Anti-TfR:GAA and anti-CD63:GAA insertions for the treatment of Pompe disease

Abstract

Nucleic acid constructs and compositions are provided that allow for the insertion of a multidomain therapeutic protein (e.g., a GAA fusion protein) coding sequence into a target genomic locus, such as an endogenous ALB locus, and / or the expression of a multidomain therapeutic protein (e.g., a GAA fusion protein) coding sequence. The nucleic acid constructs and compositions may be used in methods of incorporating a multidomain therapeutic protein (e.g., a GAA fusion protein) nucleic acid into a target genomic locus, expressing a multidomain therapeutic protein (e.g., a GAA fusion protein) in a cell, reducing glycogen accumulation, treating Pompe disease or GAA deficiency in a subject, and preventing or reducing the onset of signs or symptoms of Pompe disease in a subject (including newborn cells and subjects).

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Patent Application No. 63 / 306,040, filed February 2, 2022, and U.S. Patent Application No. 63 / 369,902, filed July 29, 2022, each of which is incorporated herein by reference in its entirety.

[0002] Reference to a sequence listing submitted as an XML file via EFS WEB The sequence listing set forth in file 590204SEQLIST.xml is 1.16 megabytes, was created on January 26, 2023, and is incorporated herein by reference. [Background technology]

[0003] Gene therapy has long been recognized as having enormous potential in terms of how human diseases are approached and treated. Instead of relying on drugs or surgery, patients with underlying genetic factors can be treated by directly targeting the underlying cause. Furthermore, by targeting the underlying genetic cause, gene therapy may offer the possibility of effectively curing patients. However, the clinical application of gene therapy approaches still requires improvement in several aspects. Furthermore, treatment at an early age may present additional obstacles due to the unique environment in neonatal patients.

[0004] Pompe disease (PD), or glycogen storage disease type II, is a monogenic lysosomal disorder caused by deficient activity of the enzyme lysosomal acid α-glucosidase (GAA). GAA deficiency leads to the accumulation of its substrate, glycogen, in the lysosomes of cells in tissues, including skeletal and cardiac muscles. This abnormal accumulation of glycogen in muscle fibers leads to progressive damage to muscle tissue, with symptoms that can include cardiac hypertrophy, mild to severe muscle weakness, and ultimately death due to cardiac or respiratory failure. Infantile-onset PD (IOPD) is associated with GAA activity less than 1% of normal. IOPD is severe, affecting visceral organs, muscles, and the central nervous system (CNS). Late-onset PD (LOPD) is associated with GAA activity between 2% and 40% of normal. Late-onset PD (LOPD) is less severe and primarily involves respiratory and skeletal muscles.

[0005] The only approved therapy for PD is enzyme replacement therapy (ERT). Recombinant human (rh) GAA is delivered to patients by intravenous infusion every two weeks. While ERT has been highly successful in treating cardiac symptoms of PD, skeletal muscle and the CNS remain minimally treated by ERT. The primary mechanism by which rhGAA reaches lysosomes is through uptake by the cation-independent mannose 6-phosphate (M6P) receptor (CIMPR), which binds to M6P on rhGAA. However, CI-MPR expression in skeletal muscle is very low, and rhGAA is poorly mannose 6-phosphorylated. Furthermore, CI-MPR can be misdirected to autophagosomes in diseased cells rather than lysosomes, but large amounts of the drug are also taken up by the liver, an organ that does not have major pathology in PD. ERT does not cross the blood-brain barrier. Additionally, PD may require treatment at a young age, which presents additional obstacles due to the unique environment in newborns and young patients. Summary of the Invention

[0006] Nucleic acid constructs and compositions are provided that enable insertion of a multidomain therapeutic protein (e.g., a GAA fusion protein) coding sequence into a target genomic locus, such as the endogenous ALB locus, and / or expression of a multidomain therapeutic protein (e.g., a GAA fusion protein) coding sequence. The nucleic acid constructs and compositions can be used in methods for incorporating or inserting a multidomain therapeutic protein (e.g., a GAA fusion protein) nucleic acid into a target genomic locus in a cell or population of cells or a subject, for expressing a multidomain therapeutic protein (e.g., a GAA fusion protein) in a cell or population of cells or a subject, for reducing glycogen accumulation in a cell or population of cells or a subject, for treating Pompe disease or GAA deficiency in a subject, and for preventing or reducing the onset of signs or symptoms of Pompe disease in subjects diagnosed with Pompe disease, including subjects with reduced GAA activity or expression, and including neonatal subjects. In some embodiments, the cell, population of cells, or subject is a neonatal cell, population of neonatal cells, or neonatal subject.

[0007] In one aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in a neonatal cell or population of neonatal cells, where the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to the neonatal cell or population of neonatal cells: (a) a nucleic acid construct comprising a coding sequence for the multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase, where the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, where the nuclease agent targets a nuclease target site in the target genomic locus, where the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In one aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase into a target genomic locus in a neonatal cell or population of neonatal cells. Some such methods include administering to a neonatal cell or population of neonatal cells: (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In another aspect, methods are provided for expressing a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase in a neonatal cell or population of neonatal cells, wherein the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain.In another aspect, provided is a method of expressing a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase from a target genomic locus in a neonatal cell or population of neonatal cells, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to a neonatal cell or population of neonatal cells (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase in a neonatal cell or population of neonatal cells. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase from a targeted genomic locus in a neonatal cell or population of neonatal cells.Some such methods include administering to a neonatal cell or population of neonatal cells (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus. In some such methods, the neonatal cell is a liver cell, or the population of neonatal cells is a liver cell population. In some such methods, the neonatal cell is a hepatocyte, or the population of neonatal cells is a hepatocyte population. In some such methods, the neonatal cell is a human cell, or the population of neonatal cells is a human cell population. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 24 weeks of birth. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 12 weeks of birth. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 8 weeks of birth. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 4 weeks of birth. In some such methods, the neonatal cells are in vitro or ex vivo, or the population of neonatal cells is in vitro or ex vivo. In some such methods, the neonatal cells are in vivo in a neonatal subject, or the population of neonatal cells is in vivo in a neonatal subject.

[0008] In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in a neonatal cell or population of neonatal cells of a neonatal subject, where the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase, where the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, where the nuclease agent targets a nuclease target site in the target genomic locus, where the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in neonatal cells or a population of neonatal cells of a neonatal subject. Some such methods include administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In another aspect, methods are provided for expressing a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase protein in neonatal cells or a population of neonatal cells in a neonatal subject, wherein the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain.In another aspect, provided is a method of expressing a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase protein from a target genomic locus in a neonatal cell or population of neonatal cells of a neonatal subject, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, where the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase protein is expressed from the modified target genomic locus. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase protein in neonatal cells or a population of neonatal cells of a neonatal subject. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase protein from a target genomic locus in a neonatal cell or population of neonatal cells of a neonatal subject.Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus. In some such methods, the expressed multidomain therapeutic protein is delivered to and internalized by skeletal muscle and cardiac tissues of the subject. In some such methods, the neonatal cells are liver cells, or the population of neonatal cells is a population of liver cells. In some such methods, the neonatal cells are hepatocytes, or the population of neonatal cells is a population of hepatocytes. In some such methods, the neonatal cells are human cells or the population of neonatal cells is a population of human cells. In some such methods, the subject has Pompe disease.

[0009] In another aspect, methods are provided for treating lysosomal α-glucosidase deficiency in a neonatal subject in need thereof. Some such methods include administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising the delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus. Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus. In another aspect, a method of reducing glycogen accumulation in a tissue of a neonatal subject in need of treatment is provided.Some such methods include administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain; and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, wherein the nuclease agent cleaves the nuclease target site, wherein the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and wherein the multidomain therapeutic protein comprising the delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus and reduces glycogen accumulation in the tissue. Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus to reduce glycogen accumulation in the tissue. In some such methods, the subject has Pompe disease. In another aspect, a method of treating Pompe disease in a neonatal subject in need thereof is provided.Some such methods include administering to a neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain; and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; the nuclease agent cleaves the nuclease target site; the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus; and the multidomain therapeutic protein comprising the delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby treating Pompe disease. Some such methods include administering to a neonatal subject (a) a nucleic acid construct including a coding sequence for a multidomain therapeutic protein including a CD63-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein including a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby treating Pompe disease. In some such methods, the Pompe disease is infantile-onset Pompe disease. In some such methods, the method results in a therapeutically effective level of circulating the multidomain therapeutic protein or lysosomal α-glucosidase in the subject. In some such methods, the method reduces glycogen accumulation in skeletal muscle tissue, cardiac tissue, or central nervous system tissue of the subject. In some such methods, the method reduces glycogen accumulation in skeletal muscle tissue and cardiac tissue of the subject. In some such methods, the method results in a reduction of glycogen levels in the skeletal muscle tissue and / or cardiac tissue of the subject that is comparable to wild-type levels at the same age.In some such methods, the method improves muscle strength in the subject or prevents loss of muscle strength in the subject compared to a control subject. In some such methods, the method results in the subject having muscle strength comparable to wild-type levels at the same age.

[0010] In another aspect, methods are provided for preventing or reducing the onset of signs or symptoms of Pompe disease in a neonatal subject in need thereof. Some such methods include administering to the neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising the delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject. Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site at a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject. In some such methods, the Pompe disease is infantile-onset Pompe disease. In some such methods, the Pompe disease is late-onset Pompe disease. In some such methods, the method results in a therapeutically effective level of circulating multidomain therapeutic protein or lysosomal α-glucosidase in the subject. In some such methods, the method prevents or reduces glycogen accumulation in skeletal muscle, cardiac, or central nervous system tissue of the subject.In some such methods, the method prevents or reduces glycogen accumulation in skeletal muscle and cardiac tissue of the subject.

[0011] In some such methods, the neonatal subject is a human subject within 24 weeks of birth. In some such methods, the neonatal subject is a human subject within 12 weeks of birth. In some such methods, the neonatal subject is a human subject within 8 weeks of birth. In some such methods, the neonatal subject is a human subject within 4 weeks of birth.

[0012] In some such methods, the method results in increased expression of the multidomain therapeutic protein in the subject compared to a method comprising administering an episomal expression vector encoding the multidomain therapeutic protein to a control subject. In some such methods, the method results in increased serum levels of the multidomain therapeutic protein in the subject compared to a method comprising administering an episomal expression vector encoding the multidomain therapeutic protein to a control subject. In some such methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, at least about 9 μg / mL, or at least about 10 μg / mL. In some such methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 2 μg / mL or at least about 5 μg / mL. In some such methods, the methods result in serum levels of the multidomain therapeutic protein in the subject of about 2 μg / mL to about 30 μg / mL or about 2 μg / mL to about 20 μg / mL. In some such methods, the methods result in serum levels of the multidomain therapeutic protein in the subject of about 5 μg / mL to about 30 μg / mL or about 5 μg / mL to about 20 μg / mL. In some such methods, the methods achieve a lysosomal α-glucosidase activity level of at least about 40% of normal, at least about 45% of normal, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of normal. In some such methods, the subject has infantile-onset Pompe disease and the methods achieve a lysosomal α-glucosidase activity level of at least about 1% or greater than about 1% of normal. In some such methods, the subject has late-onset Pompe disease and the method achieves a lysosomal α-glucosidase activity level that is at least about 40% of normal, or greater than about 40% of normal.In some such methods, the methods increase lysosomal α-glucosidase activity by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% over the subject's baseline lysosomal α-glucosidase activity. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at the peak level of expression measured in the subject 6 months after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at the peak level of expression measured in the subject 1 year after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 6 months after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 2 years after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 2 years after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 6 months after administration.

