In another aspect, the IGF-1 polypeptide is a wild-type form of IGF-1. In some embodiments, the wild-type form of IGF-1 comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 1. In some embodiments, the wild-type form of IGF-1 comprises SEQ ID NO: 1.

TABLE 1

IGF-1 Signaling Modulators

SEQ

ID

Name
Target
Fragment
Sequence
NO

IGF-1
IGF1R
70 aa
GPETLCGAELVDALQFVCGDRGFYFNKPTG
1

peptide
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSA

IGF-1
IGF1R
IGF-1-Hole
GPETLCGAELVDALQFVCGDRGFYFNKPTG
2

IgG1 N297G
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSAGGGGSGGGGSEPKSSDKTHTC

PPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYGSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VCTLPPSRDELTKNQVSLSCAVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLVSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

IGF-1
IGF1R
IGF-1-Hole
GPETLCGAELVDALQFVCGDRGFYFNKPTG
46

of 1790.3
YGSSSRAPQTGIVDECCFRSCDLRRLEMYCA

PLKPAKSAGGGGSGGGGSEPKSSDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYGSTYRVVSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQPREPQVC

TLPPSRDELTKNQVSLSCAVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLVSKLT

VDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK

IGF-1
IGF1R
IGF-1-Hole
GPETLCGAELVDALQFVCGDRGFLFNKPTG
47

of 1790.4
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSAGGGGSGGGGSEPKSSDKTHTC

PPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYGSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VCTLPPSRDELTKNQVSLSCAVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLVSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

IGF-1
IGF1R
IGF-1-Hole
GPETLCGAELVDALQFVCGDRGFYFNKPTG
48

of 1790.5
AGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSAGGGGSGGGGSEPKSSDKTHTC

PPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYGSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VCTLPPSRDELTKNQVSLSCAVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLVSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

IGF-1
IGF1R
IGF-1-Hole
GPETLCGAELVDALQFVCGDRGFYFNKPTG
49

of 1790.6
YGSSSRAPQTGIVDECCFRSCDLRRLEMYCA

PLKPAKSAGGGGSGGGGSEPKSSDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYGSTYRVVSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQPREPQVC

TLPPSRDELTKNQVSLSCAVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLVSKLT

VDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK

IGF-1
IGF1R
IGF-1-Hole
GPETLCGAELVDALQFVCGDRGFLFNKPTG
50

of 1790.7
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSAGGGGSGGGGSEPKSSDKTHTC

PPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYGSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VCTLPPSRDELTKNQVSLSCAVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLVSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

IGF1-
IGF1R
IGF1Ea
GPETLCGAELVDALQFVCGDRGFYFNKPTG
3

Ea

peptide
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSARSVRAQRHTDMPKTQKEVHLK

NASRGSAGNKNYRM

IGF1-
IGF1R
IGF1Eb
GPETLCGAELVDALQFVCGDRGFYFNKPTG
4

Eb

peptide
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSARSVRAQRHTDMPKTQKYQPPS

TNKNTKSQRRKGWPKTHPGGEQKEGTEASL

QIRGKKKEQRREIGSRNAECRGKKGK

IGF1-
IGF1R
IGF1Ec
GPETLCGAELVDALQFVCGDRGFYFNKPTG
5

Ec

peptide
YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSARSVRAQRHTDMPKTQKYQPPS

TNKNTKSQRRKGSTFEERK

IGF1-
IGF1R
IGF1 R37x
GPETLCGAELVDALQFVCGDRGFYFNKPTG
6

R37x

YGSSSRAPQTGIVDECCFRSCDLRRLEMYCA

PLKPAKSA

IGF1-
IGF1R
IGF1Ea
GPETLCGAELVDALQFVCGDRGFYFNKPTG
7

Ea

peptide
YGSSSRAPQTGIVDECCFRSCDLRRLEMLCA

R37x

PLKPAKSARSVRAQRHTDMPKTQKEVHLKN

ASRGSAGNKNYRM

IGF1-
IGF1R
IGF1Eb
GPETLCGAELVDALQFVCGDRGFYFNKPTG
8

Eb

peptide
YGSSSRAPQTGIVDECCFRSCDLRRLEMYCA

R37x

PLKPAKSARSVRAQRHTDMPKTQKYQPPST

NKNTKSQRRKGWPKTHPGGEQKEGTEASLQ

IRGKKKEQRREIGSRNAECRGKKGK

IGF1-
IGF1R
IGF1Ec
GPETLCGAELVDALQFVCGDRGFYFNKPTG
9

Ec

peptide
YGSSSRAPQTGIVDECCFRSCDLRRLEMYCA

R37x

PLKPAKSARSVRAQRHTDMPKTQKYQPPST

NKNTKSQRRKGSTFEERK

Des
IGF1R
Des 1-3
TLCGAELVDALQFVCGDRGFYFNKPTGYGS
10

1-3

IGF1
SSRRAPQTGIVDECCFRSCDLRRLEMYCAPL

IGF1

KPAKSA

IGF1
IGF1R
IGF1 E3R
GPRTLCGAELVDALQFVCGDRGFYFNKPTG
11

E3R

YGSSSRRAPQTGIVDECCFRSCDLRRLEMYC

APLKPAKSA

In another aspect, a signaling pathway modulator modulates a growth hormone (GH) signaling pathway. The GH signaling pathway modulator can modulate a growth hormone receptor (GHIR). The GH signaling pathway modulator can comprise a GHIR activator. In certain instances, the GH signaling pathway modulator comprises an GH polypeptide. For example, the GH polypeptide can comprise an GH polypeptide with a decreased ability to activate the GHIR relative to endogenous GH. Decreased activity can be assessed in a functional assay such as those described herein. In some instances, the GH polypeptide comprises a variant of GH. An GH variant can have, for example, an amino acid sequence of SEQ ID NO: 12 or 13, or an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 12 or 13 (Table 2). In some instances, an GH variant comprises about one, about two, about three, about four, or about five amino acid modifications of an amino acid sequence of SEQ ID NO: 12 or 13.

In another aspect, a signaling pathway modulator modulates a basic fibroblast growth factor (bFGF) signaling pathway. The bFGF signaling pathway modulator can modulate a fibroblast growth factor receptor (FGFR). The bFGF signaling pathway modulator can comprise a FGFR activator. In certain instances, the bFGF signaling pathway modulator comprises an bFGF polypeptide. For example, the bFGF polypeptide can comprise an bFGF polypeptide with a decreased ability to activate the FGFR relative to endogenous bFGF. Decreased activity can be assessed in a functional assay such as those described herein. In some instances, the bFGF polypeptide comprises a variant of bFGF. An bFGF variant can have, for example, an amino acid sequence any one of SEQ ID NOS: 14-17, or an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to any one of SEQ ID NOS: 14-17 (Table 2). In some instances, an bFGF variant comprises about one, about two, about three, about four, or about five amino acid modifications of an amino acid sequence of any one of SEQ ID NOS: 14-17.

In another aspect, a signaling pathway modulator modulates an interleukin 4 (IL4) signaling pathway. The IL4 signaling pathway modulator can modulate an interleukin 4 receptor (IL4R). The IL4 signaling pathway modulator can comprise a IL4R activator. In certain instances, the IL4 signaling pathway modulator comprises an IL4 polypeptide. For example, the IL4 polypeptide can comprise an IL4 polypeptide with a decreased ability to activate the IL4R relative to endogenous IL4. Decreased activity can be assessed in a functional assay such as those described herein. In some instances, the IL4 polypeptide comprises a variant of IL4. An IL4 variant can have, for example, an amino acid sequence any one of SEQ ID NOS: 18-21, or an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to any one of SEQ ID NOS: 18-21 (Table 2). In some instances, an IL4 variant comprises about one, about two, about three, about four, or about five amino acid modifications of an amino acid sequence of any one of SEQ ID NOS: 18-21.

