCTGTGATCCGGGCCCGCCCCCTAGCGGCCGGGG

AGGGAGGGGCCGGGTCCGCGGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCC

GGGAATGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGC

TGCTGCTGCTGGGGGGATCACAGACCATTTCTTTCTTTCGGCCAGGCTGAGGCCCT

GACGTGGATGGGCAAACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGTCCGTG

TTCCATCCTCCACGCACCCCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTTCTT GTGCATGACGCCCTGCTCTGGGGAGCGTCTGGCGCGATCTCTGCCTGCTTACTCG GGAAATTTGCTTTTGCCAAACCCGCTTTTTCGGGGATCCCGCGCCCCCCTCCTCAC TTGCGCTGCTCTCGGAGCCCCAGCCGGCTCCGCCCGCTTCGGCGGTTTGGATATTT

ATTGACCTCGTCCTCCGACTCGCTGACAGGCTACAGGACCCCCAACAACCCCAAT CCACGTTTTGGATGCACTGAGACCCCGACATTCCTCGGTATTTATTGTCTGTCCCC ACCTAGGACCCCCACCCCCGACCCTCGCGAATAAAAGGCCCTCCATCTGCCCAAA GCTCTGGA(SEQ ID NO: 24).

[000297] In some embodiments, oligonucleotides may comprise a region of complementarity to a sequence set forth as follows, which is an example mouse DMPK gene sequence (Gene ID 13400; NM_001190490.1).

GAACTGGCCAGAGAGACCCAAGGGATAGTCAGGGACGGGCAGACATGCAGCTAG GGTTCTGGGGCCTGGACAGGGGCAGCCAGGCCCTGTGACGGGAAGACCCCGAGC TCCGGCCCGGGGAGGGGCCATGGTGTTGCCTGCCCAACATGTCAGCCGAAGTGCG

GCTGAGGCAGCTCCAGCAGCTGGTGCTGGACCCAGGCTTCCTGGGACTGGAGCCC CTGCTCGACCTTCTCCTGGGCGTCCACCAGGAGCTGGGTGCCTCTCACCTAGCCCA GGACAAGTATGTGGCCGACTTCTTGCAGTGGGTGGAGCCCATTGCAGCAAGGCTT

AAGGAGGTCCGACTGCAGAGGGATGATTTTGAGATTTTGAAGGTGATCGGGCGTG GGGCGTTCAGCGAGGTAGCGGTGGTGAAGATGAAACAGACGGGCCAAGTGTATG CCATGAAGATTATGAATAAGTGGGACATGCTGAAGAGAGGCGAGGTGTCGTGCT TCCGGGAAGAAAGGGATGTATTAGTGAAAGGGGACCGGCGCTGGATCACACAGC TGCACTTTGCCTTCCAGGATGAGAACTACCTGTACCTGGTCATGGAATACTACGT GGGCGGGGACCTGCTAACGCTGCTGAGCAAGTTTGGGGAGCGGATCCCCGCCGA GATGGCTCGCTTCTACCTGGCCGAGATTGTCATGGCCATAGACTCCGTGCACCGG CTGGGCTACGTGCACAGGGACATCAAACCAGATAACATTCTGCTGGACCGATGTG GGCACATTCGCCTGGCAGACTTCGGCTCCTGCCTCAAACTGCAGCCTGATGGAAT GGTGAGGTCGCTGGTGGCTGTGGGCACCCCGGACTACCTGTCTCCTGAGATTCTG CAGGCCGTTGGTGGAGGGCCTGGGGCAGGCAGCTACGGGCCAGAGTGTGACTGG TGGGCACTGGGCGTGTTCGCCTATGAGATGTTCTATGGGCAGACCCCCTTCTACG CGGACTCCACAGCCGAGACATATGCCAAGATTGTGCACTACAGGGAACACTTGTC GCTGCCGCTGGCAGACACAGTTGTCCCCGAGGAAGCTCAGGACCTCATTCGTGGG CTGCTGTGTCCTGCTGAGATAAGGCTAGGTCGAGGTGGGGCAGACTTCGAGGGTG

CCACGGACACATGCAATTTCGATGTGGTGGAGGACCGGCTCACTGCCATGGTGAG CGGGGGCGGGGAGACGCTGTCAGACATGCAGGAAGACATGCCCCTTGGGGTGCG CCTGCCCTTCGTGGGCTACTCCTACTGCTGCATGGCCTTCAGAGACAATCAGGTCC CGGACCCCACCCCTATGGAACTAGAGGCCCTGCAGTTGCCTGTGTCAGACTTGCA AGGGCTTGACTTGCAGCCCCCAGTGTCCCCACCGGATCAAGTGGCTGAAGAGGCT GACCTAGTGGCTGTCCCTGCCCCTGTGGCTGAGGCAGAGACCACGGTAACGCTGC AGCAGCTCCAGGAAGCCCTGGAAGAAGAGGTTCTCACCCGGCAGAGCCTGAGCC GCGAGCTGGAGGCCATCCGGACCGCCAACCAGAACTTCTCCAGCCAACTACAGG AGGCCGAGGTCCGAAACCGAGACCTGGAGGCGCATGTTCGGCAGCTACAGGAAC GGATGGAGATGCTGCAGGCCCCAGGAGCCGCAGCCATCACGGGGGTCCCCAGTC CCCGGGCCACGGATCCACCTTCCCATCTAGATGGCCCCCCGGCCGTGGCTGTGGG CCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGTCACCTGCTGCTCCCT GCCAGGATCCCTAGGCCTGGCCTATCCGAGGCGCGTTGCCTGCTCCTGTTCGCCG CTGCTCTGGCTGCTGCCGCCACACTGGGCTGCACTGGGTTGGTGGCCTATACCGG CGGTCTCACCCCAGTCTGGTGTTTCCCGGGAGCCACCTTCGCCCCCTGAACCCTAA GACTCCAAGCCATCTTTCATTTAGGCCTCCTAGGAAGGTCGAGCGACCAGGGAGC GACCCAAAGCGTCTCTGTGCCCATCGCGCCCCCCCCCCCCCCCCACCGCTCCGCTC CACACTTCTGTGAGCCTGGGTCCCCACCCAGCTCCGCTCCTGTGATCCAGGCCTGC CACCTGGCGGCCGGGGAGGGAGGAACAGGGCTCGTGCCCAGCACCCCTGGTTCC TGCAGAGCTGGTAGCCACCGCTGCTGCAGCAGCTGGGCATTCGCCGACCTTGCTT TACTCAGCCCCGACGTGGATGGGCAAACTGCTCAGCTCATCCGATTTCACTTTTTC ACTCTCCCAGCCATCAGTTACAAGCCATAAGCATGAGCCCCCTATTTCCAGGGAC ATCCCATTCCCATAGTGATGGATCAGCAAGACCTCTGCCAGCACACACGGAGTCT TTGGCTTCGGACAGCCTCACTCCTGGGGGTTGCTGCAACTCCTTCCCCGTGTACAC GTCTGCACTCTAACAACGGAGCCACAGCTGCACTCCCCCCTCCCCCAAAGCAGTG TGGGTATTTATTGATCTTGTTATCTGACTCACTGACAGACTCCGGGACCCACGTTT TAGATGCATTGAGACTCGACATTCCTCGGTATTTATTGTCTGTCCCCACCTACGAC CTCCACTCCCGACCCTTGCGAATAAAATACTTCTGGTCTGCCCTAAA (SEQ ID NO: 25). In some embodiments, an oligonucleotide may comprise a region of complementarity to DMPK gene sequences of multiple species, e.g., selected from human, mouse and non-human species (e.g., cynomolgus monkey).