[0013] In some such methods, the subject is a human subject.

[0014] In some such methods, the method further comprises assessing pre-existing AAV immunity in the neonatal subject prior to administering the nucleic acid construct to the subject. In some such methods, the pre-existing AAV immunity is pre-existing AAV8 immunity. In some such methods, assessing pre-existing AAV immunity comprises assessing immunogenicity using a total antibody immunoassay or a neutralizing antibody assay.

[0015] In some such methods, the nucleic acid construct is administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is not administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered before the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered after the nuclease agent or one or more nucleic acids encoding the nuclease agent.

[0016] In some such methods, the CD63-binding delivery domain is fused to the lysosomal α-glucosidase protein via a peptide linker. In some such methods, the coding sequence for the CD63-binding delivery domain is codon-optimized or CpG-depleted. In some such methods, the coding sequence for the CD63-binding delivery domain is codon-optimized and CpG-depleted. In some such methods, the CD63-binding delivery domain comprises an anti-CD63 antigen binding protein. In some such methods, the CD63-binding delivery domain comprises an anti-CD63 antibody, antibody fragment, or single-chain variable fragment (scFv). In some such methods, the CD63-binding delivery domain is a single-chain variable fragment (scFv). In some such methods, the scFv comprises the sequence set forth in SEQ ID NO: 183. In some such methods, the scFv consists of the sequence set forth in SEQ ID NO: 183. In some such methods, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192, and optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186. In some such methods, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192 and encodes an scFv comprising SEQ ID NO: 183; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186 and encodes an scFv comprising SEQ ID NO: 183.In some such methods, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 183, and optionally the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 183. In some such methods, the scFv coding sequence comprises the sequence set forth in any of SEQ ID NOs: 184-192, and optionally the scFv coding sequence comprises the sequence set forth in SEQ ID NO: 186. In some such methods, the scFv coding sequence consists of the sequence set forth in any of SEQ ID NOs: 184-192; optionally, the scFv coding sequence consists of the sequence set forth in SEQ ID NO:186.

[0017] In some such methods, the lysosomal α-glucosidase lacks the lysosomal α-glucosidase signal peptide and propeptide. In some such methods, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized or CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173.In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such methods, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176. In some such methods, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0018] In some such methods, the lysosomal α-glucosidase lacks the lysosomal α-glucosidase signal peptide and propeptide. In some such methods, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized or CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182, and optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173.In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such methods, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176. In some such methods, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0019] In some such methods, the coding sequence of the multidomain therapeutic protein is codon-optimized or CpG-depleted. In some such methods, the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted. In some such methods, the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193. In some such methods, the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 193. In some such methods, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:194-202; optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:196; and optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:736.In some such methods, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202, wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193; and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 99% identical to SEQ ID NO: 196. and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193.In some such methods, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOS: 194-202, is codon-optimized, and is CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193; and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identical to SEQ ID NO: 196. , at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and is codon-optimized and CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193. In some such methods, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 194-202, and optionally the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736. In some such methods, the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in any of SEQ ID NOs: 194-202, optionally, the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 196, and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736.

[0020] In some such methods, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence of the multidomain therapeutic protein. In some such methods, the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence of the multidomain therapeutic protein. In some such methods, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence of the multidomain therapeutic protein and the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence of the multidomain therapeutic protein. In some such methods, the nucleic acid construct does not comprise homology arms. In some such methods, the nucleic acid construct is inserted into the target genomic locus via non-homologous end joining. In some such methods, the nucleic acid construct comprises homology arms. In some such methods, the nucleic acid construct is inserted into the target genomic locus via homology directed repair. In some such methods, the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein. In some such methods, the coding sequence of the multidomain therapeutic protein is operably linked to a promoter, optionally a liver-specific promoter. In some such methods, the nucleic acid construct is single-stranded DNA or double-stranded DNA. In some such methods, the nucleic acid construct is single-stranded DNA. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise homology arms.

[0021] In some such methods, the nucleic acid construct is in a nucleic acid vector or lipid nanoparticle. In some such methods, the nucleic acid construct is in a nucleic acid vector. In some such methods, the nucleic acid vector is a viral vector. In some such methods, the nucleic acid vector is an adeno-associated viral (AAV) vector, and optionally, the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally, the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 160, and optionally, the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 160. In some such methods, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such methods, the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. In some such methods, the AAV vector is a recombinant AAV8 (rAAV8) vector. In some such methods, the AAV vector is a single-stranded rAAV8 vector. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the nucleic acid construct is CpG-depleted.

[0022] In some such methods, the target genomic locus is an albumin gene, and optionally, the albumin gene is a human albumin gene. In some such methods, the nuclease target site is within intron 1 of the albumin gene. In some such methods, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) (i) a Cas protein or a nucleic acid encoding a Cas protein, and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0023] In some such methods, the nuclease agent comprises (a) a Cas protein or a nucleic acid encoding a Cas protein, and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the guide RNA target sequence is within intron 1 of the albumin gene. In some such methods, the albumin gene is a human albumin gene. In some such methods, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any of SEQ ID NOs: 30-61, and optionally, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the DNA-targeting segment comprises any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment comprises any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the DNA-targeting segment consists of any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment consists of any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the guide RNA comprises any of SEQ ID NOs: 62-125, optionally the guide RNA comprises any of SEQ ID NOs: 68, 100, 62, 94, 65, 97, 73, and 105. In some such methods, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides set forth in SEQ ID NO:36.In some such methods, the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 36. In some such methods, the DNA-targeting segment comprises SEQ ID NO: 36. In some such methods, the DNA-targeting segment consists of SEQ ID NO: 36. In some such methods, the guide RNA comprises SEQ ID NO: 68 or 100.

[0024] In some such methods, the method comprises administering a guide RNA in the form of RNA. In some such methods, the guide RNA comprises at least one modification. In some such methods, the at least one modification comprises a 2'-O-methyl modified nucleotide. In some such methods, the at least one modification comprises an internucleotide phosphorothioate bond. In some such methods, the at least one modification comprises a modification in one or more of the first five nucleotides at the 5' end of the guide RNA. In some such methods, the at least one modification comprises a modification in one or more of the last five nucleotides at the 3' end of the guide RNA. In some such methods, the at least one modification comprises a phosphorothioate bond between the first four nucleotides at the 5' end of the guide RNA. In some such methods, the at least one modification comprises a phosphorothioate bond between the last four nucleotides at the 3' end of the guide RNA. In some such methods, the at least one modification comprises a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA. In some such methods, the at least one modification comprises 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA. In some such methods, the at least one modification comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA, and (iv) a 2'-O-methyl modified nucleotide in the last three nucleotides at the 3' end of the guide RNA. In some such methods, the guide RNA is a single guide RNA (sgRNA).In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, and wherein the guide RNA comprises (i) phosphorothioate linkages between the first four nucleotides at the 5' end of the guide RNA, (ii) phosphorothioate linkages between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0025] In some such methods, the Cas protein is a Cas9 protein. In some such methods, the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein. In some such methods, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such methods, the Cas protein comprises the sequence set forth in SEQ ID NO: 11. In some such methods, the nucleic acid encoding the Cas protein is codon-optimized for expression in mammalian or human cells. In some such methods, the method comprises administering a nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such methods, the mRNA encoding the Cas protein comprises at least one modification. In some such methods, the mRNA encoding the Cas protein is modified to comprise modified uridines at one or more or all uridine positions. In some such methods, the modified uridine is pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such methods, the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such methods, the modified uridine is pseudouridine. In some such methods, the mRNA encoding the Cas protein is fully substituted with pseudouridine. In some such methods, the mRNA encoding the Cas protein comprises a 5' cap. In some such methods, the mRNA encoding the Cas protein comprises a polyadenylation sequence.In some such methods, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12. In some such methods, the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence. In some such methods, the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence.

[0026] In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a polyadenylation sequence. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0027] In some such methods, the Cas protein or a nucleic acid encoding the Cas protein and the guide RNA or one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such methods, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such methods, the cationic lipid is lipid A((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate). In some such methods, the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some such methods, the helper lipid is cholesterol. In some such methods, the stealth lipid is PEG2k-DMG. In some such methods, the cationic lipid is lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such methods, the lipid nanoparticles comprise four lipids in the following molar ratio: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0028] In some such methods, the albumin gene is a human albumin gene, the method comprises administering a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 68 or 100, the method comprises administering a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 226, 225, or 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0029] In some such methods, the albumin gene is a human albumin gene, the method comprises administering a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, the method comprises administering a nucleic acid encoding a Cas protein, The acid comprises an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such methods, the albumin gene is a human albumin gene, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the method comprises administering a Cas protein wherein the nucleic acid comprises an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a polyadenylation sequence, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0030] In another aspect, a neonatal cell or population of neonatal cells produced by any of the above methods is provided. In another aspect, a cell or population of cells produced by any of the above methods is provided. In another aspect, a neonatal cell or population of neonatal cells is provided that comprises a nucleic acid construct inserted into a target genomic locus, the nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase inserted into the target genomic locus, optionally the delivery domain being a CD63-binding delivery domain or a TfR-binding delivery domain. In another aspect, a neonatal cell or population of neonatal cells is provided that comprises a nucleic acid construct inserted into a target genomic locus, the nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase inserted into the target genomic locus. In some such neonatal cells or populations of neonatal cells, the neonatal cells are liver cells, or the population of neonatal cells is a population of liver cells. In some such neonatal cells or populations of neonatal cells, the neonatal cells are hepatocytes or the population of neonatal cells is a population of hepatocytes. In some such neonatal cells or populations of neonatal cells, the neonatal cells are human cells or the population of neonatal cells is a population of human cells. In some such neonatal cells or populations of neonatal cells, the neonatal cells or population of neonatal cells are from a human neonatal subject within 24 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells or population of neonatal cells are from a human neonatal subject within 12 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells or population of neonatal cells are from a human neonatal subject within 8 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells or population of neonatal cells are from a human neonatal subject within 4 weeks of birth.In some such neonatal cells or populations of neonatal cells, the neonatal cells are in vitro or ex vivo, or the population of neonatal cells is in vitro or ex vivo. In some such neonatal cells or populations of neonatal cells, the neonatal cells are in vivo in a subject, or the population of neonatal cells is in vivo. In some such neonatal cells or populations of neonatal cells, a multidomain therapeutic protein is expressed.

