Veterinary composition and methods for non-surgical neutering and castration

1. A composition useful for non-surgical sterilization of non-human mammals, said composition comprising an adeno-associated virus (AAV) vector having an AAV capsid having packaged therein nucleic acid sequences comprising an AAV 5′ inverted terminal repeat (ITR), a sequence encoding a polypeptide which comprises a functional portion of an antibody which specifically binds and neutralizes gonadotropin releasing hormone (GnRH), wherein the functional portion of the antibody has a heavy chain variable amino acid sequence of:

(a) murine, SEQ ID NO: 12: QIQLVQSGPELKKPGETVKISCK

ASGYPFT-NYGMNWVKQAPGKGLKWMGWINTYTGEPACADD

FRGRFA(I)SLETSA(R)TAYLQINNL(I)NEDTATYFCARTGGGRYN

YGMDYWGQGTSVTVSS;

(b) canine, SEQ ID NO: 14: EVQLVESGGDLVKPAGSLRLS

CVA-SGYPFTNYGMNWVRQAPGKGLQWVAWINTYTGEPA

CADDFRGRFTISRDNAKRTLYLQMNSLIAEDTAVYYCAKTGG

GRYNYGMDYWGHGTSLFVSS; and

(c) feline, SEQ ID NO: 16:

DVQLMESGGDLVKPGGSLRLTCVAS-

GYPFTNYGMNWVRQAPGKGLQWVAWINTYTGEPACADDFRG

RFTISRDNAKRTLYLQMNSLITEDTATYYCTRTGGGRYNYGMDYW

GQGALVTVSS,

or a sequence having about 97% to about 99% identity with one of (a) to (c).

2. The composition according to claim 1 , wherein the polypeptide is an anti-GnRH antibody, immunoadhesin, a functional portion of the antibody which neutralizes GnRH, or a fusion protein comprising a functional portion of the antibody which neutralizes GnRH.

3. The composition according to claim 2 , wherein the functional portion of the antibody which neutralizes GnRH is selected from a Fab, Fab′, scFv, or a heavy chain variable domain.

4. The composition according to claim 1 , wherein:

(a) the heavy chian variable amino acid sequence is  

the murine sequence and is encoded by: SEQ ID NO: 13:

CAGATCCAGTTGGTGCAGTCTGGACCTGAACTGAAGAAGCCTGGAGAGAC

AGTCAAGATCTCCTGCAAGGCTTCTGGATATCCCTTCACAAACTATGGAA

TGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGG

ATAAACACCTACACTGGAGAGCCAGCATGTGCTGATGACTTCAGGGGACG

GTTTGCCATCTCTTTGGAAACCTCCGCCAGAACTGCCTATTTGCAGATCA

ACAACCTCATAAATGAGGACACGGCAACATATTTCTGTGCAAGAACGGGG

GGTGGTAGGTACAACTATGGTATGGACTATTGGGGTCAAGGAACCTCAGT

CACCGTCTCCTCA, or

(b) the heavy chain variable amino acid sequence is  

the canine sequence and is encoded by: SEQ ID NO: 15:

gaggtgcagctggtggagagcggcggggatctggtcaagcctgccggcag

cctgagactgagctgcgtggcaagcggctaccccttcacaaattatggca

tgaactgggtgcgacaggcccctggcaagggcctgcagtgggtggcctgg

ataaacacctacaccggggaaccagcatgtgcagatgacttcagaggccg

cttcaccatatctcgagacaatgctaagcggaccctgtatctgcagatga

actcactgattgcagaggacacagcagtgtactactgtgccaagacagga

ggagggcgctacaactatggcatggactactggggccacggaacgagcct

gtttgtctcatctgcgtcgaccacagccccctctgtgttccccctggccc

cttcctgtgggtcaacctaggcagcacagtggccctggcgtgtcttgtgt

ctggctacttccctgaacctgtgacagtcagctggaacagcggaagcctg

acctctggagtgcacaccttccccagtgtcctgcaaagctcaggcctgca

cagcctgtcaagtatggtgacagtgcccagtagcaggtggccttctgaaa

cctttacctgcaacgtggtgcaccctgcatccaacaccaaagtggataag

cctgttttcaatgagtgcagatgcacagatacacctccctgccctgtgcc

tgagcctctgggaggaccatcagtcctgatcttccctccaaagcctaagg

atatcctgcggatcaccagaacccccgaggtcacctgtgtcgtcctggat

ctgggccgggaagatcctgaagtgcagattagctggtttgtggacggcaa

ggaagtgcacacagctaagacccaatcccgggagcagcagttcaatggca

cctaccgggtggtctctgtcctgcccatcgagcaccaagattggctgaca

ggcaaagagtttaagtgccgagtcaaccacatagatcttccctcccctat

tgagcggaccatctccaaggcacgggggcgagcgcacaaaccctctgtct

atgtgctgcctccctctcccaaagaattgagctctagcgatacagtgtca

atcacctgcctgatcaaggacttctacccccctgacattgatgttgaatg

gcaatcaaatgggcagcaagaaccagagagaaaacacagaatgacccctc

cacagctggatgaggacgggtcctactttctgtactctaaactttccgtg

gacaagagcagatggcagcagggagaccattcacctgtgcggtcatgcac

gagacactgcaaaaccactacacagatctgtccttgagccactcacctgg

caag;

or (c) the heavy chain variable amino acid sequence 

is the feline sequence and is encoded by:

SEQ ID NO: 17:

gacgtgcagctgatggagtctgggggcgacctagtcaagcctggggggtc

cctgcggcttacgtgtgtggcaagtgggtaccccttcaccaactatggaa

tgaactgggtcagacaggcccctggaaaaggcctgcagtgggtggcctgg

atcaacacctatacaggagaacctgcctgtgcagatgactttagaggccg

attcaccatttcaagagataacgcgaagcgaaccttgtacttacagatga

actccctgatcacagaagacacagcaacctactactgtacccggacagga

gggggccgctacaactatggcatggactactgggggcaaggagcactggt

gacagtctcatctgcgtcgaccacagccccctagtgaccccctggcccct

tcttgtggaaccacctctggagcgacagtggctctggcgtgccttgtcct

ggggtacttccctgaacctgtgaccgtcagctggaactccggagcactga

catctggagtgcacacctttcctgcggtcctgcaagcttccggcctgtac

tcactgtccagcatggtgactgtgccttcttcaagatggctgtctgacac

gttcacctgcaatgtggcgcaccaccttcaaacacaaaggtcgataagac

cgtgagaaagacagaccacccccctggcccaaagccctgcgactgtccta

agtgcccccctcctgaaatgagggcggccccagcatcttcatattccccc

ctaagcccaaagacaccttgagtatctctcgaacaccagaagtcacctgc

ctggtggtggacctaggccctgatgactagatgtgcaaataacctggttc

gtggacaacacccaggtgtacaccgccaaaacctccccaagagaggagca

gttcaactccacctatcgggtcgttagtgtgctgcccattctgcaccaag

actggctgaaaggcaaggagttcaagtgcaaggtcaatagcaaatcactg

ccctatcccattgaaagaaccattagcaaggccaagggacagccccacga

acctcaggtgtatgtgctgccacctgcccaggaagagctcagccgcaaca

aggtctctgtgacctgcctgatcaagtccttccaccctcctgacatagca

gtggagtgggaaataacaggacagcctgagcctgaaaacaactaccgcac

cacccctccccaactggactccgatggaacctactttgtctactctaaga

gtagtggatcgaagccactggcaaaggggcaacacctacacctgctctgt

cagccacgaagccctgcacagccaccacacccaaaagtccctgacccaga

gccccggaaag

or a sequence having about 97% to about 99% identity with one of (a) to (c).

5. The composition according to claim 1 , wherein the anti-GnRH antibody or functional portion thereof comprises non-canine complementarity determining regions (CDRs) in canine immunoglobulin constant framework regions.

6. The composition according to claim 5 , wherein sequences encoding the anti-GnRH antibody or functional portion thereof is optimized for canine delivery.

7. The composition according to claim 1 , wherein the antibody is an IgG protein.

8. The composition according to claim 1 , wherein the AAV has a capsid which is selected from the group consisting of AAV1, AAV8, and AAV9.

9. A composition useful for non-surgical sterilization of non-human mammals, said composition comprising an adeno-associated virus (AAV) vector having an AAV capsid having packaged therein nucleic acid sequences comprising an AAV 5′ inverted terminal repeat (ITR), a sequence encoding a polypeptide which comprises a functional portion of an antibody which specifically binds and neutralizes gonadotropin releasing hormone (GnRH), wherein the functional portion of the antibody has a light chain variable amino acid sequence of:

(a) murine, SEQ ID NO: 6:

QIVLTQSPAIMSASPGEKVTITCSATSSVSYIHWFQQKPGTSPKLWIYST

SNLASGVP(V)RFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPPTFG

GGTKLEIKR; or

(b) canine, SEQ ID NO: 8:

DIVMTQTPLSLSVSPGEPASISCSATSSVSYIHWYLQKAGQSPRLLPEST

SNLASGVPVRFSGSGSGTDFTLRIGRVEAEDAGIYYCQQRSSYPPTFGQG

TRLEVRR, or

(c) feline, SEQ ID NO: 10:

DIVMTQTPLSLSVTPGEPASISCSATSSVSYIHWYLQKPGQSPRRLIYST

SNLASGVPVRFSGSGSGTDFTLRISRVEADDVGVYYCQQRSSYPPTFGPG

TKLEIKR;

or a sequence having about 97% to about 99% identity with one of (a) to (c).