[000298] In some embodiments, the oligonucleotide may comprise a region of complementarity to a mutant form of DMPK, for example, a mutant form as reported in Botta A. et al. “The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients.” J Med Genet. 2008 Oct;45(10):639-46.; and Machuca-Tzili L. et al. “Clinical and molecular aspects of the myotonic dystrophies: a review.” Muscle Nerve. 2005 Jul;32(l):l-18.; the contents of each of which are incorporated herein by reference in their entireties.

[000299] In some embodiments, an oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK. In some embodiments, the oligonucleotide targeting DMPK is any one of the antisense oligonucleotides targeting DMPK as described in US Patent Application Publication US20160304877A1, published on October 20, 2016, entitled “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression,” incorporated herein by reference). In some embodiments, the DMPK targeting oligonucleotide targets a region of the DMPK gene sequence as set forth in Genbank accession No. NM_001081560.2 (SEQ ID NO: 24) or as set forth in Genbank accession No.

NG_009784.1.

[000300] In some embodiments, a DMPK targeting oligonucleotide provided herein comprises a nucleotide sequence comprising a region complementary to a target region that is at least 8 continuous nucleotides (e.g., at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20 or more continuous nucleotides) of SEQ ID NO: 24.

[000301] In some embodiments, a DMPK targeting oligonucleotide provided herein is 10-35 (e.g., 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 13- 18, 14-17, 15-18, 20-30, 15-17, 27-30, 25-35, or 30-35) nucleotides in length. In some embodiments, a DMPK targeting oligonucleotide provided herein is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, optionally 15-30, or 16 nucleotides in length. In some embodiments, a DMPK targeting oligonucleotide provided herein is 16 nucleotides in length.

[000302] In some embodiments, a DMPK targeting oligonucleotide provided herein comprises a region of complementarity of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) consecutive nucleotides to a DMPK RNA.

[000303] In some embodiments, a DMPK targeting oligonucleotide provided herein comprises a region of complementarity of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) consecutive nucleotides to a DMPK sequence as set forth in SEQ ID NO: 24 or 25.

[000304] In some embodiments, a DMPK targeting oligonucleotide provided herein comprises a region of complementarity of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides to a target sequence as set forth in SEQ ID NO: 22 (TGACTGGTGGGCGCTG). In some embodiments, an oligonucleotide useful for targeting DMPK comprises at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides of a sequence as set forth in SEQ ID NO: 21 (CAGCGCCCACCAGUCA). In some embodiments, an oligonucleotide useful for targeting DMPK comprises the nucleotide sequence of SEQ ID NO: 21.

[000305] In some embodiments, the DMPK targeting oligonucleotide comprises a 5’-X- Y-Z-3’ configuration. An oligonucleotide comprising a 5’-X-Y-Z-3’ configuration can refer to a chimeric antisense compound in which a gap region having a plurality of nucleosides that support RNase H cleavage is positioned between flanking regions having one or more nucleosides, wherein the nucleosides comprising the gap region are chemically distinct from the nucleoside or nucleosides comprising the flanking regions. In some embodiments, an oligonucleotide described herein (e.g., a DMPK-targeting oligonucleotide described herein) comprises a 5'-X-Y-Z-3' configuration, with X and Z as flanking regions around a gap region Y. In some embodiments, the gap region Y comprises one or more 2’ -deoxyribonucleosides. In some embodiments, each nucleoside in the gap region Y is a 2’ -deoxyribonucleoside, and neither the flanking region X nor the flanking region Z contains any 2’ -deoxyribonucleosides. [000306] In some embodiments, the gap region Y comprises a continuous stretch of 6 or more 2’ -deoxyribonucleosides, which are capable of recruiting an RNAse, such as RNAse H. In some embodiments, the oligonucleotide binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid. In some embodiments, the flanking regions X and Z each comprise one or more modified nucleosides. In some embodiments, flanking regions X and Z each comprise one or more high-affinity modified nucleosides, e.g., one to six high-affinity modified nucleosides. Examples of high affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2’ -MOE, 2'-O-Me, 2’-F) or 2’ -4’ bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the flanking regions X and Z may be of 1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. The flanking regions X and Z may be of similar length or of dissimilar lengths. In some embodiments, the gap region Y may comprise a nucleotide sequence of 5-20 nucleotides, 5-15 nucleotides, 5-12 nucleotides, or 6-10 nucleotides in length.

[000307] In some embodiments, the gap region Y comprises one or more unmodified intemucleoside linkages. In some embodiments, one or both flanking regions X and Z each independently comprise phosphorothioate intemucleoside linkages (e.g., phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the gap region Y and two flanking regions X and Z each independently comprise modified intemucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

[000308] In some embodiments, the gap region Y in the gapmer is 5-20 nucleosides in length. For example, the gap region Y may be 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides in length. In some embodiments, the gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, each nucleoside in the gap region Y is a 2’ -deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2’-deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region Y is a modified nucleoside (e.g., a 2’ modified nucleoside such as those described herein). In some embodiments, one or more cytosines in the gap region Y are optionally 5-methyl-cytosines. In some embodiments, each cytosine in the gap region Y is a 5-methyl-cytosine.

[000309] In some embodiments, the flanking region X of the oligonucleotide (X in the 5'-X-Y-Z-3' configuration) and the flanking region Z of the oligonucleotide (Z in the 5'-X-Y- Z-3' configuration) are independently 1-20 nucleosides long. For example, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide may be independently 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 1-2, 2-5, 2-7, 3-5, 3-7, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides long. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides long. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide are of the same length. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide are of different lengths. In some embodiments, the flanking region X of the oligonucleotide is longer than the flanking region Z of the oligonucleotide. In some embodiments, the flanking region X of the oligonucleotide is shorter than the flanking region Z of the oligonucleotide.

[000310] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide) comprises a 5'-X-Y-Z-3' configuration of 5-10-5, 4-12-4, 3-14-3, 2- 16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3- 8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1,

2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1,

2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4,

4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6,

6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8,

8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1,

2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5,

5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7,

7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1, 2-17-2, 1-16-4,

4-16-1, 2-16-3, 3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3, 1-14-6, 6-14-1, 2-14-5, 5-14-2, 3-14-4,

4-14-3, 1-13-7, 7-13-1, 2-13-6, 6-13-2, 3-13-5, 5-13-3, 4-13-4, 1-12-8, 8-12-1, 2-12-7, 7-12-2,

3-12-6, 6-12-3, 4-12-5, 5-12-4, 2-11-8, 8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4, 5-11-5, 1-20-1,

1-19-2, 2-19-1, 1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1, 2-17-3, 3-17-2, 1-16-5, 2-16-4, 4-16-2,

3-16-3, 1-15-6, 6-15-1, 2-15-5, 5-15-2, 3-15-4, 4-15-3, 1-14-7, 7-14-1, 2-14-6, 6-14-2, 3-14-5,

5-14-3, 4-14-4, 1-13-8, 8-13-1, 2-13-7, 7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4, 2-12-8, 8-12-2,

3-12-7, 7-12-3, 4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3, 4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1,

1-20-2, 2-20-1, 1-20-3, 3-19-1, 2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2,

3-17-3, 1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6, 6-15-2, 3-15-5,

5-15-3, 4-15-4, 1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6, 6-14-3, 4-14-5, 5-14-4, 2-13-8, 8-13-2,

3-13-7, 7-13-3, 4-13-6, 6-13-4, 5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8, 8-12-3, 4-

12-7, 7-12-4, 5-12-6, 6-12-5, 4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6,

6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4,

1-15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3,

4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7,

7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6. The numbers indicate the number of nucleosides in X, Y, and Z regions, respectively, in an oligonucleotide comprising the 5’-X- Y-Z-3’ configuration.