In another aspect, a signaling pathway modulator modulates an activin type II receptor (ActRII). The signaling pathway modulator can comprise an ActRII inhibitor. In certain instances, the signaling pathway modulator comprises bimagrumab. In some instances, the antibody or antibody fragment comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 22, a VH CDR2 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 23, and a VH CDR3 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 24. In some instances, the antibody or antibody fragment comprises a VH CDR1 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 22, a VH CDR2 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 23, and a VH CDR3 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 24. In some instances, the antibody or antibody fragment comprises a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 25, a VL CDR2 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL CDR3 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 27. In some instances, the antibody or antibody fragment comprises a VL CDR1 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 25, a VL CDR2 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 26, and a VL CDR3 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 27. In some instances, the antibody or antibody fragment comprises a variable heavy (VH) chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 28. In some instances, the antibody or antibody fragment comprises a VH chain comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 28. In some instances, the antibody or antibody fragment comprises a variable light (VL) chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 29. In some instances, the antibody or antibody fragment comprises a VL chain comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 29. In some instances, the antibody or antibody fragment comprises an amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 30 (Table 2). In some instances, the antibody or antibody fragment comprises about one, about two, about three, about four, or about five amino acid modifications of an amino acid sequence set forth in SEQ ID NO: 30.

TABLE 2

Other Signaling Modulators

SEQ ID

Name
Target
Fragment
Sequence
NO

GH
GHR
Cargo
FPTIPLSRLFDNAMLRAHRLHQLA
12

Polypeptide
FDTYQEFEEAYIPKEQKYSFLQNP

QTSLCFSESIPTPSNREETQQKSNL

ELLRISLLLIQSWLEPVQFLRSVFA

NSLVYGASDSNVYDLLKDLEEGI

QTLMGRLEDGSPRTGQIFKQTYS

KFDTNSHNDDALLKNYGLLYCFR

KDMDKVETFLRIVQCRSVEGSCG

F

GH
GHR
GH-Hole
FPTIPLSRLFDNAMLRAHRLHQLA
13

Heavy Chain
FDTYQEFEEAYIPKEQKYSFLQNP

QTSLCFSESIPTPSNREETQQKSNL

ELLRISLLLIQSWLEPVQFLRSVFA

NSLVYGASDSNVYDLLKDLEEGI

QTLMGRLEDGSPRTGQIFKQTYS

KFDTNSHNDDALLKNYGLLYCFR

KDMDKVETFLRIVQCRSVEGSCG

FGGGGSGGGGSEPKSSDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSHEDPEVK

FNWYVDGVEVHNAKTKPREEQY

GSTYRVVSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQ

PREPQVCTLPPSRDELTKNQVSLS

CAVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLVSKLTVD

KSRWQQGNVFSCSVMHEALHNH

YTQKSLSLSPGK

bFGF
FGFR
Cargo
PALPEDGGSGAFPPGHFKDPKRL
14

Polypeptide-1
YCKNGGFFLRIHPDGRVDGVREK

SDPHIKLQLQAEERGVVSIKGVCA

NRYLAMKEDGRLLASKCVTDECF

FFERLESNNYNTYRSRKYTSWYV

ALKRTGQYKLGSKTGPGQKAILF

LPMSAKS

bFGF
FGFR
bFGF Hole
PALPEDGGSGAFPPGHFKDPKRL
15

Heavy Chain-
YCKNGGFFLRIHPDGRVDGVREK

1
SDPHIKLQLQAEERGVVSIKGVCA

NRYLAMKEDGRLLASKCVTDECF

FFERLESNNYNTYRSRKYTSWYV

ALKRTGQYKLGSKTGPGQKAILF

LPMSAKSGGGGSGGGGSEPKSSD

KTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNAKTK

PREEQYGSTYRVVSVLTVLHQDW

LNGKEYKCKVSNKALPAPIEKTIS

KAKGQPREPQVCTLPPSRDELTK

NQVSLSCAVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

bFGF
FGFR
Cargo
PALPEDGGSGAFPPGHFKDPKRL
16

Polypeptide-2
YCKNGGFFLRIHPDGRVDGVREK

SDPHIKLQLQAEERGVVSIKGVCA

NRYLAMKEDGRLLASKCVTDECF

FFERLESNNYNTYRSRKYTSWYV

ALKRTGQYKLGSKTGPGQKAILF

LPMSAKS

bFGF
FGFR
bFGF Hole
GSCPPCPAPELLGGPSVFLFPPKPK
17

Heavy Chain-
DTLMISRTPEVTCVVVDVSHEDPE

2
VKFNWYVDGVEVHNAKTKPREE

QYGSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAK

GQPREPQVCTLPPSRDELTKNQVS

LSCAVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLVSKLT

VDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGGGGGSGGGGS

PALPEDGGSGAFPPGHFKDPKRL

YCKNGGFFLRIHPDGRVDGVREK

SDPHIKLQLQAEERGVVSIKGVCA

NRYLAMKEDGRLLASKCVTDECF

FFERLESNNYNTYRSRKYTSWYV

ALKRTGQYKLGSKTGPGQKAILF

LPMSAKS

IL4
IL4R
Cargo
HKCDITLQEIIKTLNSLTEQKTLCT
18

Polypeptide-1
ELTVTDIFAASKNTTEKETFCRAA

TVLRQFYSHHEKDTRCLGATAQQ

FHRHKQLIRFLKRLDRNLWGLAG

LNSCPVKEANQSTLENFLERLKTI

MREKYSKCSS

IL4
IL4R
IL4 Hole
HKCDITLQEIIKTLNSLTEQKTLCT
19

Heavy Chain-
ELTVTDIFAASKNTTEKETFCRAA

1
TVLRQFYSHHEKDTRCLGATAQQ

FHRHKQLIRFLKRLDRNLWGLAG

LNSCPVKEANQSTLENFLERLKTI

MREKYSKCSSGGGGSGGGGSEPK

SSDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDV

SHEDPEVKFNWYVDGVEVHNAK

TKPREEQYGSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVCTLPPSRDEL

TKNQVSLSCAVKGFYPSDIAVEW

ESNGQPENNYKTTPPVLDSDGSFF

LVSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

IL4
IL4R
Cargo
HKCDITLQEIIKTLNSLTEQKTLCT
20

Polypeptide-2
ELTVTDIFAASKNTTEKETFCRAA

TVLRQFYSHHEKDTRCLGATAQQ

FHRHKQLIRFLKRLDRNLWGLAG

LNSCPVKEANQSTLENFLERLKTI

MREKYSKCSS

IL4
IL4R
IL4 Hole
GSCPPCPAPELLGGPSVFLFPPKPK
21

Heavy Chain-
DTLMISRTPEVTCVVVDVSHEDPE

2
VKFNWYVDGVEVHNAKTKPREE

QYGSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAK

GQPREPQVCTLPPSRDELTKNQVS

LSCAVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLVSKLT

VDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGGGGGSGGGGS

HKCDITLQEIIKTLNSLTEQKTLCT

ELTVTDIFAASKNTTEKETFCRAA

TVLRQFYSHHEKDTRCLGATAQQ

FHRHKQLIRFLKRLDRNLWGLAG

LNSCPVKEANQSTLENFLERLKTI

MREKYSKCSS

bimagrumab
ActRII
VH CDR1
GYTFTSSY
22

VH CDR2
INPVSGST
23

VH CDR3
GGWFDY
24

bimagrumab
ActRII
VL CDR1
SSDVGSYNY
25

VL CDR2
GVSK
26

VL CDR3
GTFAGGSYYGV
27

bimagrumab
ActRII
VH
QVQLVQSGAEVKKPGASVKVSC
28

KASGYTFTSSYINWVRQAPGQGL

EWMGTINPVSGSTSYAQKFQGRV

TMTRDTSISTAYMELSRLRSDDTA

VYYCARGGWFDYWGQGTLVTVS

S

bimagrumab
ActRII
VL
QSALTQPASVSGSPGQSITISCTGT
29

SSDVGSYNYVNWYQQHPGKAPK

LMIYGVSKRPSGVSNRFSGSKSGN

TASLTISGLQAEDEADYYCGTFA

GGSYYGVFGGGTKLTVL

bimagrumab
ActRII
bimagrumab-
DIVMTQGALPNPVPSGESVSITCR
30

Hole Heavy
SSKSLLYSDGKTYLNWYLQRPGQ

Chain
SPQLLIYWMSTRASGVSDRFSGSG

SGTDFTLKISGVEAEDVGVYYCQ

QGLEFPDTFGAGTKLELKGGGGS

GGGGSGGGGSQVQLVQSGAEVK

KPGASVKVSCKASGYTFTSSYIN

WVRQAPGQGLEWMGTINPVSGS

TSYAQKFQGRVTMTRDTSISTAY

MELSRLRSDDTAVYYCARGGWF

DYWGQGTLVTVSSASTKGPSVFP

LAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSCD

KTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNAKTK

PREEQYGSTYRVVSVLTVLHQDW

LNGKEYKCKVSNKALPAPIEKTIS

KAKGQPREPQVCTLPPSRDELTK

NQVSLSCAVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

bimagrumab
ActRII
Cargo
DIVMTQGALPNPVPSGESVSITCR
44

Polypeptide
SSKSLLYSDGKTYLNWYLQRPGQ

SPQLLIYWMSTRASGVSDRFSGSG

SGTDFTLKISGVEAEDVGVYYCQ

QGLEFPDTFGAGTKLELKGGGGS

GGGGSGGGGSQVQLVQSGAEVK

KPGASVKVSCKASGYTFTSSYIN

WVRQAPGQGLEWMGTINPVSGS

TSYAQKFQGRVTMTRDTSISTAY

MEELSRLRSDDTAVYYCARGGWF

DYWGQGTLVTVSSASTKGPSVFP

LAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSCD

KTHT

In one aspect, the antigen binding domain binds to an epitope expressed in muscle tissue. In some instances, the antigen binding domain binds to an epitope expressed in a muscle cell. In some instances, the antigen binding domain binds to an epitope expressed in a mammalian muscle satellite cell. In some instances, the antigen binding domain binds to an epitope expressed in a mammalian skeletal muscle cell. In some instances, the antigen binding domain binds to an epitope expressed in a mammalian cardiac muscle cell. In some instances, the antigen binding domain binds to an epitope expressed in a mammalian muscle fiber.