[0031] In some such neonatal cells or populations of neonatal cells, the CD63-binding delivery domain is fused to a lysosomal α-glucosidase protein via a peptide linker. In some such neonatal cells or populations of neonatal cells, the coding sequence for the CD63-binding delivery domain is codon-optimized or CpG-depleted. In some such neonatal cells or populations of neonatal cells, the coding sequence for the CD63-binding delivery domain is codon-optimized and CpG-depleted. In some such neonatal cells or populations of neonatal cells, the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein. In some such neonatal cells or populations of neonatal cells, the CD63-binding delivery domain comprises an anti-CD63 antibody, antibody fragment, or single-chain variable fragment (scFv). In some such neonatal cells or populations of neonatal cells, the CD63-binding delivery domain is a single-chain variable fragment (scFv). In some such neonatal cells or populations of neonatal cells, the scFv comprises the sequence set forth in SEQ ID NO: 183. In some such neonatal cells or populations of neonatal cells, the scFv consists of the sequence set forth in SEQ ID NO: 183. In some such neonatal cells or populations of neonatal cells, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192, and optionally the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186.In some such neonatal cells or populations of neonatal cells, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192 and encodes an scFv comprising SEQ ID NO: 183; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186 and encodes an scFv comprising SEQ ID NO: 183. In some such neonatal cells or populations of neonatal cells, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 183; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 183. In some such neonatal cells or populations of neonatal cells, the scFv coding sequence comprises the sequence set forth in any of SEQ ID NOs: 184-192. Optionally, the scFv coding sequence comprises the sequence set forth in SEQ ID NO: 186. In some such neonatal cells or populations of neonatal cells, the scFv coding sequence consists of the sequence set forth in any of SEQ ID NOs: 184 to 192. Optionally, the scFv coding sequence consists of the sequence set forth in SEQ ID NO: 186.

[0032] In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase lacks a lysosomal α-glucosidase signal peptide and propeptide. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is codon-optimized or CpG-depleted. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such neonatal cells or populations of neonatal cells, the lysosomal alpha-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal alpha-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176.In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 174-182 and 205-212 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO: 173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such neonatal cells or populations of neonatal cells, the lysosomal alpha-glucosidase coding sequence comprises a sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal alpha-glucosidase coding sequence comprises a sequence set forth in SEQ ID NO: 176.In some such neonatal cells or populations of neonatal cells, the lysosomal alpha-glucosidase coding sequence consists of any of the sequences set forth in SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal alpha-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0033] In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase lacks a lysosomal α-glucosidase signal peptide and propeptide. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is codon-optimized or CpG-depleted. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such neonatal cells or populations of neonatal cells, the lysosomal alpha-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 174-182, and optionally, the lysosomal alpha-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176.In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 174-182 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO: 173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 174-182, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO: 173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO: 173. In some such neonatal cells or populations of neonatal cells, the lysosomal alpha-glucosidase coding sequence comprises a sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal alpha-glucosidase coding sequence comprises a sequence set forth in SEQ ID NO: 176.In some such neonatal cells or populations of neonatal cells, the lysosomal alpha-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal alpha-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0034] In some such neonatal cells or populations of neonatal cells, the coding sequence for the multidomain therapeutic protein is codon-optimized or CpG-depleted. In some such neonatal cells or populations of neonatal cells, the coding sequence for the multidomain therapeutic protein is codon-optimized and CpG-depleted. In some such neonatal cells or populations of neonatal cells, the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193. In some such neonatal cells or populations of neonatal cells, the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 193. In some such neonatal cells or populations of neonatal cells, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:194-202, and optionally the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:196, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:736.In some such neonatal cells or populations of neonatal cells, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202, wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193; and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202. 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193.In some such neonatal cells or populations of neonatal cells, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202, is codon-optimized, and is CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193; and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202, is codon-optimized, and is CpG-depleted, and and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and wherein the coding sequence for the multidomain therapeutic protein is codon-optimized and CpG-depleted, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193. In some such neonatal cells or populations of neonatal cells, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 194-202, and optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736. In some such neonatal cells or populations of neonatal cells, the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in any of SEQ ID NOs: 194-202, optionally the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 196, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736.

[0035] In some such neonatal cells or populations of neonatal cells, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein. In some such neonatal cells or populations of neonatal cells, the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein. In some such neonatal cells or populations of neonatal cells, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein and the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein. In some such neonatal cells or populations of neonatal cells, the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, and the coding sequence for the multidomain therapeutic protein is operably linked to an endogenous promoter at the target genomic locus. In some such neonatal cells or populations of neonatal cells, the coding sequence for the multidomain therapeutic protein is operably linked to a promoter, optionally a liver-specific promoter. In some such neonatal cells or populations of neonatal cells, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, and wherein the coding sequence for the multidomain therapeutic protein is operably linked to an endogenous promoter at the target genomic locus.

[0036] In some such neonatal cells or populations of neonatal cells, the target genomic locus is an albumin gene, and optionally, the albumin gene is a human albumin gene. In some such neonatal cells or populations of neonatal cells, the nuclease target site is within intron 1 of the albumin gene.

[0037] In another aspect, a composition is provided comprising a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase, wherein the lysosomal α-glucosidase coding sequence is CpG-depleted relative to the wild-type lysosomal α-glucosidase coding sequence, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. In another aspect, a composition is provided comprising a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase, wherein the lysosomal α-glucosidase coding sequence is CpG-depleted relative to the wild-type lysosomal α-glucosidase coding sequence. In some such compositions, the CD63-binding delivery domain is fused to the lysosomal α-glucosidase protein via a peptide linker. In some such compositions, the lysosomal α-glucosidase lacks the lysosomal α-glucosidase signal peptide and propeptide. In some such compositions, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such compositions, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such compositions, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such compositions, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176.In some such compositions, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such compositions, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such compositions, the lysosomal α-glucosidase coding sequence comprises a sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence comprises a sequence set forth in SEQ ID NO: 176.In some such compositions, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0038] In another aspect, a composition is provided comprising a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase, wherein the lysosomal α-glucosidase coding sequence is CpG-depleted relative to the wild-type lysosomal α-glucosidase coding sequence. In some such compositions, the CD63-binding delivery domain is fused to the lysosomal α-glucosidase protein via a peptide linker. In some such compositions, the lysosomal α-glucosidase lacks the lysosomal α-glucosidase signal peptide and propeptide. In some such compositions, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such compositions, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such compositions, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such compositions, the lysosomal alpha-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182, and optionally, the lysosomal alpha-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176.In some such compositions, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such compositions, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such compositions, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176. In some such compositions, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0039] In some such compositions, the coding sequence for the CD63-binding delivery domain is codon-optimized or CpG-depleted. In some such compositions, the coding sequence for the CD63-binding delivery domain is codon-optimized and CpG-depleted. In some such compositions, the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein. In some such compositions, the CD63-binding delivery domain comprises an anti-CD63 antibody, antibody fragment, or single-chain variable fragment (scFv). In some such compositions, the CD63-binding delivery domain is a single-chain variable fragment (scFv). In some such compositions, the scFv comprises the sequence set forth in SEQ ID NO: 183. In some such compositions, the scFv consists of the sequence set forth in SEQ ID NO: 183. In some such compositions, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192, and optionally the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186. In some such compositions, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192 and encodes an scFv comprising SEQ ID NO: 183; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186 and encodes an scFv comprising SEQ ID NO: 183.In some such compositions, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 184-192, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 183; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 183. In some such compositions, the scFv coding sequence comprises the sequence set forth in any of SEQ ID NOs: 184-192, and optionally, the scFv coding sequence comprises the sequence set forth in SEQ ID NO: 186. In some such compositions, the scFv coding sequence consists of the sequence set forth in any of SEQ ID NOs: 184-192, and optionally, the scFv coding sequence consists of the sequence set forth in SEQ ID NO:186.

[0040] In some such compositions, the coding sequence of the multidomain therapeutic protein is codon-optimized or CpG-depleted. In some such compositions, the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted. In some such compositions, the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193. In some such compositions, the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 193. In some such compositions, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:194-202; optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:196; and optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:736.In some such compositions, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202, wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193; and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 99% identical to SEQ ID NO: 196. and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193.In some such compositions, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 194-202, is codon-optimized, and is CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193; and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identical to SEQ ID NO: 196. , at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and is codon-optimized and CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 736, and the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 193. In some such compositions, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 194-202. Optionally, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736. In some such compositions, the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in any of SEQ ID NOs: 194-202, optionally the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 196, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736.

[0041] In some such compositions, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein. In some such compositions, the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein. In some such compositions, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein and the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein. In some such compositions, the nucleic acid construct does not comprise a homology arm. In some such compositions, the nucleic acid construct comprises a homology arm. In some such compositions, the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein. In some such compositions, the nucleic acid construct is single-stranded or double-stranded DNA. In some such compositions, the nucleic acid construct is single-stranded DNA. In some such compositions, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise homology arms.

[0042] In some such compositions, the nucleic acid construct is in a nucleic acid vector or lipid nanoparticle. In some such compositions, the nucleic acid construct is in a nucleic acid vector. In some such compositions, the nucleic acid vector is a viral vector. In some such compositions, the nucleic acid vector is an adeno-associated viral (AAV) vector, and optionally, the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally, the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 160, and optionally, the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 160. In some such compositions, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such compositions, the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. In some such compositions, the AAV vector is a recombinant AAV8 (rAAV8) vector. In some such compositions, the AAV vector is a single-stranded rAAV8 vector. In some such compositions, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such compositions, the nucleic acid construct is CpG-depleted.

[0043] In some such compositions, the composition further comprises a nuclease agent that targets a nuclease target site in the target genomic locus. In some such compositions, the target genomic locus is an albumin gene, and optionally, the albumin gene is a human albumin gene. In some such compositions, the nuclease target site is within intron 1 of the albumin gene. In some such compositions, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) (i) a Cas protein or a nucleic acid encoding a Cas protein, and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0044] In some such compositions, the nuclease agent comprises (a) a Cas protein or a nucleic acid encoding a Cas protein, and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such compositions, the guide RNA target sequence is within intron 1 of the albumin gene. In some such compositions, the albumin gene is a human albumin gene. In some such compositions, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any of SEQ ID NOs: 30-61, and optionally, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the DNA-targeting segment comprises any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment comprises any of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the DNA-targeting segment consists of any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment consists of any of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the guide RNA comprises any of SEQ ID NOs: 62-125, optionally the guide RNA comprises any of SEQ ID NOs: 68, 100, 62, 94, 65, 97, 73, and 105. In some such compositions, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides set forth in SEQ ID NO:36.In some such compositions, the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 36. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 36. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 36. In some such compositions, the guide RNA comprises SEQ ID NO: 68 or 100.

[0045] In some such compositions, the guide RNA is in the form of RNA. In some such compositions, the guide RNA comprises at least one modification. In some such compositions, the at least one modification comprises a 2'-O-methyl modified nucleotide. In some such compositions, the at least one modification comprises an internucleotide phosphorothioate bond. In some such compositions, the at least one modification comprises a modification in one or more of the first five nucleotides at the 5' end of the guide RNA. In some such compositions, the at least one modification comprises a modification in one or more of the last five nucleotides at the 3' end of the guide RNA. In some such compositions, the at least one modification comprises a phosphorothioate bond between the first four nucleotides at the 5' end of the guide RNA. In some such compositions, the at least one modification comprises a phosphorothioate bond between the last four nucleotides at the 3' end of the guide RNA. In some such compositions, the at least one modification comprises a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA. In some such compositions, at least one modification comprises 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA. In some such compositions, at least one modification comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA, and (iv) a 2'-O-methyl modified nucleotide in the last three nucleotides at the 3' end of the guide RNA. In some such compositions, the guide RNA is a single guide RNA (sgRNA).In some such compositions, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 100, and the guide RNA comprises (i) phosphorothioate linkages between the first four nucleotides at the 5' end of the guide RNA, (ii) phosphorothioate linkages between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0046] In some such compositions, the Cas protein is a Cas9 protein. In some such compositions, the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein. In some such compositions, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such compositions, the Cas protein comprises the sequence set forth in SEQ ID NO: 11. In some such compositions, the nucleic acid encoding the Cas protein is codon-optimized for expression in mammalian or human cells. In some such compositions, the composition comprises a nucleic acid encoding the Cas protein, and the nucleic acid comprises an mRNA encoding the Cas protein. In some such compositions, the mRNA encoding the Cas protein comprises at least one modification. In some such compositions, the mRNA encoding the Cas protein is modified to include modified uridines at one or more or all uridine positions. In some such compositions, the modified uridine is pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such compositions, the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such compositions, the modified uridine is pseudouridine. In some such compositions, the mRNA encoding the Cas protein is fully substituted with pseudouridine. In some such compositions, the mRNA encoding the Cas protein comprises a 5' cap. In some such compositions, the mRNA encoding the Cas protein comprises a polyadenylation sequence. In some such compositions, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12.In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence. In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence.