[000311] In some embodiments, one or more nucleosides in the flanking region X of the oligonucleotide (X in the 5'-X-Y-Z-3' configuration) or the flanking region Z of the oligonucleotide (Z in the 5'-X-Y-Z-3' configuration) are modified nucleosides (e.g., high- affinity modified nucleosides). In some embodiments, the modified nucleoside (e.g., high- affinity modified nucleosides) is a 2’ -modified nucleoside. In some embodiments, the 2’- modified nucleoside is a 2’ -4’ bicyclic nucleoside or a non-bicyclic 2’ -modified nucleoside. In some embodiments, the high-affinity modified nucleoside is a 2’-4’ bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2’ -modified nucleoside (e.g., 2’ -fluoro (2’-F), 2’-O- methyl (2’-O-Me), 2’-O-methoxyethyl (2’-MOE), 2’-O-aminopropyl (2’-O-AP), 2’-O- dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2’-O-N-methylacetamido (2’-0-NMA)). [000312] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-X-Y-Z-3' configuration, wherein X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X (the 5’-most position is position 1) is a non-bicyclic 2’- modified nucleoside (e.g., 2’-M0E or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2’deoxyribonucleoside. In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein)comprises a 5'-X-Y-Z-3' configuration, wherein X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in Z (the 5’-most position is position 1) is a non- bicyclic 2’-modified nucleoside (e.g., 2’-M0E or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2’deoxyribonucleoside. In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-X- Y-Z-3' configuration, wherein X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X and at least one of positions but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in Z (the 5’-most position is position 1) is a non-bicyclic 2’-modified nucleoside (e.g., 2’-M0E or 2’- O-Me), wherein the rest of the nucleosides in both X and Z are 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2’deoxyribonucleoside.

[000313] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide) is 10-20 nucleosides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides) in length, comprises a region of complementarity to at least 8 consecutive nucleosides (e.g., at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 consecutive nucleosides) of SEQ ID NO: 22 (TGACTGGTGGGCGCTG), and comprises a 5’-X-Y-Z-3’ configuration, wherein X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in X is a 2’-modified nucleoside (e.g., 2’- MOE modified nucleoside, 2’-0-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6-10 (e.g., 6, 7, 8, 9, or 10) linked 2’ -deoxyribonucleosides, wherein each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in Z is a 2’- modified nucleoside (e.g., 2’- MOE modified nucleoside, 2’-O-Me modified nucleoside, LNA, cEt, or ENA).

[000314] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises at least 8 consecutive nucleosides (e.g., at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 consecutive nucleosides consecutive nucleosides) of the nucleotide sequence of SEQ ID NO: 21 (CAGCGCCCACCAGUCA), and comprises a 5’-X-Y-Z-3’ configuration, wherein X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in X is a 2’ -modified nucleoside (e.g., 2’ -MOE modified nucleoside, 2’-O-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6-10 (e.g., 6, 7, 8, 9, or 10) linked 2’- deoxyribonucleosides, wherein each cytosine in Y is optionally and independently a 5-methyl- cytosine; and Z comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in Z is a 2’ -modified nucleoside (e.g., 2’ -MOE modified nucleoside, 2’-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide may independently and optionally be replaced with an uracil base (U), and each U may independently and optionally be replaced with a T.

[000315] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises the nucleotide sequence of SEQ ID NO: 21 and comprises a 5’-X-Y-Z-3’ configuration, wherein X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in X is a 2’ -modified nucleoside (e.g., 2’-MOE modified nucleoside, 2’-O-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6-10 (e.g., 6, 7, 8, 9, or 10) linked 2’ -deoxyribonucleosides, wherein each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in Z is a 2’- modified nucleoside (e.g., 2’-MOE modified nucleoside, 2’-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide may independently and optionally be replaced with an uracil base (U), and each U may independently and optionally be replaced with a T.

[000316] In some embodiments, X comprises at least one 2’ -4’ bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2’-modified nucleoside e.g., 2’-MOE modified nucleoside or 2’-O-Me modified nucleoside, and/or (e.g., and) Z comprises at least one 2’ -4’ bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2’- modified nucleoside (e.g., 2’-M0E modified nucleoside or 2’-0-Me modified nucleoside). [000317] In some embodiments, the 2’ -4’ bicyclic nucleoside is selected from LNA, cEt, and ENA nucleosides. In some embodiments, the non-bicyclic 2’ -modified nucleoside is a 2’- MOE modified nucleoside or a 2’-OMe modified nucleoside.

[000318] In some embodiments, the nucleosides of the oligonucleotides are joined together by phosphorothioate intemucleoside linkages, phosphodiester intemucleoside linkages or a combination thereof. In some embodiments, the oligonucleotide comprises only phosphorothioate internucleoside linkages joining each nucleoside (i.e., the oligonucleotide comprises a fully phosphorothioate backbone). In some embodiments, the oligonucleotide comprises at least one phosphorothioate intemucleoside linkage. In some embodiments, the oligonucleotide comprises a mix of phosphorothioate and phosphodiester internucleoside linkages. In some embodiments, the oligonucleotide comprises only phosphorothioate intemucleoside linkages joining each pair of 2’ -deoxyribonucleosides and a mix of phosphorothioate and phosphodiester intemucleoside linkages joining the remaining nucleosides.

[000319] In some embodiments, the oligonucleotide comprises a 5’-X-Y-Z-3’ configuration of LLEE-(D)8-EELL, wherein “E” is a 2’-M0E modified ribonucleoside; “L” is LNA; “D” is a 2’ -deoxy ribonucleoside; and “10” or “8” is the number of 2’- deoxyribonucleosides in Y, and wherein the oligonucleotide comprises phosphorothioate intemucleoside linkages, phosphodiester intemucleoside linkages or a combination thereof. [000320] In some embodiments, each cytidine (e.g., a 2’ -modified cytidine) in X and/or Z of the oligonucleotide is optionally and independently a 5-methyl-cytidine, and/or each uridine (e.g., a 2’ -modified uridine) in X and/or Z is optionally and independently a 5-methyl- uridine.

[000321] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5’-X-Y-Z-3’ configuration and comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an LNA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’-

Il l 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage.

[000322] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a structure of the formula (le):

[000323] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) can be in salt form, e.g., as sodium, potassium, or magnesium salts.

[000324] In some embodiments, the 5’ or 3’ nucleoside (e.g., terminal nucleoside) of the oligonucleotide is conjugated to an amine group, optionally via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer and the 5’ or 3’ nucleoside of the oligonucleotide. In some embodiments, the 5’ or 3’ nucleoside (e.g., terminal nucleoside) of an oligonucleotide described herein is covalently linked to a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R^A)-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR^A-, - NR^AC(=O)-, -NR^AC(=O)R^A-, -C(=O)R^A-, -NR^AC(=O)O-, -NR^AC(=O)N(R^A)-, -0C(=0)-, - 0C(=0)0-, -OC(=O)N(R^A)-, -S(0)2NR^A-, -NR^AS(0)2-, or a combination thereof; each R^A is independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is a substituted or unsubstituted alkylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted heteroarylene, -O-, -N(R^A)-, or -C(=0)N(R^A)2, or a combination thereof.

[000325] In some embodiments, the 5’ or 3’ nucleoside of the oligonucleotide is conjugated to a compound of the formula -NH2-(CH2)_n-, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, a phosphodiester linkage is present between the compound of the formula NH2-(CH2)_n- and the 5’ or 3’ nucleoside of the oligonucleotide. In some embodiments, a compound of the formula NH2- (CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-l -hexanol (NH2- (CH₂)₆-OH) and the 5’ phosphate of the oligonucleotide.

[000326] In some embodiments, the oligonucleotide is conjugated to a targeting agent, e.g., a muscle targeting agent such as an anti-TfRl antibody, e.g., via an amine group of a lysine of the targeting agent.