The antigen binding domain can be, for example, an antibody or an antigen binding fragment. When the targeting moiety is the antigen binding fragment, the antigen binding fragment can be, for example, a fragment antigen-binding (Fab) domain or a single chain variable fragment (scFv). In some instances, the antigen binding domain comprises a VH complementarity determining region 1 (VH CDR1) comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 31, a VH CDR2 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 32, and a VH CDR3 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 33. In some instances, the antigen binding domain comprises a VH CDR1 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 31, a VH CDR2 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 32, and a VH CDR3 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 33. In some instances, the antigen binding domain comprises a VL CDR1 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 34, a VL CDR2 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 35, and a VL CDR3 comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 36. In some instances, the antigen binding domain comprises a VL CDR1 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 34, a VL CDR2 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 35, and a VL CDR3 comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 36. In some instances, the antigen binding domain comprises a variable light (VL) chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 38, and a variable heavy (VH) chain knob comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 37. In some instances, the antigen binding domain comprises a variable light (VL) chain comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 38, and a variable heavy (VH) chain knob comprising an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 37 (Table 3).

TABLE 3

Antigen Binding Domain

SEQ

Name
Target
Fragment
Sequence
ID NO

Anti-ITGA7
ITGA7
VH CDR1
GFTFSNYD
31

VH CDR2
ISYDGSRN
32

VH CDR3
TTADNYWFAY
33

Anti-ITGA7
ITGA7
VL CDR1
KSLLYSDGKTY
34

VL CDR2
WMS
35

VL CDR3
CQQGLEFPDT
36

Anti-ITGA7
ITGA7
VH
EVQLVESGGGLVQPGRSMKVS
37

CAASGFTFSNYDMAWVRQAPT

KGLEWVASISYDGSRNYYRDSV

KGRFTISRDNAKSTLYLQMDSL

RSEDTATYYCTTADNYWFAYW

GQGTLVTVSS

Anti-ITGA7
ITGA7
VL
DIVMTQGALPNPVPSGESVSITC
38

RSSKSLLYSDGKTYLNWYLQRP

GQSPQILIYWMSTRASGVSDRFS

GSGSGTDFTLKISGVEAEDVGV

YYCQQGLEFPDTFGAGTKLELK

Anti-ITGA7
ITGA7
Heavy
EVQLVESGGGLVQPGRSMKVS
39

chain: Knob
CAASGFTFSNYDMAWVRQAPT

KGLEWVASISYDGSRNYYRDSV

KGRFTISRDNAKSTLYLQMDSL

RSEDTATYYCTTADNYWFAYW

GQGTLVTVSSASTKGPSVFPLAP

SSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKS

CDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVD

VSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYGSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLP

PCRDELTKNQVSLWCLVKGFYP

SDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQ

QGNVFSCSVMHEALHNHYTQK

SLSLSPGK

Anti-ITGA7
ITGA7
Light chain
DIVMTQGALPNPVPSGESVSITC
40

RSSKSLLYSDGKTYLNWYLQRP

GQSPQLLIYWMSTRASGVSDRF

SGSGSGTDFTLKISGVEAEDVG

VYYCQQGLEFPDTFGAGTKLEL

KRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Anti-ITGA7
ITGA7
Heavy
EVQLVESGGGLVQPGRSMKVS
51

chain: Knob
CAASGFTFSNYDMAWVRQAPT

of 1790.3
KGLEWVASISYDGSRNYYRDSV

KGRFTISRDNAKSTLYLQMDSL

RSEDTATYYCTTADNYWFAYW

GQGTLVTVSSASTKGPSVFPLAP

SSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKS

CDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVD

VSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYGSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLP

PCRDELTKNQVSLWCLVKGFYP

SDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQ

QGNVFSCSVMHEALHNHYTQK

SLSLSPGK

Anti-ITGA7
ITGA7
Light chain
DIVMTQGALPNPVPSGESVSITC
52

of 1790.3
RSSKSLLYSDGKTYLNWYLQRP

GQSPQLLIYWMSTRASGVSDRF

SGSGSGTDFTLKISGVEAEDVG

VYYCQQGLEFPDTFGAGTKLEL

KRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Anti-ITGA7
ITGA7
Heavy
EVQLVESGGGLVQPGRSMKVS
53

chain: Knob
CAASGFTFSNYDMAWVRQAPT

of 1790.4
KGLEWVASISYDGSRNYYRDSV

KGRFTISRDNAKSTLYLQMDSL

RSEDTATYYCTTADNYWFAYW

GQGTLVTVSSASTKGPSVFPLAP

SSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKS

CDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVD

VSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYGSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLP

PCRDELTKNQVSLWCLVKGFYP

SDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQ

QGNVFSCSVMHEALHNHYTQK

SLSLSPGK

Anti-ITGA7
ITGA7
Light chain
DIVMTQGALPNPVPSGESVSITC
54

of 1790.4
RSSKSLLYSDGKTYLNWYLQRP

GQSPQLLIYWMSTRASGVSDRF

SGSGSGTDFTLKISGVEAEDVG

VYYCQQGLEFPDTFGAGTKLEL

KRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Anti-ITGA7
ITGA7
Heavy
EVQLVESGGGLVQPGRSMKVS
55

chain: Knob
CAASGFTFSNYDMAWVRQAPT

of 1790.5
KGLEWVASISYDGSRNYYRDSV

KGRFTISRDNAKSTLYLQMDSL

RSEDTATYYCTTADNYWFAYW

GQGTLVTVSSASTKGPSVFPLAP

SSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKS

CDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVD

VSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYGSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLP

PCRDELTKNQVSLWCLVKGFYP

SDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQ

QGNVFSCSVMHEALHNHYTQK

SLSLSPGK

Anti-ITGA7
ITGA7
Light chain
DIVMTQGALPNPVPSGESVSITC
56

of 1790.5
RSSKSLLYSDGKTYLNWYLQRP

GQSPQLLIYWMSTRASGVSDRF

SGSGSGTDFTLKISGVEAEDVG

VYYCQQGLEFPDTFGAGTKLEL

KRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Anti-ITGA7
CD47
Heavy
QVQLQESGPGLVKPSGTLSLTC
57

chain: Knob
AVSGVSIRSINWWNWVRQPPG

of 1790.6
KGLEWIGEIYHSGSTNYNPSLKS

RVTISVDKSKNQFSLKLNSVTA

ADTAVYYCARDGGIAVTDYYY

YGLDVWGQGTTVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVD

KKVEPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTP

EVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYGST

YRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQP

REPQVYTLPPCRDELTKNQVSL

WCLVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

Anti-ITGA7
CD47
Light chain
EIVLTQSPATLSLSPGERATLSC
58

of 1790.6
RASESVSSNLAWYQQKPGQAP

RLLIYGAFNRATGIPARFSGSGS

GTDFTLTISSLEPEDFAVYYCQQ

RSDWFTFGGGTKVEIKRTVAAP

SVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSG

NSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

Anti-ITGA7
CD47
Heavy
QVQLQESGPGLVKPSGTLSLTC
59

chain: Knob
AVSGVSIRSINWWNWVRQPPG

of 1790.7
KGLEWIGEIYHSGSTNYNPSLKS

RVTISVDKSKNQFSLKLNSVTA

ADTAVYYCARDGGIAVTDYYY

YGLDVWGQGTTVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVD

KKVEPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTP

EVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYGST

YRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQP

REPQVYTLPPCRDELTKNQVSL

WCLVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

Anti-ITGA7
CD47
Light chain
EIVLTQSPATLSLSPGERATLSC
60

of 1790.7
RASESVSSNLAWYQQKPGQAP

RLLIYGAFNRATGIPARFSGSGS

GTDFTLTISSLEPEDFAVYYCQQ

RSDWFTFGGGTKVEIKRTVAAP

SVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSG

NSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

The signaling pathway modulator and the targeting moiety can be in any orientation. In one non-limiting embodiment, the signaling pathway modulator is connected to an amino terminus of the targeting moiety. In another non-limiting embodiment, the signaling pathway modulator is connected to a carboxy terminus of the targeting moiety.