[0047] In some such compositions, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 68 or 100, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12. In some such compositions, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such compositions, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence. In some such compositions, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a polyadenylation sequence.In some such compositions, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0048] In some such compositions, the Cas protein or a nucleic acid encoding the Cas protein and the guide RNA or one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such compositions, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such compositions, the cationic lipid is lipid A((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate). In some such compositions, the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some such compositions, the helper lipid is cholesterol. In some such compositions, the stealth lipid is PEG2k-DMG. In some such compositions, the cationic lipid is lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such compositions, the lipid nanoparticles comprise four lipids in the following molar ratio: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0049] In some such compositions, the albumin gene is a human albumin gene, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 68 or 100, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG. In some such compositions, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such compositions, the albumin gene is a human albumin gene, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid is and a 5' cap and a polyadenylation sequence; wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the guide RNA is in the form of RNA, the guide RNA comprises SEQ ID NO: 100, and the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition encodes a Cas protein. the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such compositions, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises any of SEQ ID NOs: 194-202, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 196, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 736, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0050] In some such compositions, the composition is for use in a method of inserting a nucleic acid encoding a multidomain therapeutic protein into a target genomic locus in a cell or population of cells. In some such compositions, the composition is for use in a method of expressing a multidomain therapeutic protein from a target genomic locus in a cell or population of cells. In some such compositions, the composition is for use in a method of expressing a multidomain therapeutic protein in a cell or population of cells. In some such compositions, the composition is for use in a method of inserting a nucleic acid encoding a multidomain therapeutic protein into a target genomic locus in a cell or population of cells of a subject. In some such compositions, the composition is for use in a method of expressing a multidomain therapeutic protein from a target genomic locus in a cell or population of cells of a subject. In some such compositions, the composition is for use in a method of expressing a multidomain therapeutic protein in a cell or population of cells of a subject. In some such compositions, the cells are neonatal cells and the population of cells is a population of neonatal cells. In some such compositions, the cells are not neonatal cells and the population of cells is not a population of neonatal cells. In some such compositions, the composition is for use in a method of treating lysosomal α-glucosidase deficiency in a subject in need thereof. In some such compositions, the composition is for use in a method of reducing glycogen accumulation in tissues of a subject in need thereof. In some such compositions, the composition is for use in a method of treating Pompe disease in a subject in need thereof. In some such compositions, the composition is for use in a method of preventing or reducing the onset of signs or symptoms of Pompe disease in a newborn subject in need thereof. In some such compositions, the subject is a newborn subject. In some such compositions, the subject is not a newborn subject.

[0051] In another aspect, a cell comprising any of the above compositions is provided. In some such cells, the nucleic acid construct is integrated into a target genomic locus and the multidomain therapeutic protein is expressed from the target genomic locus, or the nucleic acid construct is integrated into intron 1 of the endogenous albumin locus and the multidomain therapeutic protein is expressed from the endogenous albumin locus. In some such cells, the cell is a human cell. In some such cells, the cell is a liver cell. In some such cells, the liver cell is a hepatocyte. In some such cells, the cell is a neonatal cell. In some such cells, the neonatal cell is from a human neonatal subject within 24 weeks of birth. In some such cells, the neonatal cell is from a human neonatal subject within 12 weeks of birth. In some such cells, the neonatal cell is from a human neonatal subject within 8 weeks of birth. In some such cells, the neonatal cell is from a human neonatal subject within 4 weeks of birth. In some such cells, the cell is not a neonatal cell. In some such cells, the cell is in vivo. In some such cells, the cells are in vitro or ex vivo.

[0052] In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in a cell or population of cells, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods comprise administering any of the above compositions to a cell or population of cells, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, a nuclease agent cleaving a nuclease target site in the target genomic locus, and the nucleic acid construct being inserted into the target genomic locus. In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in a cell or population of cells. Some such methods comprise administering any of the above compositions to a cell or population of cells, wherein the nuclease agent cleaves a nuclease target site in the target genomic locus, and the nucleic acid construct being inserted into the target genomic locus. In another aspect, methods are provided for expressing in a cell or population of cells a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods comprise administering any of the above compositions to a cell or population of cells, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, and wherein the coding sequence for the multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct and expressed in the cell or population of cells. In another aspect, methods are provided for expressing in a cell or population of cells a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase from a target genomic locus, optionally wherein the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain.Some such methods include administering any of the above-described compositions to a cell or population of cells, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, the nuclease agent cleaves the nuclease target site in the target genomic locus, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and a multidomain therapeutic protein comprising the delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase in a cell or population of cells. Some such methods include administering any of the above-described compositions to a cell or population of cells, where the coding sequence for the multidomain therapeutic protein is operably linked to a promoter in the nucleic acid construct and expressed in the cell or population of cells. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase from a target genomic locus in a cell or population of cells. Some such methods include administering any of the above compositions to a cell or population of cells, wherein the nuclease agent cleaves the nuclease target site in the target genomic locus, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus. In some such methods, the cell is a liver cell or the population of cells is a population of liver cells. In some such methods, the cell is a hepatocyte or the population of cells is a population of hepatocytes. In some such methods, the cell is a human cell or the population of cells is a population of human cells. In some such methods, the cell is a neonatal cell or the population of cells is a population of neonatal cells. In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 24 weeks of birth.In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 12 weeks of birth. In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 8 weeks of birth. In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 4 weeks of birth. In some such methods, the cell is not a neonatal cell, or the population of cells is not a population of neonatal cells. In some such methods, the cell is in vitro or ex vivo, or the population of cells is in vitro or ex vivo. In some such methods, the cell is in vivo in a subject, or the population of cells is in vivo in a subject.

[0053] In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in a cell or population of cells of a subject, where the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods comprise administering to a subject any of the above-described compositions, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, a nuclease agent cleaving a nuclease target site in the target genomic locus, and the nucleic acid construct being inserted into the target genomic locus. In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in a cell or population of cells of a subject. Some such methods comprise administering to a subject any of the above-described compositions, where optionally the nuclease agent cleaves a nuclease target site in the target genomic locus, and the nucleic acid construct being inserted into the target genomic locus. In another aspect, provided are methods for expressing a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase protein in a cell or population of cells of a subject, where the delivery domain is optionally a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods comprise administering any of the above compositions to a subject, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, and the coding sequence for the multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct and expressed in the cell. In another aspect, provided are methods for expressing a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase protein from a target genomic locus in a cell or population of cells of a subject, where optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain.Some such methods include administering any of the above-described compositions to a subject, wherein optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain, the nuclease agent cleaves the nuclease target site in the target genomic locus, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and a multidomain therapeutic protein comprising the delivery domain fused to a lysosomal α-glucosidase protein is expressed from the modified target genomic locus. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase protein in a cell or population of cells of a subject. Some such methods include administering any of the above-described compositions to a subject, wherein the coding sequence for the multidomain therapeutic protein is operably linked to a promoter in the nucleic acid construct and expressed in the cells. In another aspect, a method is provided for expressing a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase protein from a target genomic locus in a cell or population of cells of a subject. Some such methods include administering any of the above compositions to a subject, wherein a nuclease agent cleaves a nuclease target site in the target genomic locus, a nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus. In some such methods, the expressed multidomain therapeutic protein is delivered to and internalized by skeletal muscle tissue and cardiac tissue of the subject. In some such methods, the cells are liver cells or the population of cells is a population of liver cells. In some such methods, the cells are hepatocytes or the population of cells is a population of hepatocytes. In some such methods, the cells are human cells or the population of cells is a population of human cells. In some such methods, the cells are neonatal cells or the population of cells is a population of neonatal cells.In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 24 weeks of birth. In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 12 weeks of birth. In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 8 weeks of birth. In some such methods, the neonatal cell or population of neonatal cells is from a human neonatal subject within 4 weeks of birth. In some such methods, the cell is not a neonatal cell, or the population of cells is not a population of neonatal cells.

[0054] In another aspect, methods are provided for treating lysosomal α-glucosidase deficiency in a subject in need thereof. Some such methods include administering to a subject any of the above-described compositions, wherein a coding sequence for a multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct and expressed in the subject, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to a subject any of the above-described compositions, wherein a nuclease agent cleaves a nuclease target site in a target genomic locus, wherein the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and wherein a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to a subject any of the above-described compositions, wherein a coding sequence for a multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct, and wherein a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed in the subject. Some such methods include administering to a subject any of the above-described compositions, wherein a nuclease agent cleaves a nuclease target site in a target genomic locus, wherein the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and wherein a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus. In another aspect, a method for reducing glycogen accumulation in a tissue of a subject in need thereof is provided. Some such methods include administering to a subject any of the above-described compositions, wherein a coding sequence for a multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct, and wherein the multidomain therapeutic protein is expressed in the subject to reduce glycogen accumulation in the tissue, and optionally the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain.Some such methods include administering to a subject any of the above-described compositions, wherein a nuclease agent cleaves a nuclease target site in the target genomic locus, wherein a nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, wherein a multidomain therapeutic protein comprising a delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus to reduce glycogen accumulation in tissues, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to a subject any of the above-described compositions, wherein a coding sequence for the multidomain therapeutic protein is operably linked to a promoter in the nucleic acid construct, wherein the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed in the subject to reduce glycogen accumulation in tissues, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering any of the above-described compositions to a subject, wherein a nuclease agent cleaves a nuclease target site in the target genomic locus, wherein the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and wherein a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus to reduce glycogen accumulation in the tissue. In some such methods, the subject has Pompe disease. In another aspect, a method of treating Pompe disease in a subject in need thereof is provided. Some such methods include administering any of the above-described compositions to a subject, wherein a coding sequence for the multidomain therapeutic protein is operably linked to a promoter in the nucleic acid construct and expressed in the subject, thereby treating Pompe disease, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain.Some such methods include administering to a subject any of the above-described compositions, wherein the nuclease agent cleaves a nuclease target site in the target genomic locus, wherein the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and wherein a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby treating Pompe disease, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering to a subject any of the above-described compositions, wherein a coding sequence for the multidomain therapeutic protein is operably linked to a promoter in the nucleic acid construct, and wherein the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed in the subject, thereby treating Pompe disease. Some such methods include administering any of the above compositions to a subject, wherein the nuclease agent cleaves the nuclease target site of the target genomic locus, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby treating Pompe disease. In some such methods, Pompe disease is infantile-onset Pompe disease. In some such methods, Pompe disease is late-onset Pompe disease.