[000327] In some embodiments, it should be appreciated that methylation of the nucleobase uracil at the C5 position forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) may be equivalently identified as a thymine nucleotide or nucleoside.

[000328] In some embodiments, any one or more of the thymine bases (T’s) in any one of the oligonucleotides provided herein may independently and optionally be uracil bases (U’s), and/or any one or more of the U’s in the oligonucleotides provided herein (e.g., the oligonucleotide as set forth in SEQ ID NO: 21) may independently and optionally be T’s.

Treatment Methods

[000329] Complexes comprising an anti-TfRl antibody (e.g., a Fab) covalently linked to a molecular pay load (e.g., a DMPK targeting oligonucleotide) as described herein are effective in treating a subject having a myotonic dystrophy, e.g., DM1. In some embodiments, complexes comprise a molecular payload that is an oligonucleotide, e.g., an oligonucleotide that facilitates reduced expression or activity of DMPK (e.g., reduced level of a mutant or wild-type DMPK RNA). In some embodiments, complexes comprise a molecular payload that is an oligonucleotide, e.g., an oligonucleotide that facilitates correction of splicing defects associated with DM1. [000330] In some embodiments, a subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, a subject may have myotonic dystrophy. In some embodiments, a subject has a DMPK allele, which may optionally contain a disease-associated repeat, e.g., a CTG trinucleotide repeat expansion. In some embodiments, a subject may have a DMPK allele with an expanded disease- associated-repeat that comprises about 2-10 repeat units, about 2-50 repeat units, about 2-100 repeat units, about 50-1,000 repeat units, about 50-500 repeat units, about 50-250 repeat units, about 50-100 repeat units, about 500-10,000 repeat units, about 500-5,000 repeat units, about 500-2,500 repeat units, about 500-1,000 repeat units, or about 1,000-10,000 repeat units. In some embodiments, a subject may have myotonic dystrophy, such as DM1. In some embodiments, a subject is suffering from symptoms of DM1, e.g. muscle atrophy, muscle loss, excessive daytime sleepiness or cognitive delay. In some embodiments, a subject is not suffering from symptoms of DM1. In some embodiments, subjects have congenital myotonic dystrophy. In some embodiments, a subject is ambulant. In some embodiments, a subject is non-ambulant.

[000331] An aspect of the disclosure includes methods involving administering to a subject a formulation comprising an effective amount of complex(es) as described herein. In some embodiments, an effective amount of a pharmaceutical composition that comprises complex(es) comprising an antibody (e.g., Fab) described herein covalently linked to an oligonucleotide (e.g., a DMPK targeting oligonucleotide) described herein can be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising complex(es) as described herein may be administered by a suitable route, which may include intravenous administration, e.g., as a bolus or by continuous infusion over a period of time. In some embodiments, administration may be performed by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, or intrathecal routes. In some embodiments, a pharmaceutical composition may be in solid form, aqueous form, or a liquid form. In some embodiments, an aqueous or liquid form may be nebulized or lyophilized. In some embodiments, a lyophilized form may be reconstituted with an aqueous or liquid solution.

[000332] In some embodiments, provided are methods of and/or uses for treating a subject having a DMPK allele, which may optionally contain a disease-associated repeat, comprising administering to the subject a formulation described herein that comprises an effective amount of complex(es) described herein. In some embodiments, provided are methods of and/or uses for reducing the expression or activity of DMPK (e.g., reducing the level of a mutant or wild-type DMPK RNA, or the activity of a DMPK gene product) in a cell (e.g., a muscle cell), the methods comprising contacting the cell with a formulation described herein comprising an effective amount of complex(es) described herein. In some embodiments, the method comprises administering a lyophilized form (e.g. , lyophilized powder) of the formulation described herein, comprising reconstituting a lyophilized form of the formulation in an aqueous solution, and administering the aqueous solution of the formulation to a subject in need thereof. For example, in some embodiments, a lyophilized form of the formulation is shipped and/or stored in the lyophilized form, reconstituted at a location for administering the aqueous solution of the formulation (e.g., healthcare provider location), and administered in the reconstituted form (e.g., as an aqueous solution) by injection or intravenously, e.g., by infusion.

[000333] In some embodiments, a pharmaceutical composition is administered via sitespecific or local delivery techniques. Examples of these techniques include implantable depot sources of the complex, local delivery catheters, site specific carriers, direct injection, or direct application.

[000334] In some embodiments, a pharmaceutical composition that comprises a complex comprising a an anti-TfRl antibody (e.g., a fab) covalently linked to a molecular payload (e.g., a DMPK targeting oligonucleotide) is administered at an effective concentration that confers therapeutic effect on a subject. Effective amounts vary, as recognized by those skilled in the art, depending on the severity of the disease, unique characteristics of the subject being treated, e.g. age, physical conditions, health, or weight, the duration of the treatment, the nature of any concurrent therapies, the route of administration and related factors. These related factors are known to those in the art and may be addressed with no more than routine experimentation. In some embodiments, an effective concentration is the maximum dose that is considered to be safe for the patient. In some embodiments, an effective concentration will be the lowest possible concentration that provides maximum efficacy.

[000335] Empirical considerations, e.g., the half-life of the complex(es) in a subject, generally will contribute to determination of the concentration of pharmaceutical composition that is used for treatment. The frequency of administration may be empirically determined and adjusted to maximize the efficacy of the treatment. The efficacy of treatment may be assessed using any suitable methods, e.g., methods of evaluating treatment efficacy as described herein. In some embodiments, the efficacy of treatment may be assessed by evaluation or observation of symptoms associated with a myotonic dystrophy, e.g., muscle atrophy or muscle weakness, through measures of a subject’s self-reported outcomes, e.g. mobility, self-care, usual activities, pain/discomfort, and anxiety /depression, or by quality-of- life indicators, e.g. lifespan.

EXAMPLES

Example 1. Identification of subgroups of splice index genes

[000336] Splicing activity of a list of 22 genes in patients with Myotonic Dystrophy Type 1 (DM1) are altered by the disease.

[000337] As provided herein, there is strong interest in optimizing the signal and robustness of the DM1 splicing index and understanding how individual genes contribute to the overall DM1 splicing index. In the present study, contribution of different genes or sets of genes to the composite alternative splicing index (CASI) is assessed at different disease states from mild DM1 to severe DM1.

[000338] Without wishing to be bound by scientific theory, for genes previously identified in the DM1 splicing index, at the DNA level, CTG repeats code for hairpin CUG structures that bind MBNL1 proteins with high affinity. Due to MBNL1 loss, CELF1 is overexpressed through PKC phosphorylation and alters the splicing of different transcripts, mainly switching to embryonic isoforms. The splicing deregulation or spliceopathy induces an aberrant protein expression that provokes the loss of cell function and viability. Different transcripts from different tissues are incorrectly spliced, causing most DM1 symptoms.

[000339] First, to determine the grouping of DM1 splice index genes based on when they start to contribute to the CASI on a continuum of disease state spanning mild to severe, K-means clustering was used. Three clusters were determined to be optimal (FIG. 1A). The DM1 splice index genes were then separated, based on the distribution of per- gene ASI values across DM1 patients, into one of the three clusters, and identified as either early-stage (e.g., mild disease state), intermediate-stage (e.g., moderate disease state), or late-stage genes (e.g., severe disease state) (FIG. IB). The grouping of these clusters fit into the “timing” of the gene’s alternate splicing events over the span of disease severity of DM1 (e.g., on a continuum of mild to severe disease). FIG. 2 shows that the three groups of genes start contributing to the overall CASI at different times on the continuum of DM1 disease state from mild to severe.