In some instances, the agent can further comprise a linker. In some instances, the agent can further comprise an Fc domain. The Fc domain can be, for example, a human IgG1 Fc domain. The Fc domain can comprise a knob-in-hole. In some instances, the agent can further comprise a linker and an Fc domain.

In one non-limiting embodiment, (a) the signaling modulator comprises an amino acid sequence of any one of SEQ ID NOS: 1-30, or an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to any one of SEQ ID NOS: 1-30, wherein the C-terminus or N-terminus of the signaling pathway modulator is coupled to a GGGGSGGGGSGGGGS linker (SEQ ID NO: 41) and a hIgG1 FC domain with a hole and N297G mutation; and (b) the antigen binding domain comprising a human IgG1 heavy chain with knob mutations and a N297G mutation and a light chain. In another non-limiting embodiment, (a) the signaling modulator comprises an amino acid sequence of any one of SEQ ID NOS: 1-30, or an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to any one of SEQ ID NOS: 1-30, wherein the C-terminus or N-terminus of the signaling pathway modulator is coupled to a GGGGSGGGGS (SEQ ID NO: 42) and a hIgG1 FC domain with a hole and N297G mutation; and (b) the antigen binding domain comprising a human IgG1 heavy chain with knob mutations and a N297G mutation and a light chain. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 39. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 39. In some embodiments, the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 40. In some embodiments, the light chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in SEQ ID NO: 40 (Table 3). In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in any one of SEQ ID NOS: 51, 53, and 55. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in any one of SEQ ID NOS: 51, 53, and 55. In some embodiments, the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in any one of SEQ ID NOS: 52, 54, and 56. In some embodiments, the light chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence set forth in any one of SEQ ID NOS: 52, 54, and 56.

In one aspect, the cargo comprises a vector. The vector can be any suitable macromolecule or complex of molecules comprising a polynucleotide to be delivered to a target cell. In some embodiments, the vector is a gene editing vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a recombinant vector. In some embodiments, the vector comprises more than one vector described herein.

In one aspect, the vector is a non-viral vector. In some embodiments, the non-viral vector is capsid-free. In some embodiments, the non-viral vector is a deoxyribonucleic acid (DNA) vector. In some embodiments, the non-viral vector is a ribonucleic acid (RNA) vector. In some embodiments, the non-viral vector is obtained from a plasmid. In some embodiments, the non-viral vector is produced in a host cell.

In another aspect, the vector is a viral vector. In some embodiments, the viral vector is a replicative viral vector. In some embodiments, the viral vector is a non-replicative viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. The AAV vector can include a polynucleotide of interest (e.g., a transgene) that is flanked by AAV terminal repeat sequences (ITRs) and may be packaged into infectious viral particles. The AAV vector may be replicated when present in a host cell, such as a host cell that has been transfected with a vector encoding and expressing rap and cap gene products. In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector comprise a recombinant viral vector that comprises one or more nucleic acids described herein. In some embodiments, the viral vector is used to provide long-term gene expression. In some embodiments, the viral vector is used to provide short-term gene expression. In some embodiments, the viral vector is used to express a therapeutic molecule. In some embodiments, the therapeutic molecule is an nucleic acid, a protein, or an antibody.

In another aspect, the cargo comprises a nucleic acid. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an RNA. In some embodiments, the nucleic acid is double-stranded. In some embodiments, the RNA is an RNA interference-inducing (RNAi) molecule including, but not limited to, a siRNA, double strand RNA (dsRNA), small temporal RNA (stRNA), short-hairpin RNA (shRNA), microRNA (miRNA), and gene silencing variants thereof. In some embodiments, the nucleic acid is a double strand RNA (dsRNA). In some embodiments, the dsRNA reduces the expression of a target gene in a cell. In some embodiment, the cell is a muscle cell. In some embodiments, the reduction of the expression of the target gene treats diseases caused by the expression of the target gene.

In some embodiments, the dsRNA has a first oligonucleotide sequence and a second oligonucleotide sequence. In some embodiments, the second oligonucleotide sequence anneals to the first oligonucleotide sequence under biological conditions. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 60 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 50 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 40 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 30 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 10 nucleotides to about 20 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 60 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 50 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 40 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 20 nucleotides to about 30 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 60 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 50 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 30 nucleotides to about 40 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 60 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 40 nucleotides to about 50 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 50 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 50 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 50 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 50 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 50 nucleotides to about 60 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 60 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 60 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 60 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 60 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 70 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 70 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 70 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 80 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 80 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is from about 90 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 40 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 30 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 10 nucleotides to about 20 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 40 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 20 nucleotides to about 30 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 30 nucleotides to about 40 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 40 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 50 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 50 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 50 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 50 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 50 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 60 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 60 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 60 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 60 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 70 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 70 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 70 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 80 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 80 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is from about 90 nucleotides to about 100 nucleotides in length.

In some embodiments, the dsRNA comprises a sequence sufficiently complementary with a target sequence of a target gene. In some embodiments, the dsRNA has an asymmetric structure. In some embodiments, the dsRNA has two separate oligonucleotides that are linked by a third structure. In some embodiments, the dsRNA is a small interfering RNA (siRNA). In some embodiments, the dsRNA is a precursor of a siRNA. In some embodiments, the dsRNA is processed by Dicer to produce a siRNA. The dsRNA can be from a single RNA oligonucleotide that undergoes intramolecular annealing. In some embodiments, the dsRNA comprises at least one modification. In some embodiments, the at least one modification promotes Dicer to bind to the double-stranded RNA structure in an orientation that maximizes the double-stranded RNA structure's effectiveness in inhibiting gene expression. In some embodiments, the first and second oligonucleotides are not required to be completely complementary. In some embodiments, the siRNA is 19 nucleotides in length. In some embodiments, the siRNA is 20 nucleotides in length. In some embodiments, the siRNA is 21 nucleotides in length. In some embodiments, the siRNA is 22 nucleotides in length. In some embodiments, the siRNA is 23 nucleotides in length. In some embodiments, the siRNA is 24 nucleotides in length. In some embodiments, the siRNA is 25 nucleotides in length. In some embodiments, the siRNA is 26 nucleotides in length. In some embodiments, the siRNA is 27 nucleotides in length. In some embodiments, the siRNA is 28 nucleotides in length. In some embodiments, the siRNA is 29 nucleotides in length. In some embodiments, the siRNA is 30 nucleotides in length.

The siRNA can comprise an antisense strand and a sense strand. In some embodiments, the sense strand is from about 20 to about 30, or from about 22 to about 28 nucleotides in length. In some embodiments, the antisense strand is at least about 19 nucleotides in length. In some embodiments, the antisense strand has a modification at a 3′ end and/or 5′ end. In some embodiments, the antisense strand of the dsRNA has a 3′ overhang. In some embodiments, the antisense strand of the siRNA is modified to include from about 1 to about 9 ribonucleotides on the 5′ end to give a length of from about 20 to about 30 nucleotides.

In some embodiments, the siRNA is chemically modified. In some embodiments, the siRNA has reduced immunostimulatory activity compared to a corresponding RNA molecule not having the chemical modification. In some embodiments, the siRNA has increased serum stability compared to a corresponding RNA molecule not having the chemical modification.