[0055] In some such methods, the subject is a neonatal subject. In some such methods, the neonatal subject is a human subject within 24 weeks of birth. In some such methods, the neonatal subject is a human subject within 12 weeks of birth. In some such methods, the neonatal subject is a human subject within 8 weeks of birth. In some such methods, the neonatal subject is a human subject within 4 weeks of birth. In some such methods, the subject is not a neonatal subject. In some such methods, the method results in a therapeutically effective level of circulating multidomain therapeutic protein or lysosomal α-glucosidase in the subject. In some such methods, the method reduces glycogen accumulation in skeletal muscle tissue, cardiac tissue, or central nervous system tissue of the subject. In some such methods, the method reduces glycogen accumulation in skeletal muscle tissue and cardiac tissue of the subject. In some such methods, the method results in a reduction in glycogen levels in skeletal muscle tissue and / or cardiac tissue of the subject that is comparable to wild-type levels at the same age. In some such methods, the method improves muscle strength in the subject or prevents loss of muscle strength in the subject compared to a control subject. In some such methods, the method results in the subject having muscle strength comparable to wild-type levels at the same age.

[0056] In another aspect, methods are provided for preventing or reducing the onset of signs or symptoms of Pompe disease in a subject in need thereof. Some such methods comprise administering to a subject any of the above-described compositions, wherein a coding sequence for a multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct and expressed in the subject, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject; optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods comprise administering to a subject any of the above-described compositions, wherein a nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising the delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject; optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. Some such methods include administering any of the above-described compositions to a subject, wherein the coding sequence for the multidomain therapeutic protein is operably linked to a promoter in a nucleic acid construct, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed in the subject, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject. Some such methods include administering any of the above-described compositions to a subject, wherein the nuclease agent cleaves the nuclease target site and inserts into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a CD63-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject. In some such methods, the Pompe disease is infantile-onset Pompe disease. In some such methods, the Pompe disease is late-onset Pompe disease. In some such methods, the methods result in a therapeutically effective level of circulating multidomain therapeutic protein or lysosomal α-glucosidase in the subject.In some such methods, the method prevents or reduces glycogen accumulation in skeletal muscle tissue, cardiac tissue, or central nervous system tissue of the subject. In some such methods, the method prevents or reduces glycogen accumulation in skeletal muscle tissue, cardiac tissue, and central nervous system tissue of the subject. In some such methods, the subject is a newborn subject. In some such methods, the newborn subject is a human subject within 24 weeks of birth. In some such methods, the newborn subject is a human subject within 12 weeks of birth. In some such methods, the newborn subject is a human subject within 8 weeks of birth. In some such methods, the newborn subject is a human subject within 4 weeks of birth. In some such methods, the subject is not a newborn subject.

[0057] In some such methods, the method results in increased expression of the multidomain therapeutic protein in the subject compared to a method comprising administering an episomal expression vector encoding the multidomain therapeutic protein to a control subject. In some such methods, the method results in increased serum levels of the multidomain therapeutic protein in the subject compared to a method comprising administering an episomal expression vector encoding the multidomain therapeutic protein to a control subject. In some such methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, at least about 9 μg / mL, or at least about 10 μg / mL. In some such methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 2 μg / mL or at least about 5 μg / mL. In some such methods, the methods result in serum levels of the multidomain therapeutic protein in the subject of about 2 μg / mL to about 30 μg / mL or about 2 μg / mL to about 20 μg / mL. In some such methods, the methods result in serum levels of the multidomain therapeutic protein in the subject of about 5 μg / mL to about 30 μg / mL or about 5 μg / mL to about 20 μg / mL. In some such methods, the methods achieve lysosomal α-glucosidase activity levels of at least about 40% of normal, at least about 45% of normal, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of normal. In some such methods, the subject has infantile-onset Pompe disease and the methods achieve lysosomal α-glucosidase expression or activity levels of at least about 1% or greater than about 1% of normal. In some such methods, the subject has late-onset Pompe disease and the method achieves a lysosomal α-glucosidase expression or activity level that is at least about 40% of normal or greater than about 40% of normal.In some such methods, the methods increase lysosomal α-glucosidase activity by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% over the subject's baseline lysosomal α-glucosidase activity. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at peak levels of expression measured in the subject 6 months or 24 weeks after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at peak levels of expression measured in the subject 1 year after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 6 months or 24 weeks after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 2 years after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 2 years after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 6 months or 24 weeks after administration.

[0058] In some such methods, the method further comprises assessing pre-existing AAV immunity in the subject prior to administering the nucleic acid construct to the subject. In some such methods, the pre-existing AAV immunity is pre-existing AAV8 immunity. In some such methods, assessing pre-existing AAV immunity comprises assessing immunogenicity using a total antibody immunoassay or a neutralizing antibody assay.

[0059] In some such methods, the nucleic acid construct is administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is not administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered before the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered after the nuclease agent or one or more nucleic acids encoding the nuclease agent.

[0060] In some such methods, the subject is a human subject. In some such methods, the subject is a newborn subject. In some such methods, the newborn subject is a human subject within 24 weeks of birth. In some such methods, the newborn subject is a human subject within 12 weeks of birth. In some such methods, the newborn subject is a human subject within 8 weeks of birth. In some such methods, the newborn subject is a human subject within 4 weeks of birth. In some such methods, the subject is not a newborn subject.

[0061] Nucleic acid constructs and compositions are provided that enable insertion of a multidomain therapeutic protein (e.g., a GAA fusion protein) coding sequence into a target genomic locus, such as the endogenous ALB locus, and / or expression of a multidomain therapeutic protein (e.g., a GAA fusion protein) coding sequence. The nucleic acid constructs and compositions can be used in methods for incorporating or inserting a multidomain therapeutic protein (e.g., a GAA fusion protein) nucleic acid into a target genomic locus in a cell or population of cells or a subject, for expressing a multidomain therapeutic protein (e.g., a GAA fusion protein) in a cell or population of cells or a subject, for reducing glycogen accumulation in a cell or population of cells or a subject, for treating Pompe disease or GAA deficiency in a subject, and for preventing or reducing the onset of signs or symptoms of Pompe disease in subjects diagnosed with Pompe disease, including neonatal cells and neonatal subjects, such as subjects with reduced GAA activity or expression. In some embodiments, the cells, population of cells, or subject are neonatal cells, populations of neonatal cells, or neonatal subjects.

[0062] In one aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase into a target genomic locus in a neonatal cell or population of neonatal cells. Some such methods include administering to a neonatal cell or population of neonatal cells (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In another aspect, methods are provided for expressing a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase in a neonatal cell or population of neonatal cells. In another aspect, methods are provided for expressing a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase from a target genomic locus in a neonatal cell or a population of neonatal cells. Some such methods include administering to the neonatal cell or population of neonatal cells (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus. In some such methods, the neonatal cell is a liver cell, or the population of neonatal cells is a liver cell. In some such methods, the neonatal cells are hepatocytes or the population of neonatal cells is a population of hepatocytes, hi some such methods, the neonatal cells are human cells or the population of neonatal cells is a population of human cells.In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 24 weeks of birth. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 12 weeks of birth. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 8 weeks of birth. In some such methods, the neonatal cells or population of neonatal cells are from a human neonatal subject within 4 weeks of birth. In some such methods, the neonatal cells are in vitro or ex vivo, or the population of neonatal cells is in vitro or ex vivo. In some such methods, the neonatal cells are in vivo in a neonatal subject, or the population of neonatal cells is in vivo in a neonatal subject.

[0063] In another aspect, methods are provided for inserting a nucleic acid encoding a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase into a target genomic locus in neonatal cells or a population of neonatal cells of a neonatal subject. Some such methods include administering to the neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In another aspect, methods are provided for expressing a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase protein in neonatal cells or a population of neonatal cells of a neonatal subject. In another aspect, methods are provided for expressing a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase protein from a target genomic locus in a neonatal cell or population of neonatal cells in a neonatal subject. Some such methods include administering to the neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site within a target gene at the target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus. In some such methods, the expressed multidomain therapeutic protein is delivered to and internalized by skeletal muscle, heart, and central nervous system tissues of the subject, hi some such methods, the neonatal cells are liver cells, or the population of neonatal cells is a population of liver cells.In some such methods, the neonatal cells are hepatocytes or the population of neonatal cells is a population of hepatocytes, hi some such methods, the neonatal cells are human cells or the population of neonatal cells is a population of human cells.

[0064] In another aspect, methods for treating lysosomal α-glucosidase deficiency in a neonatal subject in need thereof are provided. Some such methods include administering to the neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus. In another aspect, methods for reducing glycogen accumulation in tissues of a neonatal subject in need thereof are provided. Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus, reducing glycogen accumulation in the tissue. In some such methods, the subject has Pompe disease. In another aspect, a method of treating Pompe disease in a neonatal subject in need thereof is provided.Some such methods include administering to a neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby treating Pompe disease. In some such methods, the Pompe disease is infantile-onset Pompe disease.

[0065] In some such methods, the method results in a therapeutically effective level of circulating multidomain therapeutic protein or lysosomal α-glucosidase in the subject. In some such methods, the method reduces glycogen accumulation in skeletal muscle tissue, heart tissue, or central nervous system tissue of the subject. In some such methods, the method reduces glycogen accumulation in skeletal muscle tissue, heart tissue, and central nervous system tissue of the subject. In some such methods, the method results in a reduction in glycogen levels in skeletal muscle tissue, heart tissue, and / or central nervous system tissue of the subject that is comparable to wild-type levels at the same age. In some such methods, the method improves muscle strength in the subject or prevents muscle strength loss in the subject compared to a control subject. In some such methods, the method results in the subject having muscle strength comparable to wild-type levels at the same age.

[0066] In another aspect, methods are provided for preventing or reducing the onset of signs or symptoms of Pompe disease in a neonatal subject in need thereof. Some such methods include administering to the neonatal subject (a) a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase, and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to lysosomal α-glucosidase is expressed from the modified target genomic locus, thereby preventing or reducing the onset of signs or symptoms of Pompe disease in the subject. In some such methods, the Pompe disease is infantile-onset Pompe disease. In some such methods, the Pompe disease is late-onset Pompe disease. In some such methods, the method results in a therapeutically effective level of circulating multidomain therapeutic protein or lysosomal α-glucosidase in the subject. In some such methods, the method prevents or reduces glycogen accumulation in skeletal muscle tissue, heart tissue, or central nervous system tissue of the subject. In some such methods, the method prevents or reduces glycogen accumulation in skeletal muscle tissue, heart tissue, and central nervous system tissue of the subject. In some such methods, the newborn subject is a human subject within 24 weeks of birth. In some such methods, the newborn subject is a human subject within 12 weeks of birth. In some such methods, the newborn subject is a human subject within 8 weeks of birth. In some such methods, the newborn subject is a human subject within 4 weeks of birth.