[000340] Muscleblind Eike Splicing Regulator 1 (MBEN1) binding affinity was considered as a potential indicator of whether a gene is likely to start contributing to the overall CASI at a certain disease stage (e.g., mild or severe). Table 4 shows previously identified binding affinities of ATP2A1 and INSR to MBNE1. ATP2A1, one of the most abundant proteins in skeletal muscle, identified as a gene that starts to contribute to the overall CASI when disease is severe (FIG. 2), shows higher binding affinity to MBNL1 than INSR, which has been identified as a gene that starts to contribute to the overall CASI even when DM1 is mild (FIG. 2). According to the GTEx bulk RNA-seq database, expression of genes that contribute to the overall CASI when disease is severe may have greater muscle specificity compared to genes that contribute to the overall CASI when disease is mild (see also, FIG. 3). [000341] The different sets of genes that have different MBNL binding affinity can also indicate treatment efficacy more accurately at different disease state, relative to a splicing index with the 22 identified genes from the natural history study. FIG. 4A shows that genes with higher MBNL binding affinity are better indicators for therapeutic efficacy when disease is at a severe state. FIG. 4B shows that genes with lower MBNL binding affinity are better indicators for therapeutic efficacy when disease is at a mild state.

Table 4. Reported Experimental Evidence of Different MBNL Binding Affinity for Splice

Index Genes

Example 2. Matching of subgroups of splice index genes to disease severity enhances dynamic range in 3 month change in CASI

[000342] Baseline (BL) and 3-month (3M) longitudinal samples from 34 patients with DM1 were analyzed to determine changes in the composite alternative splicing index (CASI) for all 22 genes between baseline and the 3-month follow-up. Significant changes in CASI were observed over this time frame. Patients were then stratified into one of three clusters (mild-disease state, “Mild”; moderate disease state, “Moderate”; and severe disease state, “Severe”) based on baseline CASI (Mild = 0 - 0.25, Moderate = 0.25 - 0.65 and Severe = 0.65 - 1.0) and significant changes were only found in the Moderate group (see FIG. 5). [000343] Patient stratification was integrated with gene subgroup information and recalibrated the composite score (CASI) based on specific subsets of genes within each severity stratum. In mild-disease state patients, the composite score based on all 22 genes was not significant, but a score based on early-stage genes showed significant changes. Similarly, for severe disease state patients, late-stage genes were most informative, while intermediatestage genes were most informative for moderate disease state patients.

[000344] Based on these observations, combining patient severity subgroups with splice index gene subgroups can enhance the detection of splice index changes in longitudinal assessments. This approach is particularly beneficial in therapeutic intervention studies, where treatment may alter a patient's disease severity by modulating MBNL1 sequestration over time, thus changing which gene subgroups are most informative. To implement this, the CASI must be recalculated at each time point, and patients should be reassigned to the appropriate disease severity subgroup. Based on this reassignment, the corresponding gene subgroup should be selected to calculate changes in the splice index. By recalculating disease severity at each time point and adjusting the gene subsets used for analysis, it ensures that the most relevant biomarkers are captured as treatment progresses.

ADDITIONAL EMBODIMENTS

AO. A method comprising determining a composite measure of splicing activity for a set of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state-associated genes in a nucleic acid sample obtained from a subject.

A0.1 The method of embodiment AO, wherein the composite measure of splicing activity is based on an inclusion or an exclusion of an RNA segment in the one or more RNA transcripts of each gene of the set of disease state-associated genes in the nucleic acid sample.

A0.2 The method of embodiment AO.1, wherein the RNA segment is an exonic region of the one or more RNA transcripts of each gene of the set of disease state- associated genes.

AO.3 The method of any one of embodiments A0-A0.2, wherein the composite measure of splicing activity is based on alternative splicing indices.

Al. A method comprising: (a) determining an alternative splicing index (ASI) for each gene of the set of disease state-associated genes based on the detected splicing events of one or more RNA transcripts of the gene detected in a nucleic acid sample obtained from a subject, wherein the splicing events are detected using nucleic acid hybridization assays; and

A2 The method of embodiment Al, further comprising obtaining the nucleic acid sample from the subject; subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events; and obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events.

A3. The method of embodiment Al or embodiment A2, wherein the CASI determined in step (b) for the set of disease state- associated genes is above a first threshold CASI value.

A4. The method of embodiment A3, wherein each gene of the set of disease state- associated genes comprises a sequence exhibiting a lower binding affinity to MBNL1, relative to a set of genes with a CASI of below the first threshold CASI value.

A5. The method of embodiment A3 or embodiment A4, wherein the set of disease state- associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and 0PA1.

A6. The method of embodiment A3 or embodiment A4, wherein the set of disease state- associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, 0PA1, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1.

A7. The method of any one of embodiments A1-A6, wherein the subject has a repeat expansion disease that is at a mild to moderate state.

A8. The method of embodiment Al or embodiment A2, wherein the CASI determined in step (b) for the set of disease state- associated genes is below a second threshold CASI value. A9. The method of embodiment A8, wherein each gene of the set of disease state- associated genes comprises a sequence exhibiting a higher binding affinity to MBNL1, relative to a set of genes with a CASI of above the second threshold CASI value.

A10. The method of embodiment A8 or embodiment A9, wherein the set of disease state- associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

Al l. The method of embodiment A8 or embodiment A9, wherein the set of disease state- associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, BINI, RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SOS1, CLCN1, and MBNL1.

A12. The method of any one of embodiments A8-A11, wherein the subject has a repeat expansion disease that is at a moderate to severe state.

A13. The method of embodiment Al or embodiment A2, wherein the CASI determined in step (b) for the set of disease state- associated genes is above a third threshold CASI value.

A14. The method of embodiment A13, wherein each gene of the set of disease state- associated genes comprises a sequence exhibiting a higher binding affinity to MBNL1, relative to a set of genes with a CASI of above the second threshold CASI value.

A15. The method of embodiment A13 or embodiment A 14, wherein the set of disease state- associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

A16. The method of any one of embodiments A13-A15, wherein the subject has a repeat expansion disease that is at a severe state.

A17. The method of any one of embodiments A1-A16, wherein the nucleic acid sample is obtained from a tissue sample obtained from the subject.

A18. The method of embodiment A17, wherein the tissue sample is a muscle biopsy sample. A19. The method of any one of embodiments A1-A17, wherein the nucleic acid sample is obtained from a blood sample obtained from the subject.

A20. The method of any one of embodiments A1-A19, wherein the repeat expansion disease is a muscle disease or disorder.

A21. The method of embodiment A20, wherein the muscle disease or disorder is myotonic dystrophy.

A22. The method of embodiment A21, wherein the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

A23. The method of any one of embodiments A1-A22, wherein the subject is undergoing treatment or has been treated for the repeat expansion disease.

A24. The method of any one of embodiments A1-A22, wherein the subject will be treated for the repeat expansion disease.

A25. The method of any one of embodiments A1-A24, wherein the nucleic acid hybridization assays comprise RT-PCR and/or sequencing.

A26. The method of any one of embodiments A1-A25, wherein the CASI determined in step (b) is between a value of 0 and 1.

A27. The method of any one of embodiments A1-A26, wherein the ASI determined in step (a) is between a value of 0 and 1.

A28. The method of any one of embodiments A1-A27, wherein the repeat expansion disease is associated with spliceopathy.

BO. A method comprising determining composite measures of splicing activity for at least two sets of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the sets of disease state-associated genes in a nucleic acid sample obtained from a subject; and evaluating the disease state of the subject based on the composite measures of splicing activity.

Bl. A method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease, the method comprising:

B2. The method of embodiment Bl, further comprising obtaining the nucleic acid sample from the subject; subjecting the nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events; and obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events.

B3. The method of embodiment B 1 or embodiment B2, wherein the first set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and 0PA1.