In some embodiments, the siRNA is a shRNA. The shRNA can function as an siRNA. The shRNA can have double-stranded hairpin-like structure for increased stability. In some embodiments, the hairpin-like structure is from about 1 to about 30, from about 1 to about 24, or from about 1 to about 10, nucleotides in length. The sequence of the hairpin-like structure can include nucleotide residues unrelated to the target gene. In some embodiments, the hairpin-like structure is 5′-UU-3′. In some embodiments, the hairpin-like structure includes non-nucleotide moieties. In some embodiments, the hairpin-like structure does not include any non-nucleotides moieties. The shRNA can also comprise RNAs with stem-loop structures that contain mismatches and/or bulges. In some embodiments, the hairpin-like structure comprises nucleotides, non-nucleotides, or combinations of nucleotides and non-nucleotides.

In another aspect, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is an antisense oligonucleotide (ASO). In some embodiments, the ASO comprises a sequence sufficiently complementary with a messenger RNA (mRNA) of a target gene of a target cell. In some embodiments, the target cell is a muscle cell. In some embodiments, the ASO targets a pre-mRNA. In some embodiments, the ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety. In some embodiments, the ASO is a morpholino. In some embodiments, the ASO comprises at least one modified sugar moiety. In some embodiments, the ASO is from 8 to 50 nucleotides in length. In some embodiments, the ASO is from 8 to 40 nucleotides in length. In some embodiments, the ASO is from 8 to 35 nucleotides in length. In some embodiments, the ASO is from 8 to 30 nucleotides in length. In some embodiments, the ASO is from 8 to 25 nucleotides in length. In some embodiments, the ASO is from 8 to 20 nucleotides in length. In some embodiments, the ASO is from 8 to 15 nucleotides in length. In some embodiments, the ASO is from 9 to 50 nucleotides in length. In some embodiments, the ASO is from 9 to 40 nucleotides in length. In some embodiments, the ASO is from 9 to 35 nucleotides in length. In some embodiments, the ASO is from 9 to 30 nucleotides in length. In some embodiments, the ASO is from 9 to 25 nucleotides in length. In some embodiments, the ASO is from 9 to 20 nucleotides in length. In some embodiments, the ASO is from 9 to 15 nucleotides in length. In some embodiments, the ASO is from 10 to 50 nucleotides in length. In some embodiments, the ASO is from 10 to 40 nucleotides in length. In some embodiments, the ASO is from 10 to 35 nucleotides in length. In some embodiments, the ASO is from 10 to 30 nucleotides in length. In some embodiments, the ASO is from 10 to 25 nucleotides in length. In some embodiments, the ASO is from 10 to 20 nucleotides in length. In some embodiments, the ASO is from 10 to 15 nucleotides in length. In some embodiments, the ASO is from 11 to 50 nucleotides in length. In some embodiments, the ASO is from 11 to 40 nucleotides in length. In some embodiments, the ASO is from 11 to 35 nucleotides in length. In some embodiments, the ASO is from 11 to 30 nucleotides in length. In some embodiments, the ASO is from 11 to 25 nucleotides in length. In some embodiments, the ASO is from 11 to 20 nucleotides in length. In some embodiments, the ASO is from 11 to 15 nucleotides in length. In some embodiments, the ASO is from 12 to 50 nucleotides in length. In some embodiments, the ASO is from 12 to 40 nucleotides in length. In some embodiments, the ASO is from 12 to 35 nucleotides in length. In some embodiments, the ASO is from 12 to 30 nucleotides in length. In some embodiments, the ASO is from 12 to 25 nucleotides in length. In some embodiments, the ASO is from 12 to 20 nucleotides in length. In some embodiments, the ASO is from 12 to 15 nucleotides in length.

In another aspect, the cargo comprises a lipid nanoparticle (LNP). The LNP comprises a lipid formulation that can be used to deliver a vector to a target site of interest (e.g., cell, tissue, organ, and the like).

In some embodiments, the nucleic acid is encapsulated in the LNP. In some embodiments, the LNP comprises a lipid selected from the group consisting of an ionizable or cationic lipid, a helper phospholipid, a conjugated lipid, a PEG-conjugated lipid, a cholesterol-based lipid, and a combination thereof. In some embodiments, the ionizable lipid is ALC-0315, SM-102, or MC-3. In some embodiments, the cholesterol-based lipid is cholesterol or derivatives thereof. In some embodiments, the helper phospholipid is DSPC. In some embodiments, the conjugated lipid is PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, or mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid. In some embodiments, the PEG-conjugated lipid is ALC-0159. In some embodiments, the PEG-lipid conjugate is PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof.

In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the LNP comprises a non-cationic lipid. In some embodiments, the non-cationic lipid is a neutral uncharged, zwitterionic, or anionic lip. In some embodiments, the non-ionic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), di stearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOP 5), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), di stearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine.

In some embodiments, the ionizable lipid is present at a molar ratio of 35-55%. In some embodiments, the PEG-conjugated lipid is present at a molar ratio of 0.25% to 3%. In some embodiments, the helper phospholipid is present at a molar ratio of 5% to 20%. In some embodiments, the cholesterol-based lipid is present at a molar ratio of 20% to 45%. In some embodiments, the cationic lipid may be present in an amount of from about 10% by weight of LNP to about 85% by weight of the lipid nanoparticle, or from about 50% by weight of LNP to about 75% by weight of the LNP. All lipid molar ratios described here are relative to the total lipid content of the LNP. Molar ratios of lipids in the LNP, for example the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid, can be varied as needed.

In some embodiments, the LNP does not comprise any phospholipids. In some embodiments, the LNP comprises one or more additional compounds. In some embodiments, the additional compounds are administered separately. In some embodiments, the additional compounds can be included in the LNP provided herein. In some embodiments, the additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.

In another aspect, the cargo comprises a drug. In yet another aspect, the cargo comprises two or more types of drugs. In some embodiment, the drug is a therapeutic agent for muscle diseases. In some embodiments, the drug is conjugated to the antigen-binding domain via a linker. In some embodiments, the linker is cleavable. In some embodiments, the linker is cleavable by a cleaving agent that is present in an intracellular environment (e.g., within a lysosome or endosome or caveolea). In some embodiments, the linker contains peptide (e.g. oligopeptide, or di-, tri-, tetra- or penta-peptide containing linkers). In some embodiments, the linker comprises a valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide. In some embodiments, the drug conjugated to the antigen-binding domain functions as a targeted drug delivery system. In some embodiments, the conjugation of the drug to the antigen-binding domain is site-specific. In some embodiments, the conjugation of the drug to the antigen-binding domain is not site-specific. In some embodiments, the drug is a prodrug that is activated upon administration to a subject. In some embodiments, the drug is a small molecule drug. In some embodiments, the drug is a peptide drug. In some embodiments, the drug is a protein drug.

In some embodiments, the drug is a toxin. In some embodiments, the toxin is a cytotoxic molecule. In some embodiments, the toxin is a radionuclide. In some embodiments, the toxin is a chemotherapeutic drug. In some embodiments, the toxin is an anticancer agent. In some embodiments, the anticancer agent is adriamycin, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, methotrexate, 5-fluorouracil, aclacinomycin, nitrogen mustard, cyclophosphamide, bleomycin, daunorubicin, doxorubicin, vincristine, vinblastine, or a combination thereof. In some embodiments, the toxin is cell membrane permeable. In some embodiments, the toxin is dianthin or saporin. In some embodiments, the toxin is Pseudomonas aeruginosa exotoxin (PE) or a cytotoxic fragment thereof (e.g., PE38). In some embodiments, the toxin is monomethyl auristatin E (MMAE) or MMAF.

In another aspect, the cargo comprises an immune modulator. In some embodiments, immune modulator is immunosuppressant. In some embodiments, the immune modulator decreases an activity of an immune system. In some embodiments, immune modulator is immunostimulatory. In some embodiments, the immune modulator increases an activity of an immune system. In some embodiments, the immune modulator is an interleukin (IL). In some embodiments, the interleukin is an IL-10. In some embodiments, the interleukin is an IL-35. In some embodiments, the interleukin is an IL-4. In some embodiments, the interleukin is an IL-13. In some embodiments, the interleukin is an IL-27. In some embodiments, the interleukin is an IL-37. In some embodiments, the interleukin is a combination of interleukins provided herein. In some embodiments, the immune modulator is a chemokine. In some embodiments, the chemokine is CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, or a combination thereof.

In another aspect, provided herein is a pharmaceutical composition comprising (i) any of the agents and (ii) one or more pharmaceutically acceptable excipient(s).

Functional Assays

The agents described herein can be tested in any in vitro and/or in vitro functional assay, such as those described below.

In Vitro Functional Assay

Ligand binding properties of amino acid substituted variants of human insulin-like growth factor I (IGF-I) are analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-IR), respectively. Ligand binding kinetic rates are determined using BIAcore biosensor interaction analysis technology. Secondary structure content of the IGF-I variants is estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations.