[0067] In some such methods, the method results in increased expression of the multidomain therapeutic protein in the subject compared to a method comprising administering an episomal expression vector encoding the multidomain therapeutic protein to a control subject. In some such methods, the method results in increased serum levels of the multidomain therapeutic protein in the subject compared to a method comprising administering an episomal expression vector encoding the multidomain therapeutic protein to a control subject. In some such methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, at least about 9 μg / mL, or at least about 10 μg / mL. In some such methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 2 μg / mL or at least about 5 μg / mL. In some such methods, the methods result in serum levels of the multidomain therapeutic protein in the subject of about 2 μg / mL to about 30 μg / mL or about 2 μg / mL to about 20 μg / mL. In some such methods, the methods result in serum levels of the multidomain therapeutic protein in the subject of about 5 μg / mL to about 30 μg / mL or about 5 μg / mL to about 20 μg / mL. In some such methods, the methods achieve lysosomal α-glucosidase activity levels of at least about 40% of normal, at least about 45% of normal, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of normal. In some such methods, the subject has infantile-onset Pompe disease and the methods achieve lysosomal α-glucosidase expression or activity levels of at least about 1% or greater than about 1% of normal. In some such methods, the subject has late-onset Pompe disease and the method achieves a lysosomal α-glucosidase expression or activity level that is at least about 40% of normal or greater than about 40% of normal.In some such methods, the methods increase lysosomal α-glucosidase activity by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% over the subject's baseline lysosomal α-glucosidase activity. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at peak levels of expression measured in the subject 6 months or 24 weeks after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at peak levels of expression measured in the subject 1 year after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 6 months or 24 weeks after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 50% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 2 years after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 2 years after administration. In some such methods, the expression or activity of the multidomain therapeutic protein is at least 60% of the expression or activity of the multidomain therapeutic protein at its peak level of expression measured in the subject 6 months or 24 weeks after administration.

[0068] In some such methods, the method further comprises assessing pre-existing AAV immunity in the neonatal subject prior to administering the nucleic acid construct to the subject. In some such methods, the pre-existing AAV immunity is pre-existing AAV8 immunity. In some such methods, assessing pre-existing AAV immunity comprises assessing immunogenicity using a total antibody immunoassay or a neutralizing antibody assay.

[0069] In some such methods, the nucleic acid construct is administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is not administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered before the nuclease agent or one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered after the nuclease agent or one or more nucleic acids encoding the nuclease agent.

[0070] In some such methods, the TfR-binding delivery domain is fused to a lysosomal α-glucosidase protein via a peptide linker. In some such methods, the coding sequence for the TfR-binding delivery domain is codon-optimized or CpG-depleted. In some such methods, the coding sequence for the TfR-binding delivery domain is codon-optimized and CpG-depleted. In some such methods, the TfR-binding delivery domain comprises an anti-TfR antigen binding protein. In some such methods, the anti-TfR antigen binding protein comprises (i) HCDR1, HCDR2, and HCDR3 of a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, or 527 (or a variant thereof). and / or (ii) an HCVR comprising a CDR3, and / or (iii) an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, ​​392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, or 532 (or a variant thereof). In some such methods, the anti-TfR antigen binding protein comprises: (1) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); (2) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); (3) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof);and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); (4) an HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); (5) an HCVR comprising HCDR1 and HCDR2 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof). and HCDR3, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); (6) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); (7) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof). (8) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR, and LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); (9) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof), and LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); (10) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (10) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); (11) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof);(11) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 322 (or a variant thereof); (12) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 327 (or a variant thereof), and LCDR1 and LCDR2 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 332 (or a variant thereof). and LCDR3; (13) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 337 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 342 (or a variant thereof); (14) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 347 (or a variant thereof), and an amino acid sequence set forth in SEQ ID NO: 352 (or a variant thereof). (15) an LCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 357 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 362 (or a variant thereof); (16) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 367 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 372; (17) LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 377 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof); (18) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 387 (or a variant thereof);and LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 392 (or a variant thereof), (19) HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 397 (or a variant thereof), and LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 402 (or a variant thereof), (20) HCDR1, HCDR2, and LCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 407 (or a variant thereof). (21) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 417 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 422 (or a variant thereof); (22) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 427 (or a variant thereof). (21) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 432 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 432 (or a variant thereof); (22) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof); (23) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof); (24) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 447; (24) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 452, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 453; (25) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 457, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 462;(26) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 467 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 472 (or a variant thereof); (27) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 477 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 482 (or a variant thereof); (28) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 487 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 492 (or a variant thereof); (29) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 497 (or a variant thereof), and (30) an LCVR comprising an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 507 (or a variant thereof), and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 512 (or a variant thereof); (31) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 517 (or a variant thereof), and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 522 (or a variant thereof); (32) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 527 (or a variant thereof), and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 532 (or a variant thereof). In some such methods, the anti-TfR antigen binding protein comprises: (1) an HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (or a variant thereof);and an LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof), or (2) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 457 (or a variant thereof), and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 462 (or a variant thereof). (a) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof), as well as an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); (b) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof), as well as an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); (c) an LCVR comprising an amino acid sequence set forth in SEQ ID NO: 238 (d) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof), and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); (d) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof), and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); (e) HCDR1 having the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof);(f) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof), and LCVRs comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof); (g) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), and the amino acid sequence set forth in SEQ ID NO: 279 (h) HCVRs comprising an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof), and LCVRs comprising an LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof), and LCVRs comprising an LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof), an LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof), and an LCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof); (i) HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof), and an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof);and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof); (j) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof); (k) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an amino acid sequence set forth in SEQ ID NO: 320 (or (l) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 328 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 329 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 330 (or a variant thereof), and LCVRs comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 333 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 334 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 335 (or a variant thereof); (m) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 338 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 339 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 340 (or a variant thereof);and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 343 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 344 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 345 (or a variant thereof); (n) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 348 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 349 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 350 (or a variant thereof), and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 353 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 354 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 355 (or a variant thereof); (o) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 358 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 359 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 360 (or a variant thereof), and an amino acid sequence set forth in SEQ ID NO: 363 (p) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 368 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 369 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 370 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 373 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 374 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 375 (or a variant thereof); (q) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 378 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 379 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 380 (or a variant thereof), and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 383 (or a variant thereof);(r) an LCVR comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 384 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 385 (or a variant thereof); (r) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 388 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 389 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 390 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 393 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 394 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 395 (or a variant thereof); (s) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 398 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 399 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 400 (or a variant thereof), as well as an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 403 (or a variant thereof), and the amino acid sequence set forth in SEQ ID NO: 404 (t) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 409 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 410 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 413 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 414 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 415 (or a variant thereof); (u) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 418 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 419 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 420 (or a variant thereof), as well as an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 423 (or a variant thereof), and an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 424 (or a variant thereof);and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 425 (or a variant thereof); (v) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 428 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 429 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 430 (or a variant thereof), and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 433 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 434 (or a variant thereof), and the amino acid set forth in SEQ ID NO: 435; (w) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 438 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 439 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 444 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 445 (or a variant thereof); (x) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 448 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 449 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 450 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 453 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 454 (or a variant thereof), and an amino acid sequence set forth in SEQ ID NO: 455 (or a variant thereof). (y) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 458 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 459 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 460 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 463 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 464 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 465 (or a variant thereof); (z) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 468 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 469 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 470 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 473 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 474 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 475 (or a variant thereof);(aa) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 478 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 479 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 480 (or a variant thereof), as well as an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 483 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 484 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 485 (or a variant thereof); (ab) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 488 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 489 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 490 (or a variant thereof), as well as an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 493 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 494 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 495 (or a variant thereof); (ac) an amino acid sequence set forth in SEQ ID NO: 498 (a) an HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 499 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 500 (or a variant thereof), and an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 503 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 504 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 505 (or a variant thereof); (ad) an HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 508 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 509 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 510 (or a variant thereof); and an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 513 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 514 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 515 (or a variant thereof); (ae) an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 518 (or a variant thereof);HCVRs comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 519 (or a variant thereof) and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 520 (or a variant thereof), as well as LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 523 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 524 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 525 (or a variant thereof), and / or (af) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 528 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 529 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 530 (or a variant thereof), and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 533 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 534 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 535 (or a variant thereof). In some such methods, the anti-TfR antigen binding protein comprises (a) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 438 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 439 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 444 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 445 (or a variant thereof); (b) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 458 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 459 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 460 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 463 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 464 (or a variant thereof), and an LCVR3 comprising the amino acid sequence set forth in SEQ ID NO: 465 (or a variant thereof). In some such methods, the anti-TfR antigen binding protein(i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); (iv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); (v) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); (vi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); (vii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (viii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); (ix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); (x) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); (xi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 322 (or a variant thereof); (xii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 327 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 332 (or a variant thereof); (xiii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 337 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 342 (or a variant thereof);(xiv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 347 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 352 (or a variant thereof); (xv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 357 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 362 (or a variant thereof); (xvi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 367 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 372 (or a variant thereof); (xvii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 377 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof); (xviii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 387 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 392 (or a variant thereof); (xix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 397 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 402 (or a variant thereof); (xx) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 407 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 412 (or a variant thereof), (xxi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 417 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 422 (or a variant thereof), (xxii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 427 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 432 (or a variant thereof), (xxiii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof), (xxiv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 447 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 452 (or a variant thereof), (xxv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 457 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 462 (or a variant thereof), (xxvi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 467 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 472 (or a variant thereof),(xxvii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 477 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 482 (or a variant thereof); (xxviii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 487 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 492 (or a variant thereof); (xxix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 497 (or a variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 502 (or a variant thereof); (xxx) an amino acid sequence set forth in SEQ ID NO: 507 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 512 (or variant thereof), (xxxi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 517 (or variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 522 (or variant thereof), and / or (xxxii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 527 (or variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 532 (or variant thereof). In some such methods, the anti-TfR antigen binding protein comprises (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (or variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 442 (or variant thereof), or (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 457 (or variant thereof) and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 462 (or variant thereof). In some such methods, the TfR-binding delivery domain comprises an anti-TfR antibody, antibody fragment, or single-chain variable fragment (scFv). In some such methods, the TfR-binding delivery domain is a single-chain variable fragment (scFv), and optionally the multidomain therapeutic protein comprises domains arranged in the following orientation: N'-heavy chain variable region-light chain variable region-lysosomal α-glucosidase-C' or N'-light chain variable region-heavy chain variable region-lysosomal α-glucosidase-C', and optionally the scFv and lysosomal α-glucosidase are linked via a peptide linker, optionally -(GGGGS) m-(SEQ ID NO: 600), where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and optionally the scFv variable regions are connected by a peptide linker, optionally -(GGGGS) m (SEQ ID NO: 600), where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some such methods, the multidomain therapeutic protein comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), as well as lysosomal α-glucosidase, V H , V L , and lysosomal α-glucosidase, which are: (i) V L -V H -lysosomal α-glucosidase, (ii) V H -V L -lysosomal α-glucosidase, (iii) V L -[(GGGGS)3]-V H -[(GGGGS)2]-lysosomal α-glucosidase, or V H -[(GGGGS)3]-V L-[(GGGGS)2]-lysosomal α-glucosidase. In some such methods, the scFv comprises the sequence set forth in any of SEQ ID NOs: 540, 549, 551, and 554, and optionally the scFv comprises the sequence set forth in SEQ ID NO: 554. In some such methods, the scFv consists of the sequence set forth in any of SEQ ID NOs: 540, 549, 551, and 554, and optionally the scFv consists of the sequence set forth in SEQ ID NO: 554. In some such methods, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 587-599. Optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 593-595, and optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 593. Optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 590-592, and optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 590.In some such methods, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 587-599 and encodes an scFv comprising any of SEQ ID NOs: 540, 549, 551, and 554. Optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 593-595 and encodes an scFv comprising SEQ ID NO: 554; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 593 and encodes an scFv comprising SEQ ID NO: 554. Optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 590-592 and encodes an scFv comprising SEQ ID NO: 551; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 590 and encodes an scFv comprising SEQ ID NO: 551. In some such methods, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 587-599, is codon-optimized, is CpG-depleted, and encodes an scFv comprising any of SEQ ID NOs: 540, 549, 551, and 554.Optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 593-595, is codon-optimized, and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 554; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 593, and the scFv coding sequence is codon-optimized and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 554. Optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 590-592, is codon-optimized, and is CpG-depleted, and encodes an scFv comprising SEQ ID NO: 551; optionally, the scFv coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 590, and the scFv coding sequence is codon-optimized, CpG-depleted, and encodes an scFv comprising SEQ ID NO: 551. In some such methods, the scFv coding sequence comprises a sequence set forth in any of SEQ ID NOs: 587-599. Optionally, the scFv coding sequence comprises the sequence set forth in any of SEQ ID NOs: 593-595, and optionally, the scFv coding sequence comprises the sequence set forth in SEQ ID NO: 593. Optionally, the scFv coding sequence comprises the sequence set forth in any of SEQ ID NOs: 590-592, and optionally, the scFv coding sequence comprises the sequence set forth in SEQ ID NO: 590. In some such methods, the scFv coding sequence consists of the sequence set forth in any of SEQ ID NOs: 587-599.Optionally, the scFv coding sequence consists of the sequence set forth in any of SEQ ID NOs: 593-595, optionally, the scFv coding sequence consists of the sequence set forth in SEQ ID NO: 593. Optionally, the scFv coding sequence consists of the sequence set forth in any of SEQ ID NOs: 590-592, optionally, the scFv coding sequence consists of the sequence set forth in SEQ ID NO: 590.