B4. The method of any one of embodiments B1-B3, wherein the second set of disease state-associated genes comprises CACNA1S, CAPZB, G0LGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

B5. The method of any one of embodiments B 1-B4, wherein the first CASI determined in step (b) for the first set of disease state- associated genes is below a first threshold CASI value. B6. The method of B5, wherein the subject is determined to have a repeat expansion disease that is at a mild state.

B7. The method of any one of embodiments B 1-B4, wherein the first CASI determined in step (b) for the first set of disease state- associated genes is above a second threshold CASI value.

B8. The method of embodiment B7, wherein the second CASI determined in step (c) for the second set of disease state-associated genes is below the second threshold CASI value.

B9. The method of any one of embodiments B 1-B4, wherein the first CASI determined in step (b) for the first set of disease state- associated genes is larger than the second CASI determined in step (c) for the second set of disease state-associated genes.

BIO. The method of embodiment B9, wherein the subject is determined to have a repeat expansion disease that is at a moderate state.

BIO. The method of any one of embodiments B1-B4, wherein the second CASI determined in step (c) for the second set of disease state-associated genes is above a third threshold CASI value.

B 11. The method of embodiment B 11 , wherein the subject is determined to have a repeat expansion disease that is at a severe state.

B12. The method of any one of embodiments Bl-Bl l, wherein the nucleic acid sample is obtained from a tissue sample obtained from the subject.

B13. The method of embodiment Bl 2, wherein the tissue sample is a muscle biopsy sample.

B14. The method of any one of embodiments Bl-Bl l, wherein the nucleic acid sample is obtained from a blood sample obtained from the subject. B 15. The method of any one of embodiments B 1-B 14, wherein the repeat expansion disease is a muscle disease or disorder.

B 16. The method of embodiment B 15, wherein the muscle disease or disorder is myotonic dystrophy.

B17. The method of embodiment Bl 6, wherein the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

Bl 8. The method of any one of embodiments B 1-B 17, further comprising treating the subject for the repeat expansion disease if the subject is determined to be at a targeted disease state.

B18.1 The method of any one of embodiments B1-B17, wherein the subject undergoes treatment with an agent that treats the repeat-expansion disease if the subject is determined to be at a targeted disease state.

B19. The method of embodiment Bl 8, wherein the repeat expansion disease is DM1 and the treating comprises administering to the subject a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

B19.1. The method of embodiment Bl 8.1, wherein the repeat expansion disease is DM1 and the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

B20. The method of any one of embodiments B1-B19.1, wherein the nucleic acid hybridization assays comprise RT-PCR and/or sequencing.

B21. The method of any one of embodiments B1-B20, wherein step (b) and step (c) are performed in any order or in parallel.

B22. The method of any one of embodiments B 1-B21, wherein the first CASI determined in step (b) and/or second CASI determined in step (c) is between a value of 0 and 1. B23. The method of any one of embodiments B 1-B22, wherein the ASI determined in step (a) is between a value of 0 and 1.

B24. The method of any one of embodiments B 1-B23, wherein the repeat expansion disease is associated with spliceopathy.

CO. A method of monitoring a subject undergoing a treatment for a repeat expansion disease, the method comprising determining, during a period of time, at least two composite measures of splicing activity for a set of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state- associated genes in a nucleic acid sample obtained from the subject; and determining the disease state of the subject based on the at least two composite measures of splicing activity to thereby monitor the subject.

Cl. A method of monitoring a subject undergoing a treatment for a repeat expansion disease, the method comprising, over a period of time:

(c) comparing the first CASI and the second CASI to thereby monitor the subject.

C2. A method of evaluating effectiveness of a treatment for a repeat expansion disease in a subject undergoing the treatment for the repeat expansion disease, the method comprising, over a period of time:

C3. The method of embodiment Cl or embodiment C2, wherein the first CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI for each gene is determined based on splicing events detected for one or more RNA transcripts for the gene in a first nucleic acid sample obtained from the subject.

C4. The method of any one of embodiments C1-C3, wherein the second CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI is determined based on splicing events detected for one or more RNA transcripts of the gene in a second nucleic acid sample obtained from the subject.

C5. The method of embodiment C4, wherein the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained from the subject after treatment.

C6. The method of embodiment C4, wherein the first nucleic acid sample is obtained from the subject earlier in time than the second nucleic acid sample during a period in which the subject is undergoing the treatment.

C7. The method of any one of embodiments C4-C6, wherein if the first CASI is larger than the second CASI, the treatment is effective.

C8. The method of any one of embodiments C1-C7, wherein the treatment comprises administering to the subject an agent that treats the repeat-expansion disease.

C8.1. The method of any one of embodiments C1-C7, wherein the subject is undergoing treatment with an agent that treats the repeat-expansion disease.

C9. The method of embodiment C8 or C8.1, wherein the repeat expansion disease is DM1 and the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

CIO. The method of any one of embodiments C1-C9, wherein the set of disease state- associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1. Cl 1. The method of any one of embodiments Cl -CIO, wherein the set of disease state- associated genes comprises RYR1, MBNL2, NFIX, CLASP 1, VPS39, BEST3, SCSI, CLCN1, and MBNL1.

C12. The method of any one of embodiments Cl-Cl 1, wherein the set of disease state- associated genes comprises CACNA1S, CAPZB, G0LGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

C13. The method of any one of embodiments C4-C12, wherein the first nucleic acid sample and/or the second nucleic acid sample is obtained from a tissue sample obtained from the subject.

C14. The method of embodiment C13, wherein the tissue sample is a muscle biopsy sample.

C15. The method of any one of embodiments C4-C12, wherein the first nucleic acid sample and/or the second nucleic acid sample is obtained from a blood sample obtained from the subject.

C16. The method of any one of embodiments C3-C15, wherein the splicing events are detected via subjecting the first nucleic acid sample and/or the second nucleic acid sample to nucleic acid hybridization assays.

C17. The method of embodiment Cl 6, wherein the nucleic acid hybridization assays comprise RT-PCR and/or sequencing.

Cl 8. The method of any one of embodiments C1-C17, wherein the first CASI determined in step (a) and/or the second CASI determined in step (b) is between a value of 0 and 1.

C19. The method of any one of embodiments C1-C18, wherein the repeat expansion disease is associated with spliceopathy.

DO.1 A computer-implemented method of processing genomic data, the method comprising: for each gene in a selected set of genes of a reference genome, obtaining measures of splicing activity based on splicing events detected across a population of individuals having the repeat expansion disease at differing disease states; and assigning genes of the selected set into different groups based on the measures of splicing activity across the population of individuals having the repeat expansion disease, wherein the different groups are associated with the differing disease states.

DO.2 The computer- implemented method of embodiment D0.1, further comprising determining scores between each gene pair in the selected set based on the measures of splicing activity across the population.

DO. A computer-implemented method of processing genomic data, the method comprising: for each gene in a selected set of genes of a reference genome, obtaining measures of splicing activity based on splicing events detected across a population of individuals having the repeat expansion disease at differing disease states; determining similarity scores between each gene pair in the selected set based on in the measures of splicing activity across the population; and assigning genes of the selected set into different groups, wherein the genes within each of the different groups are determined, based on the similarity scores, to have statistically similar variances in the measures of splicing activity across the population of individuals having the repeat expansion disease.

DI. A computer-implemented method of processing genomic data, the method comprising: obtaining sequence data of a reference genome from a database, the sequence data comprising RNA transcript characteristics associated with genes of the reference genome; selecting a set of genes of the reference genome having desired RNA transcript characteristics; for each gene in the selected set, obtaining alternative splicing indices across a population of individuals having the repeat expansion disease at differing disease states; determining similarity scores between each gene pair in the selected set based on the alternative splicing indices across the population; and assigning genes of the selected set into different groups, wherein the genes within each of the different groups are determined, based on the similarity scores, to have statistically similar variances in alternative splicing indices across the population of individuals having the repeat expansion disease.