In Vivo Functional Assay

Male mdx mice (3-6 months of age; 4-5 mice per group) are obtained. Animal care and experimental procedures are conducted in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes.

Mice are administered a negative control, an IGF-1 variant, GH variant, bFGF variant, IL4 variant, or bimagrumab, alone, an antigen binding domain alone, or an agent described herein. Groups are administered treatment once weekly for three weeks via injection (e.g., intravenous injection). Following onset of treatment, mice are monitored and weighed every second day until the conclusion of the study. Body weight and muscle mass are assessed prior to treatment, during treatment, and at the conclusion of the study.

Methods of Modulation

Provided herein is method of modulating a muscular dystrophy (MD) comprising delivering to a region adjacent to a muscle any of the agents described herein. In some instances, the administration is to a subject and the subject is a mammal. The mammal can be, for example, a human. Administering can comprise injecting the subject at a location near a skeletal muscle, a muscle satellite cell (e.g., a skeletal muscle satellite cell), a skeletal muscle, a cardiac muscle, a smooth muscle, or a muscle fiber. Injection can be any suitable route of injection including, but not limited to, intravenous (IV), subcutaneous (SQ), intramuscular (IM), intrathecal (IT) and intraperitoneal. Administration of the agent to the subject induces signaling of the IGF-1 signaling pathway, the GH signaling pathway, the bFGF signaling pathway, the IL4 signaling pathway, or inhibits ActRII. In certain instances, a pharmaceutical composition is administered to the subject, where the pharmaceutical composition comprises the agent and one or more pharmaceutically acceptable carrier(s). The term “pharmaceutically acceptable carrier” as used herein, refers to a carrier or excipient for administration of an active substance. The pharmaceutically acceptable carrier can comprise any substance or vehicle suitable for delivering the agent to the subject. The term refers to any pharmaceutical carrier that may be administered without undue toxicity. Suitable carriers may be, for example, one or more optional stabilizers, diluents, and/or excipients.

A muscular dystrophy to be treated with the methods described herein include, for example, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), a limb-girdle muscular dystrophy (LGMD), and/or a congenital muscular dystrophy.

A subject to be treated with the described methods has been diagnosed with a muscular dystrophy. Diagnosis can be based upon one or more of the following: family history; genetic testing; cardiomyopathy; calf muscle hypertrophy; cognitive impairment; delayed speech and language development; elevated serum creatine kinase; muscle loss; weakness of the face, shoulder girdle, and upper arm(s) with relative sparing of the deltoid muscles; or any combination thereof. Assessment can also include nerve conduction velocity testing (NCV) and electromyography (EMG).

In one instance, a subject treated with an agent described herein experiences at least a 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold improvement, or more, in one or more symptoms. Improvement can be in comparison to treatment with a placebo or in comparison to the same subject prior to treatment.

Certain Definitions

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein.

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

In this application, the use of “or” means “and/or” unless stated otherwise. The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

The term “about” means a range within 10% of a given value.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia (U.S.P.) or other generally recognized pharmacopeia for use in animals, including humans.

The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

The “percent sequence identity” between a reference amino acid sequence and a query amino sequence (i.e., the amino sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference amino acid sequence) is determined by optimally aligning the sequences using the Needleman-Wunsch alignment algorithm with a gap existence penalty of 11 and a gap extension penalty of 1 and comparing the sequences. The number of exact matches, divided by the total number of positions in the alignment (which corresponds with the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query amino acid sequence and the reference amino acid sequence (i.e., percent sequence identity=(# of exact matches/(total # of positions in alignment)*100). An alignment using the Needleman-Wunsch alignment algorithm (with a gap existence penalty of 11 and a gap extension penalty of 1) can be generated using the “Global Align” BLAST program available at https://blast.ncbi.nlm.nih.gov/Blast.cgi.

The “percent sequence identity” between a reference nucleic acid sequence and a query nucleic acid sequence (i.e., the nucleic acid sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference nucleic acid sequence) is determined by optimally aligning the sequences using the Needleman-Wunsch alignment algorithm (with match/mismatch scores of 2, −3, a gap existence penalty of 5, and a gap extension penalty of 2) and comparing the aligned nucleic acids. The number of exact match-es divided by the total number of nucleotides in the alignment (which corresponds with the number of nucleotides in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query nucleic acid sequence and the reference nucleic acid sequence (i.e., percent sequence identity=(# of exact matches)/(total # of nucleotides in the alignment)*100). An alignment using the Needleman-Wunsch alignment algorithm (with match/mismatch scores of 2, −3, a gap existence penalty of 5, and a gap ex-tension penalty of 2) can be generated using the “Global Align” BLAST program available at https://blast.ncbi.nlm.nih.gov/Blast.cgi.

EXAMPLES
Example 1: Methods and Procedure

The methods and procedure described herein were applied in all the Examples.

In Vitro Potency Assay (pAKT Assay)

15×10⁶DU145 cells (obtained from ATCC) frozen down at passage 5 (P5) were reseeded in a T175 culture flask containing DMEM/F12 (1:1) medium (Gibco 11320-033) supplemented with 10% FBS (Gibco 10437-028) and 10 μg/mL Gentamicin (Gibco 15710-064) (hereafter referred to as culture medium) and expanded. At 80-90% confluency, cells were trypsinized, manually counted using a hemocytometer, resuspended at 3×10⁵cells per mL in culture medium, and then reseeded in 24-well plates (Corning 3524) in 0.5 mL aliquots per well (150,000 cells/well). 14-16 hours after reseeding, the culture medium was removed, and following a wash with 1 mL PBS, cultured for 6 h in 225 μL serum-free culture medium supplemented with 0.2% BSA (Millipore A3059). 25 μL aliquots of the following growth factor dilution series were added to the cells in duplicate: 4570, 1370, 410, 125, 37, 11.1, and 3.3 nM rhIGF1 (PeproTech #100-11) (SEQ ID NO: 1) and Fusion Antibodies (comprising three polypeptide chains: SEQ ID NOS: 2, 39, and 40). After 15 min incubation at 37° C., medium was aspirated and cells lysed in 150 μL 1×PTR buffer (Extraction Buffer 5×PTR, Abcam ab193970) containing 1× Extraction Enhancer Buffer (Extraction Enhancer Buffer 50×, Abcam ab193971) and protease/phosphatase inhibitors (one Pierce mini tablet [A32959] per 10 mL 1×PTR). After 15-30 min incubation on ice, plates were vortexed, and lysates transferred to Eppendorf tubes and stored at −80° C. for assessment of IGF1R-AKT signaling strength using the Human/Mouse/Rat Phospho-Akt (S473) Pan Specific DuoSet IC ELISA (RnD #DYC887B).

C2C12 mouse muscle myoblast cells (ATCC #CRL-1772) were cultured in DMEM containing 4.5 g/L glucose (Corning 10-013-CV) supplemented with 10% FBS (Gibco 10437-028) and 10 μg/mL Gentamicin (15710-064). At 70-80% confluency, cells were trypsinized, manually counted using a hemocytometer, resuspended at 2×10⁵cells per mL in differentiation medium (DMEM (Corning 10-013-CV) supplemented with 2% horse serum (Sigma, #H1138) and 10 μg/mL Gentamicin (Gibco 15710-064), and seeded in 24-well plates (Corning 3524) in 0.5 mL aliquots per well (100,000 cells/well). After 5 days on differentiation medium C2C12 cells were washed with 1 mL PBS and then serum-starved for 6 h in 225 μL serum-free culture medium supplemented with 0.2% BSA (Millipore A3059). The remainder of the protocol is the same as described above for DU145 cells.