[0071] In some such methods, the lysosomal α-glucosidase lacks the lysosomal α-glucosidase signal peptide and propeptide. In some such methods, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized or CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173.In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and 205-212, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such methods, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176. In some such methods, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, and optionally, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0072] In some such methods, the lysosomal α-glucosidase lacks the lysosomal α-glucosidase signal peptide and propeptide. In some such methods, the lysosomal α-glucosidase comprises the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase consists of the sequence set forth in SEQ ID NO: 173. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized or CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is codon-optimized and CpG-depleted. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182, and optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176. In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176 and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173.In some such methods, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:174-182, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173; optionally, the lysosomal α-glucosidase coding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176, is codon-optimized, and CpG-depleted, and encodes a lysosomal α-glucosidase protein comprising SEQ ID NO:173. In some such methods, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176. In some such methods, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal α-glucosidase coding sequence consists of the sequence set forth in SEQ ID NO: 176.

[0073] In some such methods, the coding sequence of the multidomain therapeutic protein is codon-optimized or CpG-depleted. In some such methods, the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted. In some such methods, the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 570-573, and optionally the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573. In some such methods, the multidomain therapeutic protein consists of the sequence set forth in any of SEQ ID NOs: 570-573, and optionally the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 573. In some such methods, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 574-586. Optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:584-586; optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:584; and optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:733.Optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs:581-583; optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:581; and optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:729. In some such methods, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 574-586, and the multidomain therapeutic protein comprises a sequence set forth in any of SEQ ID NOs: 570-573.Optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 584-586, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573, and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 733, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573, and optionally the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 733, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573.Optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 581-583, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 572, and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93% identical to SEQ ID NO: 581. , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 729, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 572; optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 729, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 572. In some such methods, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 574-586, is codon-optimized, and is CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 570-573.Optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 584-586, is codon-optimized, and is CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573, and optionally the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 584-586. 7%, at least 98%, or at least 99% identical to SEQ ID NO: 733, wherein the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573, and optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 733, wherein the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 573.Optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 581-583, is codon-optimized, and is CpG-depleted, and the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 572, and optionally, the coding sequence of the multidomain therapeutic protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 581-583, is codon-optimized, and is CpG-depleted, 7%, at least 98%, or at least 99% identical to SEQ ID NO: 729, wherein the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 572, and optionally, the nucleic acid construct comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 729, wherein the coding sequence of the multidomain therapeutic protein is codon-optimized and CpG-depleted, and wherein the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 572. In some such methods, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586. Optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. Optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729.In some such methods, the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in any of SEQ ID NOs: 574-586. Optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 584, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. Optionally, the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein consists of the sequence set forth in SEQ ID NO: 581, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729. In some such methods, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein. In some such methods, the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein. In some such methods, the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein and the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein. In some such methods, the nucleic acid construct does not comprise homology arms. In some such methods, the nucleic acid construct is inserted into the target genomic locus via non-homologous end joining. In some such methods, the nucleic acid construct comprises homology arms. In some such methods, the nucleic acid construct is inserted into the target genomic locus via homology directed repair. In some such methods, the nucleic acid construct comprises a promoter that drives expression of the multidomain therapeutic protein. In some such methods, the nucleic acid construct does not comprise a promoter. In some such methods, the coding sequence for the multidomain therapeutic protein is operably linked to a promoter, and optionally, the promoter is a liver-specific promoter. In some such methods, the nucleic acid construct is single-stranded DNA or double-stranded DNA. In some such methods, the nucleic acid construct is single-stranded DNA. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, optionally the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, and optionally the multidomain therapeutic protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 574-586. The coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733, or optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, and optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729. The nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, and the nucleic acid construct does not comprise homology arms.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182, optionally the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, and optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586. The coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733, or optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729. The nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, and the nucleic acid construct does not comprise homology arms.

[0074] In some such methods, the nucleic acid construct is in a nucleic acid vector or lipid nanoparticle. In some such methods, the nucleic acid construct is in a nucleic acid vector. In some such methods, the nucleic acid vector is a viral vector. In some such methods, the nucleic acid vector is an adeno-associated viral (AAV) vector, and optionally, the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally, the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 160, and optionally, the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 160. In some such methods, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such methods, the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. In some such methods, the AAV vector is a recombinant AAV8 (rAAV8) vector. In some such methods, the AAV vector is a single-stranded rAAV8 vector.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, optionally wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182; optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584; and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160. In some such methods, the nucleic acid construct is CpG-depleted.

[0075] In some such methods, the target genomic locus is an albumin gene, and optionally, the albumin gene is a human albumin gene. In some such methods, the nuclease target site is within intron 1 of the albumin gene. In some such methods, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) (i) a Cas protein or a nucleic acid encoding a Cas protein, and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0076] In some such methods, the nuclease agent comprises (a) a Cas protein or a nucleic acid encoding a Cas protein, and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the guide RNA target sequence is within intron 1 of the albumin gene. In some such methods, the albumin gene is a human albumin gene. In some such methods, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any of SEQ ID NOs: 30-61, and optionally, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the DNA-targeting segment comprises any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment comprises any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the DNA-targeting segment consists of any of SEQ ID NOs: 30-61, optionally the DNA-targeting segment consists of any of SEQ ID NOs: 36, 30, 33, and 41. In some such methods, the guide RNA comprises any of SEQ ID NOs: 62-125, optionally the guide RNA comprises any of SEQ ID NOs: 68, 100, 62, 94, 65, 97, 73, and 105. In some such methods, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides set forth in SEQ ID NO:36.In some such methods, the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 36. In some such methods, the DNA-targeting segment comprises SEQ ID NO: 36. In some such methods, the DNA-targeting segment consists of SEQ ID NO: 36. In some such methods, the guide RNA comprises SEQ ID NO: 68 or 100.

[0077] In some such methods, the method comprises administering a guide RNA in the form of RNA. In some such methods, the guide RNA comprises at least one modification. In some such methods, the at least one modification comprises a 2'-O-methyl modified nucleotide. In some such methods, the at least one modification comprises an internucleotide phosphorothioate bond. In some such methods, the at least one modification comprises a modification in one or more of the first five nucleotides at the 5' end of the guide RNA. In some such methods, the at least one modification comprises a modification in one or more of the last five nucleotides at the 3' end of the guide RNA. In some such methods, the at least one modification comprises a phosphorothioate bond between the first four nucleotides at the 5' end of the guide RNA. In some such methods, the at least one modification comprises a phosphorothioate bond between the last four nucleotides at the 3' end of the guide RNA. In some such methods, the at least one modification comprises a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA. In some such methods, the at least one modification comprises 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA. In some such methods, the at least one modification comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA, and (iv) a 2'-O-methyl modified nucleotide in the last three nucleotides at the 3' end of the guide RNA. In some such methods, the guide RNA is a single guide RNA (sgRNA).In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, and wherein the guide RNA comprises (i) phosphorothioate linkages between the first four nucleotides at the 5' end of the guide RNA, (ii) phosphorothioate linkages between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0078] In some such methods, the Cas protein is a Cas9 protein. In some such methods, the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein. In some such methods, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such methods, the Cas protein comprises the sequence set forth in SEQ ID NO: 11. In some such methods, the nucleic acid encoding the Cas protein is codon-optimized for expression in a mammalian cell or a human cell. In some such methods, the method comprises administering a nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such methods, the mRNA encoding the Cas protein comprises at least one modification. In some such methods, the mRNA encoding the Cas protein is modified to include modified uridines at one or more or all uridine positions. In some such methods, the modified uridine is pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such methods, the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such methods, the modified uridine is pseudouridine. In some such methods, the mRNA encoding the Cas protein is fully substituted with pseudouridine. In some such methods, the mRNA encoding the Cas protein comprises a 5' cap. In some such methods, the mRNA encoding the Cas protein comprises a polyadenylation sequence. In some such methods, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12.In some such methods, the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence. In some such methods, the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence.

[0079] In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, optionally wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182; optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584; and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), wherein optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence. In some such methods, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the method comprises administering a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a polyadenylation sequence.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, optionally wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160. In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal alpha-glucosidase coding sequence comprises a sequence set forth in any of SEQ ID NOs: 174-182, and optionally, the lysosomal alpha-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176.Optionally, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586; optionally, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586; optionally, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584; optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733; or optionally, the coding sequence of the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583; , the sequence set forth in SEQ ID NO: 581, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0080] In some such methods, the Cas protein or a nucleic acid encoding the Cas protein and the guide RNA or one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such methods, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such methods, the cationic lipid is lipid A((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate). In some such methods, the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some such methods, the helper lipid is cholesterol. In some such methods, the stealth lipid is PEG2k-DMG. In some such methods, the cationic lipid is lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such methods, the lipid nanoparticles comprise four lipids in the following molar ratio: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0081] In some such methods, the albumin gene is a human albumin gene, the method comprises administering a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 68 or 100, the method comprises administering a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 226, 225, or 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, optionally wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182; optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584; and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), wherein optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0082] In some such methods, the albumin gene is a human albumin gene, the method comprises administering a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, the method comprises administering a nucleic acid encoding a Cas protein, The acid comprises an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a polyadenylation sequence, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such methods, the albumin gene is a human albumin gene, the method comprises administering a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the method comprises administering a Cas protein wherein the nucleic acid comprises an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a polyadenylation sequence, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182 and 205-212, optionally wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586, optionally wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584, and optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. or optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.In some such methods, the nucleic acid construct comprises, from 5' to 3', a splice acceptor, a coding sequence for a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the lysosomal α-glucosidase coding sequence comprises the sequence set forth in any of SEQ ID NOs: 174-182; optionally, the lysosomal α-glucosidase coding sequence comprises the sequence set forth in SEQ ID NO: 176; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 574-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 584-586; optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 584; and optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 733. optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in any of SEQ ID NOs: 581-583, optionally, the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 581, optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 729, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single-stranded rAAV8 vector, optionally, the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 160, and optionally wherein the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 160.