D2. The computer- implemented method of embodiment DI, further comprising associating each of the different groups with an indicator of a corresponding disease state of the repeat expansion disease.

D3. The computer- implemented method of embodiment DI, further comprising determining a composite alternative splicing index based on alternative splicing indices of genes from two or more different groups; and associating each of the different groups with an indicator of a corresponding disease state of the repeat expansion disease, wherein the association is based on the relative contribution of alternative splicing indices of genes of the different groups to the composite alternative splicing index in relation to the disease state of the individuals from which the alternative splicing indices are obtained.

D4. The computer-implemented method of embodiment D2 or embodiment D3, wherein the disease state is a degree of severity of the repeat expansion disease or a degree of progression of the repeat expansion disease.

D5. The computer- implemented method of any one of embodiments D1-D4, further comprising determining, based on the variance in measured alternative splicing indices, a value, n, reflecting the number of different groups.

D6. The computer-implemented method of embodiment D5, further comprising assigning genes into the n different groups using an iterative approach that minimizes the within-group variance in measured alternative splicing indices.

D7. The computer- implemented method of embodiment D4, further comprising assigning the RNA transcript characteristics to genes of the reference genome, and optionally storing the RNA transcript characteristics in the database.

D8. The computer-implemented method of any one of embodiments D1-D7, wherein the RNA transcript characteristics comprise relative splicing factor binding affinities of RNAs encoded by the genes and/or of the number of splicing factor binding elements present in RNAs encoded by the genes.

D9. The computer-implemented method any one of embodiments D1-D8, wherein the RNA transcript characteristics comprise alternative splicing isoform information.

DIO. The computer-implemented method of any one of embodiments D1-D9, further comprising, for at least each gene in the selected set, assigning one or more characteristics indicative of the alternative splicing indices across the population, the one or more characteristics comprising a variance in alternative splicing indices across the population.

Dl l. The computer- implemented method of any one of embodiments DI -DIO, wherein the repeat expansion disease is myotonic muscular dystrophy type 1 (DM1), myotonic muscular dystrophy type 2 (DM2), Fuchs endothelial corneal dystrophy (FEDC), or spinocerebellar ataxia type 8(SCA8).

D12. The computer-implemented method of any one of embodiments Dl-Dl 1, wherein the repeat expansion disease is associated with spliceopathy.

D13. The computer- implemented method of any one of embodiments DI -DI 2, wherein the repeat expansion disease is myotonic dystrophy type 1 (DM1).

D14. The computer-implemented method of any one of embodiments D1-D13, wherein the alternative splicing indices are based on normalized levels of inclusion splice isoforms and/or exclusion splice isoforms present in nucleic acid samples obtained from individuals of the population.

EO. A method of determining a disease state of a subject, the method comprising determining at least one comparator between splicing events detected for one or more RNA transcripts of each gene of one or more sets of disease state-associated genes in a nucleic acid sample obtained from the subject and determining the disease state of the subject based on the at least one comparator. EO.l. A method of monitoring a subject, the method comprising determining the disease state of the subject based on at least one comparator between splicing events detected for one or more RNA transcripts of each gene of two or more sets of disease state-associated genes in a nucleic acid sample obtained from the subject; and monitoring the subject based on splicing events detected for one or more RNA transcripts of each gene of a set of disease state- associated genes selected based on the determined disease state.

E0.2. A method of determining a disease state of a subject, the method comprising determining at least one comparator for at least two composite splicing indices (CASIs) for one or more sets of disease state-associated genes, and determining the disease state of the subject based on the at least one comparator.

E0.3. A method of monitoring a subject, the method comprising determining the disease state of the subject based on at least one comparator for composite splicing indices (CASIs) for two or more sets of disease state-associated genes; and monitoring the subject based on a CASI for a set of disease state-associated genes selected based on the determined disease state.

El. A method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease, the method comprising:

(a3) determining a first comparator between the first CASI of step (al) and the second CASI of step (a2), wherein the first set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and/or OPA1;

(bl) determining a first CASI for a second set of disease state-associated genes, (b2) determining a second CASI for the second set of disease state- associated genes, (b3) determining a second comparator between the first CASI of step (bl) and the second CASI of step (b2), wherein the second set of disease state- associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SCSI, CLCN1, and/or MBNL1; (cl) determining a first CASI for a third set of disease state-associated genes,

(c2) determining a second CASI for the third set of disease state-associated genes, (c3) determining a third comparator between the first CASI of step (cl) and the second CASI of step (c2), wherein the third set of disease state- associated genes comprises CACNA1S, CAPZB, G0LGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI; and

(d) determining the disease state of the subject based on the first comparator, second comparator, and/or third comparator.

El.l. The method of embodiment El, wherein the disease state of the subject is selected from a mild disease state, a moderate disease state, or a severe disease state.

El.2. The method of embodiment El or El.l, wherein the disease state of the subject is a mild disease state corresponding to a reference CASI for a combination of the first, second, and third sets of disease state-associated genes having a value in the range of 0 and 0.25.

El.3. The method of embodiment El or El.l, wherein the disease state of the subject is a moderate disease state corresponding to a reference CASI for a combination of the first, second, and third sets of disease state-associated genes having a value in the range of 0.25 - 0.65.

El.4. The method of embodiment El or El.l, wherein the disease state of the subject is a severe disease state corresponding to a reference CASI for a combination of the first, second, and third sets of disease state-associated genes having a value in the range of 0.65 - 1.0.

El.5. The method of any one of embodiments E1.2-E1.4, wherein the reference CASI is determined from a sample obtained from a reference subject at a time corresponding to a time at or before which a sample was obtained for determining the first CASIs in steps (al), (bl), and (cl).

E2. The method of embodiment El, wherein the first CASI in (al) and the second CASI (a2) are determined based on a first alternative splicing index (ASI) for each gene of the first set of disease state-associated genes, wherein the first CASI in (bl) and the second CASI in (b2) are determined based on a second alternative splicing index (ASI) for each gene of the second set of disease state- associated genes, and wherein the first CASI in (cl) and the second CASI in (c2) are determined based on a third alternative splicing index (ASI) for each gene of the third set of disease state-associated genes, wherein the ASI for each gene in each set of disease state-associated genes in (al), (bl), and (cl) are determined based on detected splicing events for one or more RNA transcripts of the respective genes detected in a first nucleic acid sample obtained from the subject, wherein the ASI for each gene in each set of disease state-associated genes in (a2), (b2), and (c2) are determined based on detected splicing events for one or more RNA transcripts of the respective genes detected in a second nucleic acid sample obtained from the subject, wherein the splicing events are detected using nucleic acid hybridization assays.

E3. The method of embodiment E2, further comprising obtaining the first nucleic acid sample and second nucleic acid sample from the subject; subjecting the first nucleic acid sample and second nucleic acid sample to the nucleic acid hybridization assays to detect the splicing events; and obtaining results of the nucleic acid hybridization assays indicative of the detected splicing events.

E4. The method of embodiment E2 or embodiment E3, wherein the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained during a period in which the subject is undergoing the treatment.

E5. The method of embodiment E2 or embodiment E3, wherein the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained from the subject after treatment. E6. The method of embodiment E2 or embodiment E3, wherein the first nucleic acid sample is obtained from the subject earlier in time than the second nucleic acid sample during a period in which the subject is undergoing the treatment.

E7. The method of any one of embodiments E1-E6, wherein the disease state of the subject is determined to be a mild disease state based on the first comparator, second comparator, and/or third comparator.

E8. The method of any one of embodiments E1-E6, wherein the disease state of the subject is determined to be a moderate disease state based on the first comparator, second comparator, and/or third comparator.