ELISAs were performed in 384-well plates to the manufacturers' recommendation with minor modifications. Briefly, wells of 384-well plates were coated with 25 μL 6 μg/mL phospho-Akt1 (S473) capture antibody (RnD #841692) in PBS (25 μL per 384-well), sealed and placed overnight at RT. Wells were washed 4× with 100 μL PBS containing 0.05% Tween 20, and blocked with 50 μL PBS containing 2% BSA for 1-2 h at room temperature (RT). Wells were washed 4× with 100 μL PBS containing 0.05% Tween 20 and incubated with 25 μL of lysate or P-AKT standard (RnD #841694) for 2 h at RT. Wells were washed 4× with 100 μL PBS containing 0.05% Tween 20 and then incubated with 25 μL 100 ng/mL phospho-Akt1 (S473) detection antibody (RnD #843081) diluted in PBS containing 1% BSA for 1 h at RT. Wells were washed 4× with 100 μL PBS containing 0.05% Tween 20 and incubated with 25 μL Streptavidin-HRP A (RnD #890803) diluted 1:200 in PBS containing 1% BSA for 20 min at RT. Wells were washed 4× with 100 μL PBS containing 0.05% Tween 20 and incubated with 25 μL TMB substrate solution (Abcam TMB ELISA Substrate High Sensitivity, #ab171523), and incubated for 2-20 min at RT (timing depends on the intensity of the color reaction). 12.5 μL stop solution (RnD #DY994) was added and the OD450 of each well measured using a BMG Labtech CLARIOstar® Plus Microplate Reader. EC50 values were calculated using Prism 10 software.

Potency Shift Assay

Undifferentiated wildtype (WT) and ITGA7-knockout (KO) C2C12 cells were resuspended at 0.5×10⁵cells per mL in culture medium and seeded in 24-well plates (Corning, #3524) in 0.5 mL aliquots per well (25,000 cells/well). The remainder of the protocol is the same as described above for DU145 cells, with the exception of the volume of the lysis buffer, which is 200 μL for C2C12 cells.

Animal Studies

Wild-type (WT) male C57BL/6J (Jackson Labs, Strain #000664) or C57BL/10ScSn-Dmdmdx/J (mdx) (Jackson Labs, Strain #001801) were purchased at 8 weeks of age from Jackson Laboratories. All animal studies were conducted in compliance with the protocols approved by the Explora Biolabs Institutional Animal Care and Use Committee.

In Vivo PK Analysis (Human IgG Assay)

Wild-type (WT) male C57BL/6J (Jackson Labs, Strain #000664) were purchased at 8 weeks of age and shipped to the Cavalry Biosciences vivarium facility managed by Explora Biolabs in San Francisco, CA. After at least 1 week of acclimation mice were weighed and administered custom therapeutic proteins from Fusion Antibodies, Inc. by lateral tail vein injection with insulin syringes (BD, 329424) in dose volumes of 5-10 ml/kg at dose ranges of 0.3-15 mg/kg. Individual animals were dosed for timepoints collected at 1, 4, 24, 48, 96, 144, and 240 hours post dose with 3-5 animals per timepoint. At the appropriate timepoint, animals were anesthetized with 5% isoflurane using an RC2 rodent circuit controller (922100, VetEquip) and plasma was harvested from blood collected from the inferior vena cava by centrifugation in microtainers containing Dipotassium EDTA (BD, 365974). Samples were frozen on dry ice and stored at −80 C until future analysis. For analysis, plasma samples were diluted as determined empirically for each dose level and evaluated for concentration of therapeutic proteins by a sandwich ELISA that for the humanized Fc region of therapeutic proteins (Human IgG ELISA Kit, ab195215, Abcam) according to the manufacturer's instructions. Standard curves for each therapeutic protein being analyzed were generated in sample diluent supplied with the assay at concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 ng/ml. OD450 data for standard curves and each sample was collected using a Promega GloMAX Discover Microplate Reader. Sample data was interpolated from standard curves, corrected for dilution factors, plotted and half-lives were calculated using two-phase decay in Prism 9 (Graphpad).

In Vivo Distribution (Human IgG Assay)

C57BL/10ScSn-Dmdmdx/J (mdx) (Jackson Labs, Strain #001801) or control age-matched wild-type (WT) male C57BL/6J (Jackson Labs, Strain #000664) were weighed and administered custom therapeutic proteins from Fusion Antibodies, Inc. by lateral tail vein injection with insulin syringes (BD, 329424) in dose volumes of 5-10 ml/kg at dose ranges of 0.3-15 mg/kg. Individual animals were dosed for timepoints collected at 4, 24 or 72 hours post dose with 5-8 animals per therapeutic protein/timepoint. At the appropriate timepoint animals were anesthetized with 5% isoflurane using an RC2 rodent circuit controller (922100, VetEquip) and plasma was harvested from blood collected from the inferior vena cava by centrifugation in microtainers containing Dipotassium EDTA (BD, 365974). Animals were then perfused with 20-30 ml of Dulbecco's Phosphate-Buffered Salt Solution (DPBS, Corning, 21030CV) through the heart apex. Multiple tissue were collected including some or all of the following: heart, kidney, liver, lungs, gastrocnemius muscle, diaphragm muscle, tibialis anterior muscle and forearm muscle. Tissue was weighed and flash frozen on dry ice and stored at −80 C until later analysis. Samples were homogenized 1 ml/250 mg tissue in Extraction Buffer 5×PTR (Abcam #ab193970) containing protease and phosphatase inhibitors (Thermo Scientific, A32959) using a NextAdvance Bullet Blender Gold (BB24AU) with Green, Navy or Red Eppendorf Lysis Kits (NextAdvance). Samples were diluted as determined empirically for each tissue and/or dose level and evaluated for concentration of therapeutic proteins by a sandwich ELISA that for the humanized Fc region of therapeutic proteins (Human IgG ELISA Kit, ab195215, Abcam) according to the manufacturer's instructions. Standard curves for each therapeutic protein being analyzed were generated in sample diluent supplied with the assay at concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 ng/ml. OD450 data for standard curves and each sample was collected using a Promega GloMAX Discover Microplate Reader. Sample data was interpolated from standard curves, corrected for dilution factors, plotted and IgG levels were plotted as ng/ml/sample in Prism 9 (Graphpad).

In Vivo PD Analysis

For evaluation of therapeutic protein PD, IGFR1 activation was assessed by determining relative tissue levels of phospho-AKT using the phospho-Akt (S473) Pan Specific DuoSet IC ELISA (RnD #DYC887B-2) and processing tissues as above. ELISAs were performed in 384-well plates to the manufacturers' recommendation with minor modifications. Briefly, wells of 384-well plates were coated with 25 μl 6 μg/ml phospho-Akt1 (S473) capture antibody (#841692) in PBS (25 μl per 384-well), sealed and placed overnight at RT. Wells were washed 4× with 100 μl PBS containing 0.05% Tween 20, and blocked with 50 μl PBS containing 2% BSA for 1-2 h at room temperature (RT). Wells were washed 4× with 100 μl PBS containing 0.05% Tween 20 and incubated with 25 μl of lysate or P-AKT standard for 2 h at RT. Wells were washed 4× with 100 μl PBS containing 0.05% Tween 20 and then incubated with 25 μl 100 ng/ml phospho-Akt1 (S473) detection antibody (#843081) diluted in PBS containing 1% BSA for 1 h at RT. Wells were washed 4× with 100 μl PBS containing 0.05% Tween 20 and incubated with 25 μl Streptavidin-HRP A (RnD #890803) diluted 1:200 in PBS containing 1% BSA for 20 min at RT. Wells were washed 4× with 100 μl PBS containing 0.05% Tween 20 and incubated with 25 μl TMB substrate solution (Abcam TMB ELISA Substrate High Sensitivity (ab171523; lot GR3427893-1)), and incubated for 10-20 min at RT. 12.5 μl stop solution (RnD #895926 from Ancillary Kit 2) was added. OD450 data for standard curves and each sample was collected using a Promega GloMAX Discover Microplate Reader. Sample data was interpolated from standard curves, corrected for dilution factors, plotted and pAKT levels were plotted in Prism 10 (Graphpad).

Immunohistochemistry (IHC) Assay

Muscles from Wild-type (WT) male C57BL/6J (Jackson Labs, Strain #000664) or C57BL/10ScSn-Dmdmdx/J (mdx) (Jackson Labs, Strain #001801) were isolated 4-72 hours after dosing with 3-10 mg/kg CV1707-12. Tissue was fixed in 4% paraformaldehyde overnight and switched to 30% sucrose 24 hours later. Muscles was then embedded in OCT medium and sectioned in 10-micron sections and adhered to glass slides. Tissue was then stained for hIgG using rabbit anti-human IgG followed by a secondary antibody and DAB development kit. In brief, sections were washed with PBS+0.1% Tween-20 and permeabilized with PBS+0.3% TrixonX-100. Next, slides were blocked with hydrogen peroxide for 10 minutes and then incubated with rabbit anti-human IgG (Invitrogen #31143) for 24 hours at 1:2000. Slides were then washed with PBS+0.1% Tween-20 3×, incubated with secondary antibody, anti-Rabbit IgG HRP Polymer (Vector Labs). Slides were then washed with PBS+0.1% Tween-20 3× and developed with DAB (3,3′-diaminobenzidine) substrate for 3-5 minutes at RT and rinsed in DI water for 2 minutes. Stains were then counterstained with Methyl Green for 1 minute at room temperature, rinsed in DI water, and dehydrated with 100% isopropanol two times for two minutes each. Slides were then coverslipped and imaged.