[0083] In another aspect, a neonatal cell or population of neonatal cells produced by any of the above methods is provided. In another aspect, a neonatal cell or population of neonatal cells comprising a nucleic acid construct inserted into a target genomic locus, wherein the nucleic acid construct comprises a coding sequence for a multidomain therapeutic protein comprising a TfR-binding delivery domain fused to a lysosomal α-glucosidase inserted into the target genomic locus, is provided. In some such neonatal cells or populations of neonatal cells, the neonatal cells are liver cells or the population of neonatal cells is a population of liver cells. In some such neonatal cells or populations of neonatal cells, the neonatal cells are hepatocytes or the population of neonatal cells is a population of hepatocytes. In some such neonatal cells or populations of neonatal cells, the neonatal cells are human cells or the population of neonatal cells is a population of human cells. In some such neonatal cells or populations of neonatal cells, the neonatal cells or population of neonatal cells are from a human neonatal subject within 24 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells or population of neonatal cells are from a human neonatal subject within 12 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells or populations of neonatal cells are from a human newborn subject within 8 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells or populations of neonatal cells are from a human newborn subject within 4 weeks of birth. In some such neonatal cells or populations of neonatal cells, the neonatal cells are in vitro or ex vivo, or the population of neonatal cells is in vitro or ex vivo. In some such neonatal cells or populations of neonatal cells, the neonatal cells are in vivo in a subject, or the population of neonatal cells is in vivo.

[0084] In some such neonatal cells or populations of neonatal cells, a multidomain therapeutic protein is expressed. In some such neonatal cells or populations of neonatal cells, the TfR-binding delivery domain is fused to a lysosomal α-glucosidase protein via a peptide linker. In some such neonatal cells or populations of neonatal cells, the coding sequence for the TfR-binding delivery domain is codon-optimized or CpG-depleted. In some such neonatal cells or populations of neonatal cells, the coding sequence for the TfR-binding delivery domain is codon-optimized and CpG-depleted. In some such neonatal cells or populations of neonatal cells, the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein. In some such neonatal cells or populations of neonatal cells, the anti-TfR antigen binding protein comprises (i) HCDR1 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, or 527 (or a variant thereof). and / or (ii) an HCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, ​​392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, or 532 (or a variant thereof). In some such neonatal cells or populations of neonatal cells, the anti-TfR antigen binding protein comprises: (1) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); (2) an HCDR1, HCDR2 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof);and HCDR3, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); (3) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); (4) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); (5) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); (6) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). (7) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); (8) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (9) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof);(10) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); (11) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof), and LCDR1 and LCDR2 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 322 (or a variant thereof). and LCDR3; (12) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 327 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 332 (or a variant thereof); (13) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 337 (or a variant thereof), and an amino acid sequence set forth in SEQ ID NO: 342 (or a variant thereof). (14) an LCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 347 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 352 (or a variant thereof); (15) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 357 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 362. (16) LCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 367 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 372 (or a variant thereof); (17) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 377 (or a variant thereof);and LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof), (18) HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 387 (or a variant thereof), and LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 392 (or a variant thereof), (19) HCDR1, HCDR2, and LCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 397 (or a variant thereof). (20) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 407 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 412 (or a variant thereof); (21) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 417 (or a variant thereof). (21) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 422 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 427 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 432 (or a variant thereof); (22) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 427 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 432 (or a variant thereof); (23) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (23) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 442, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 442; (24) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 447, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 452;(25) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 457 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 462 (or a variant thereof); (26) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 467 (or a variant thereof), and LCDR1 and LCDR2 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 472 (or a variant thereof). and LCDR3; (27) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 477 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 482 (or a variant thereof); (28) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 487 (or a variant thereof), and the amino acid sequence set forth in SEQ ID NO: 492 (or a variant thereof). (29) an LCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 497 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 502 (or a variant thereof); (30) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 507 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 512. (31) an LCVR comprising an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 517 (or a variant thereof), and an LCVR comprising an LCVR comprising an amino acid sequence set forth in SEQ ID NO: 522 (or a variant thereof); (32) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 527 (or a variant thereof);and an LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 532 (or a variant thereof). In some such neonatal cells or populations of neonatal cells, the anti-TfR antigen binding protein comprises (1) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 437 (or a variant thereof), and an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof), or (2) the sequence This includes HCVRs comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 457 (or a variant thereof), and LCVRs comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 462 (or a variant thereof). In some such neonatal cells or populations of neonatal cells, the anti-TfR antigen binding protein comprises: (a) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); (b) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof). 1. LCVR comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); (c) HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof), and LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); (d) HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof);and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); (e) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof), and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); (f) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof), and an HCVR comprising an amino acid sequence set forth in SEQ ID NO: 273 (g) an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof); (h) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof), and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof);(i) an LCVR comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof); (i) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof); (j) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof), as well as an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), and the amino acid sequence set forth in SEQ ID NO: 314. (k) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 323 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 324 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 325 (or a variant thereof); (l) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 328 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 329 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 330 (or a variant thereof), as well as an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 333 (or a variant thereof), and an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 334 (or a variant thereof);and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 335 (or a variant thereof); (m) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 338 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 339 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 340 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 343 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 344 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 345 (or a variant thereof); (n) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 348 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 349 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 350 (or a variant thereof), as well as an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 353 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 354 (or a variant thereof), and an amino acid sequence set forth in SEQ ID NO: 355 (or a variant thereof). (o) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 359 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 360 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 363 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 364 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 365 (or a variant thereof); (p) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 368 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 369 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 370 (or a variant thereof), as well as an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 373 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 374 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 375 (or a variant thereof);(q) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 378 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 379 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 380 (or a variant thereof), as well as an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 383 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 384 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 385 (or a variant thereof); (r) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 388 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 389 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 390 (or a variant thereof), as well as an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 393 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 394 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 395 (or a variant thereof); (s) the amino acid sequence set forth in SEQ ID NO: 398 (t) HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 409 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 410 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 413 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 414 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 415 (or a variant thereof); (u) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 418 (or a variant thereof),an HCVR comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 419 (or a variant thereof) and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 420 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 423 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 424 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 425 (or a variant thereof); (v) an HCDR comprising the amino acid sequence set forth in SEQ ID NO: 428 (or a variant thereof); (1) HCVR comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 429 (or a variant thereof) and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 430 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 433 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 434 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 435 (or a variant thereof); (w) HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 438 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 439 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 444 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 445 (or a variant thereof); (x) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 448 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 449 (y) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 458 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 459 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 460 (or a variant thereof), and LCVRs comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 463 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 464 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 465 (or a variant thereof); (z) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 468 (or a variant thereof), and an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 469 (or a variant thereof);and LCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 470 (or a variant thereof), and LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 473 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 474 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 475 (or a variant thereof); (aa) HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 478 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 479 (or a variant thereof), and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 480 (or a variant thereof); and LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 483 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 484 (or a variant thereof), and LCDR3 comprising the amino acid se...

Claims

1. A composition comprising a nucleic acid construct comprising a coding sequence for a multidomain therapeutic protein comprising a CD63-binding delivery domain fused to a lysosomal α-glucosidase, wherein the lysosomal α-glucosidase coding sequence is CpG-depleted relative to the wild-type lysosomal α-glucosidase coding sequence. Claim 2: wherein the coding sequence of the multidomain therapeutic protein is at least 99% identical to SEQ ID NO:196; The multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO:

193. The composition of claim 1.

3. 3. The composition of claim 2, wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the multidomain therapeutic protein and a polyadenylation signal or sequence downstream of the coding sequence for the multidomain therapeutic protein.

4. The composition of claim 2 , wherein the nucleic acid construct does not contain homology arms.

5. 3. The composition of claim 2, wherein the nucleic acid construct does not comprise a promoter driving expression of the multidomain therapeutic protein.

6. 3. The composition of claim 2, wherein the nucleic acid construct is single-stranded DNA or double-stranded DNA, optionally wherein the nucleic acid construct is single-stranded DNA.

7. The composition of claim 2 , wherein the nucleic acid construct is in a nucleic acid vector.

8. the nucleic acid vector is an adeno-associated virus (AAV) vector; 8. The composition of claim 7, wherein optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 160, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO:

160.

9. 9. The composition of claim 8, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector, optionally wherein the AAV vector is a single-stranded rAAV8 vector.

10. 3. The composition of claim 2, further comprising a nuclease agent that targets a nuclease target site in a target genomic locus, wherein the target genomic locus is an albumin gene, wherein the albumin gene is a human albumin gene, and optionally, the nuclease target site is within intron 1 of the albumin gene.

11. the nuclease agent (a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; and 11. The composition of claim 10, wherein (b) the composition comprises a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and the guide RNA binds to the Cas9 protein and targets the Cas9 protein to the guide RNA target sequence. (I) the DNA targeting segment comprises SEQ ID NO: 36 or the guide RNA comprises SEQ ID NO: 68 or 100, and the guide RNA comprises at least one modification; Optionally, the at least one modification comprises (i) a phosphorothioate bond between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate bond between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; (II) the nucleic acid encoding the Cas9 protein comprises an mRNA encoding the Cas9 protein; Optionally, the mRNA encoding the Cas9 protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12, and the mRNA encoding the Cas9 protein is fully substituted with N1-methyl-pseudouridines, includes a 5' cap, and includes a polyadenylation sequence. The composition of claim 11.

13. The Cas9 protein or the nucleic acid encoding the Cas9 protein, and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle; Optionally, the lipid nanoparticles comprise a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid; Optionally, the cationic lipid is lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate); Optionally, the cationic lipid is lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG; Optionally, the lipid nanoparticles comprise four lipids in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. The composition of claim 11.

14. 14. The composition of any one of claims 1 to 13 for use in a method of inserting said nucleic acid construct encoding said multidomain therapeutic protein into or expressing said multidomain therapeutic protein from a target genomic locus in a cell or population of cells of a subject.

15. A composition according to any one of claims 1 to 13 for use in a method for treating lysosomal alpha-glucosidase deficiency in a subject in need of such treatment.

16. A composition described in any one of claims 1 to 13 for use in a method for reducing glycogen accumulation in tissues of a subject in need thereof.

17. A composition according to any one of claims 1 to 13 for use in a method for treating Pompe disease in a subject in need thereof.

18. A composition according to any one of claims 1 to 13 for use in a method for preventing or reducing the onset of signs or symptoms of Pompe disease in a neonatal subject in need thereof.

19. A cell comprising the composition of any one of claims 1 to 13, optionally comprising: (I) the nucleic acid construct integrates into intron 1 of the endogenous albumin locus and the multidomain therapeutic protein is expressed from the endogenous albumin locus; and / or (II) the cell is a human cell, and / or (III) the cells are liver cells, and optionally the liver cells are hepatocytes; and / or (IV) the cells are neonatal cells; cell.

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