E9. The method of any one of embodiments E1-E6, wherein the disease state of the subject is determined to be a severe disease state based on the first comparator, second comparator, and/or third comparator.

E10. The method of any one of embodiments E1-E9, further comprising monitoring the subject undergoing the treatment according to a method described herein (e.g., a method of embodiment Cl) or evaluating effectiveness of the treatment according to a method described herein (e.g., a method of embodiment C2), wherein if the subject is determined to have a repeat expansion disease that is at a mild disease state, the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and/or OPA1, wherein if the subject is determined to have a repeat expansion disease that is at a moderate disease state, the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SCSI, CLCN1, and/or MBNL1, and wherein if the subject is determined to have a repeat expansion disease that is at a severe state, the set of disease state-associated genes comprises CACNA1S, CAPZB, G0LGA4, GFPT1, ATP2A1, ANK2, DMD, and/or BINI.

El l. The method of any one of embodiments E1-E10, wherein the nucleic acid sample is obtained from a tissue sample obtained from the subject.

E12. The method of embodiment El l, wherein the tissue sample is a muscle biopsy sample. E13. The method of any one of embodiments E1-E12, wherein the nucleic acid sample is obtained from a blood sample obtained from the subject.

El 4. The method of any one of embodiments El -El 3, wherein the repeat expansion disease is a muscle disease or disorder.

El 5. The method of embodiment El 4, wherein the muscle disease or disorder is myotonic dystrophy.

E16. The method of embodiment E15, wherein the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

E17. The method of any one of embodiments E1-E16, further comprising treating the subject for the repeat expansion disease if the subject is determined to be at a targeted disease state.

E17.1. The method of any one of embodiments E1-E16, wherein the subject undergoes treatment with an agent that treats the repeat-expansion disease if the subject is determined to be at a targeted disease state.

El 8. The method of embodiment E17, wherein the repeat expansion disease is DM1 and the treating comprises administering to the subject a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

El 8.1. The method of embodiment El 7.1, wherein the repeat expansion disease is DM1 and the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

El 9. The method of any one of embodiments El -El 8.1, wherein the nucleic acid hybridization assays comprise RT-PCR and/or sequencing.

E20. The method of any one of embodiments El -El 9, wherein the first CASI determined in step (al) and/or second CASI determined in step (a2) is between a value of 0 and 1. E21. The method of any one of embodiments E1-E20, wherein the first CASI determined in step (bl) and/or second CASI determined in step (b2) is between a value of 0 and 1.

E22. The method of any one of embodiments E1-E21, wherein the first CASI determined in step (cl) and/or second CASI determined in step (c2) is between a value of 0 and 1.

E23. The method of any one of embodiment E2-E22, wherein the first, second, and/or third ASI is between a value of 0 and 1.

E24. The method of any one of embodiments E1-E23, wherein the repeat expansion disease is associated with spliceopathy.

EQUIVALENTS AND TERMINOLOGY

[000345] The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are 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 there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure.

[000346] In addition, where features or aspects of the disclosure are described in terms of Markush groups or other grouping of alternatives, 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 or other group. [000347] It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or (e.g., and) one or more modified nucleotides and/or (e.g., and) one or more modified intemucleoside linkages and/or (e.g., and) one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

[000348] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (z.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[000349] Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

[000350] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. A method of monitoring a subject undergoing a treatment for a repeat expansion disease, the method comprising determining, during a period of time, at least two composite measures of splicing activity for a set of disease state-associated genes based on splicing events detected for one or more RNA transcripts of each gene of the set of disease state- associated genes in a nucleic acid sample obtained from the subject; and determining the disease state of the subject based on the at least two composite measures of splicing activity to thereby monitor the subject.

2. A method of monitoring a subject undergoing a treatment for a repeat expansion disease, the method comprising, over a period of time:

3. A method of evaluating effectiveness of a treatment for a repeat expansion disease in a subject undergoing the treatment for the repeat expansion disease, the method comprising, over a period of time:

4. The method of claim 2 or claim 3, wherein the first CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI for each gene is determined based on splicing events detected for one or more RNA transcripts for the gene in a first nucleic acid sample obtained from the subject.

5. The method of any one of claims 2-4, wherein the second CASI is determined based on an alternative splicing index (ASI) for each gene of the set of disease state-associated genes, wherein the ASI is determined based on splicing events detected for one or more RNA transcripts of the gene in a second nucleic acid sample obtained from the subject.

6. The method of claim 5, wherein the first nucleic acid sample is obtained from the subject before treatment and the second nucleic acid sample is obtained from the subject after treatment.

7. The method of claim 5, wherein the first nucleic acid sample is obtained from the subject earlier in time than the second nucleic acid sample during a period in which the subject is undergoing the treatment.

8. The method of any one of claims 3-7, wherein if the first CASI is larger than the second CASI, the treatment is effective.

9. The method of any one of claims 2-8, wherein the treatment comprises administering to the subject an agent that treats the repeat-expansion disease.

10. The method of claim 9, wherein the repeat expansion disease is DM1 and the agent comprises a complex comprising an anti-TfRl antibody covalently linked to an oligonucleotide that targets a DMPK RNA.

11. The method of any one of claims 2-10, wherein the set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and OPA1.

12. The method of any one of claims 2-11, wherein the set of disease state-associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SCSI, CLCN1, and MBNL1.

13. The method of any one of claims 2-12, wherein the set of disease state-associated genes comprises CACNA1S, CAPZB, GOLGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI.

14. The method of any one of claims 5-13, wherein the first nucleic acid sample and/or the second nucleic acid sample is obtained from a tissue sample obtained from the subject.

15. The method of claim 14, wherein the tissue sample is a muscle biopsy sample.

16. The method of any one of claims 5-13, wherein the first nucleic acid sample and/or the second nucleic acid sample is obtained from a blood sample obtained from the subject.

17. The method of any one of claims 4-16, wherein the splicing events are detected via subjecting the first nucleic acid sample and/or the second nucleic acid sample to nucleic acid hybridization assays.

18. The method of claim 17, wherein the nucleic acid hybridization assays comprise RT- PCR and/or sequencing.

19. The method of any one of claims 2-18, wherein the first CASI determined in step (a) and/or the second CASI determined in step (b) is between a value of 0 and 1.

20. The method of any one of claims 2-19, wherein the repeat expansion disease is associated with spliceopathy.

21. A method comprising:

22. A method of evaluating a disease state of a subject who has or is suspected of having a repeat expansion disease, the method comprising: (a) determining an alternative splicing index (ASI) for each gene of a first set of disease state-associated genes and for each gene of a second set of disease state- associated genes based on detected splicing events for one or more RNA transcripts of the respective genes detected in a nucleic acid sample obtained from a subject, wherein the splicing events are detected using nucleic acid hybridization assays; and

23. A method of determining a disease state of a subject who has or is suspected of having a repeat expansion disease, the method comprising:

(a3) determining a first comparator between the first CASI of step (al) and the second CASI of step (a2), wherein the first set of disease state-associated genes comprises CCPG1, KIF13A, INSR, CAMK2B, and 0PA1;

(b2) determining a second CASI for the second set of disease state- associated genes, (b3) determining a second comparator between the second CASI of step (bl) and the second CASI of step (b2), wherein the second set of disease state- associated genes comprises RYR1, MBNL2, NFIX, CLASP1, VPS39, BEST3, SCSI, CLCN1, and MBNL1;

(cl) determining a first CASI for a third set of disease state-associated genes, (c2) determining a second CASI for the third set of disease state-associated genes,

(c3) determining a third comparator between the first CASI of step (cl) and the second CASI of step (c2), wherein the third set of disease state- associated genes comprises CACNA1S, CAPZB, G0LGA4, GFPT1, ATP2A1, ANK2, DMD, and BINI; and