Western Blot Analysis and Quantitation

Tissue lysates were generated from 4 mice per treatment and time point. These samples were pooled and mixed 1:1 with 2× Laemmli Sample Buffer (Bio-Rad, #1610737) and betamercaptoethanol according to manufacturer's instruction and boiled for 10 minutes. Samples for kidney were further diluted 3-fold. 3.5 μL-9 μL was loaded onto a 4-15% SDS-PAGE gel (Bio-Rad, #4561086) and run at 200 V for about 45 minutes. Gels were transferred to a PVDF-type transfer membrane (Millipore, #IPVH00010) with standard western blotting procedures. Membranes were stained with Ponceau S to visualize protein loading. Primary antibody incubations were overnight at 4° C., secondary antibody incubation was for 1 h at RT. Antibodies used: P-AKTS473 1:2,000 (Cell Signaling Technology, #4060), AKT 1:1,000 (Cell Signaling Technology, #9272), GAPDH 1:2,000 (Boster, 00227-1), Goat anti-Rabbit IgG (H+L)-HRP 1:50,000 (Invitrogen, #31460). Signal was detected by incubation with chemiluminescent substrate (ThermoScientific, #34580) and autoradiography film (ECE Scientific, #E3018).

Generation of C2C12 Itga7 Knockout Cells

C2C12-ITGA7 KO cells were generated by using the Alt-R CRISPR-Cas9 System (IDT) according to the manufacturer's protocol with Itga7-targeting gRNA oligo/AltR1/rCrU rCrUrC rUrGrU rCrGrA rGrArC rUrCrA rUrArU rGrUrU rUrUrA rGrArG rCrUrA rUrGrC rU/AltR2/ and Lipofectamine CRISPRMAX transfection reagent (Thermo Fisher Scientific, #CMAX00008). Absence of MATN3 was verified by immunofluorescence staining with anti-ITGA7 (Cavalry Biosciences, #CV1707-4).

ELISAs were performed in 384-well plates to the manufacturers' recommendation with minor modifications. Briefly, wells of 384-well plates were coated with 25 μL 6 μg/mL phospho-Akt1 (S473) capture antibody (RnD #841692) in PBS (25 μL per 384-well), sealed and placed overnight at room temperature. Wells were washed 3× with 100 μL PBS containing 0.05% Tween 20, and blocked with 50 μL PBS containing 2% BSA for 1-2 h at room temperature (RT). Wells were washed 3× with 100 μL PBS containing 0.05% Tween 20 and incubated with 25 μL of lysate or P-AKT standard (RnD #841694) for 2 h at RT. Wells were washed 3× with 100 μL PBS containing 0.05% Tween 20 and then incubated with 25 μL 100 ng/mL phospho-Akt1 (S473) detection antibody (RnD #843081) diluted in PBS containing 1% BSA for 1 h at RT. Wells were washed 3× with 100 μL PBS containing 0.05% Tween 20 and incubated with 25 μL Streptavidin-HRP A (RnD #890803) diluted 1:200 in PBS containing 1% BSA for 20 min at RT. Wells were washed 3× with 100 μL PBS containing 0.05% Tween 20 and incubated with 25 μL TMB substrate solution (Abcam TMB ELISA Substrate Highest Sensitivity, #ab171522), and incubated for 2-20 min at RT (timing depends on the intensity of the color reaction). 12.5 μL stop solution (RnD #DY994) was added and the OD450 of each well measured using a BMG Labtech CLARIOstar® Plus Microplate Reader. EC₅₀values were calculated using Prism 10 software.

Hindlimb Casting

Mice were acclimated for 14 or more days following arrival. Baseline muscle function was measured and animals were assigned to balanced groups based on initial bodyweights and baseline force measurements. Mice then underwent hindlimb casting, or sham casting on one limb and were dosed with vehicle or 10 mg/kg CAV-003 every 4 days by intraperitoneal injection. After 2 weeks of casting, casts were removed, muscle function was measured again, then euthanized and necropsied for further tissue analysis.

For cast installation, mice were anesthetized by inhalation or ˜5% isoflurane. Once sedated the hindlimb was wiped with three alternating washes of povidone-iodine, then 70% ethanol, and allowed to dry. The hindlimb was then loosely wrapped in sterile surgical gauze and a custom-made plastic immobilization device, consisting of a small, tapered tube, about the diameter of the mouse leg. This was then placed on the hindlimb, with the foot in full extension, to ensure the maximal in vivo unloading of the plantar flexor group. The cast extended proximal to the knee and distal to the ankle. The immobilization device is then fixed to the hindlimb using Vetbond tissue glue. After sedation mouse was removed from anesthesia, isothermal pad and surgical field then placed in a recovery cage. Once fully ambulatory the mice were relocated back to original housing. Dietgel was provided to the animals for the duration of the study. Animals were monitored daily for signs of inflammation around the cast to ensure no impact on animal welfare. For Sham cast, animals underwent all procedures but casts were not secured with Vetbond and is removed. The plastic tube used for the casting was 7.9 mm in diameter by 25 mm in length, going from just above the knee of the mouse to just below the ankle. The limb was loosely wrapped in gauze and gently slipped into the tube, with the paw in full extension. The mouse was able to ambulate with relative ease, simply dragging the casted limb on the cage floor. This method of casting does not require specialized housing or husbandry conditions. In the event the cast needed to be replaced, it was done so immediately.

In Vivo Hindlimb Muscle Function

Muscle performance was measured in vivo with a 305 C muscle lever system (AuroraScientific Inc., Aurora, CAN). Mice were anesthetized by inhalation or ˜5% isoflurane, placed on a thermostatically controlled table where anesthesia maintenance was achieved via nose-cone (˜2-3% isofluorane). The knee was isolated by pressing a pin against the tibial head and the foot was then firmly fixed to a footplate on the motor shaft. For the plantarflexor muscle group, contractions were elicited by percutaneous electrical stimulation of the sciatic nerve. Optimal isometric twitch torque was determined by increasing the current with a minimum of 30 seconds between each contraction to avoid fatigue. A series of stimulations were then performed at increasing frequency of stimulation (0.2 ms pulse, 500 ms train duration): 1, 20, 40, 50, 60, 80, 100, 150 Hz and maximal peak isometric force was collected and plotted.

Body Mass and Composition

Mice were subjected to Time Domain (TD)-NMR (LF90 Minispec, Bruker, Spring, TX, USA) to determine body composition of fat, lean mass, and fluid in conscious mice under randomly fed conditions. Following body mass measurements, mice were placed into a Plexiglas sample holder (90 mm in diameter and 250 mm in length), with ventilation holes provided at both ends and around the tube circumference, and inserted into the 0.5 T magnet bore. All measurements were performed in triplicate, and the average was reported. The mice are then returned to their cages upon completion of the measurements.

FSHD Mouse Model

Female FLExDUX4.CRE mice were assigned to one of two balanced groups based on body weight. Mice were dosed with vehicle or 10 mg/kg CAV-003 every 4 days for 4 weeks by intraperitoneal injection. One group of littermate CRE WT mice served as a reference control and were dosed with vehicle every 4 days for 4 weeks by intraperitoneal injection. At the end of the study, in vivo hindlimb muscle function was measured as described above and animals were euthanized for tissue collection.

Example 2. In Vitro Potency of ITGA7-Targeted IGF-1

The in vitro potency of ITGA7-targeted IGF-1 (comprising three polypeptide chains: SEQ ID NOS: 2, 39, and 40) was tested in both of non-muscle cells (DU145) and differentiated muscle cells (C2C12) via pAKT assay. The half maximal effective concentration (EC50) of IGF-1 in DU145 cells was 4.6 nM and the EC50 of ITGA7-targeted IGF-1 in DU145 cells was 30.5 nM. The half maximal effective concentration (EC50) of IGF-1 in C2C12 cells was 7.8 nM and the EC50 of ITGA7-targeted IGF-1 in C2C12 cells was reduced to 7.1 nM. It indicated that ITGA7-targeted IGF-1 was more potent in differentiated muscle cells compared to non-muscle cells, as shown in