10. The composition according to claim 9 , wherein:

(a) the light chain variable amino acid sequence is  

the murine sequence and is enclosed by:

SEQ ID NO: 7:

CAAATTGTTCTCACCCAGTCTCCAGCCATCATGTCTGCATCTCCAGGGGA

GAAGGTCACCATAACCTGCAGTGCCACTCAAGTGTAAGTTACATACACTG

GTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACAT

CCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGG

ACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCAC

TTATTACTGCCAGCAAAGGAGTAGTTACCCACCCACGTTCGGAGGGGGGA

CCAACTGGAAATAAAACGVHCAGATCCAGTTGGTGCAGTCTGGACCTGAA

CTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATA

TCCCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGG

GTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAGCATGT

GCTGATGACTTCAGGGGACGGTTTGCCATCTCTTTGGAAACCTCCGCCAG

ACTGCCTATTTGCAGATCAACAACCTCATAAATGAGGACACGGCAACATA

TTTCTGTGCAAGAACGGGGGGTGGTAGGTACAACTATGGTATGGACTATT

GGGGTCAAGGAACCTCAGTCACCGTCTCCTCA, or

(b) the light chain variable amino acid sequence is 

the canine sequence and is encoded by: 

SEQ ID NO: 9:

GATATAGTGATGACCCAAACCCCTCTAAGCCTATCTGTCTCCCCTGGGGA

GCCGGCGAGTATCAGCTGCAGCGCCACCAGCAGCGTGTCATATATCCACT

GGTACCTGCAAAAGGCTGGACAGTCCCCTAGACTTCTGCCCGAAAGCACA

TCTAACCTGGCCAGCGGGGTCCCTGTGAGGTTTAGTGGGAGTTAGCCTGT

CTAGCACCCTCACCATGAGCAGCACAGAGTACTTGAGTCATGAGCTGTAT

AGCTGTGAGATTACCCACAAGAGTCTGCCAAGCACCCTTATAAAAAGTTT

CCAGCGATCTGAGTGT; or

(c) the light chain variable amino acid sequence is 

the feline sequence and is encoded by: SEQ ID NO: 11:

GACATCGTGATGACCCAAACCCCTCTGAGCCTGTCCGTCACCCCCGGGGA

GCCCGCCAGCATAAGCTGCTCCGCTACCAGCTCCGTTAGCTACATTCACT

GGTATCTGCAAAAGCCTGGCCAGAGCCCTAGGCGACTGATCTATAGCACC

TCCAACCTGGCCTCTGGTGTGCCAGTGCGCTTCTCTGGGTCTGGCAGCGG

GACCGACTTTACCCTGAGGATCTCCAGAGTGGAGGCTGATGATGTGGGGG

TGTACTACTGCCAGCAGAGGAGCAGCTATCCTCCTACCTTTGGCCCCGGC

ACCAAGCTGGAGATAAAGAGGAGTGATGCCCAGCCCAGCGTGTTTCTGTT

CCAACCTTCTCTGGATGAGCTGCACACCGGGAGCGCCTCTATAGTGTGTA

TTCTGAATGATTTCTATCCCAAAGAAGTTAATGTCAAGTGGAAGGTGGAT

GGGGTGGTCCAGAACAAGGGCATCCAGGAAAGCACGACCGAACAGAACTC

CAAGGACTCCACATATTCTCTGAGTAGTACCCTGACCATGAGTAGCACCG

AATACCAGAGTCACGAGAAATTCAGCTGCGAGGTGACCCACAAGAGCTTG

GCCAGCACCCTAGTGAAGAGCTTTAACCGAAGCGAGTGCCAGCGAGAA;

or a sequence having about 97% to about 99% identity with one of (a) to (c).

11. The composition according to claim 9 , wherein the polypeptide is an anti-GnRH antibody, immunoadhesin, a functional portion of the antibody which neutralizes GnRH, or a fusion protein comprising a functional portion of the antibody which neutralizes GnRH.

12. The composition according to claim 11 , wherein the functional portion of the antibody which neutralizes GnRH is selected from a Fab, Fab', scFv, or a heavy chain variable domain.

13. The composition according to claim 9 , wherein the anti-GnRH antibody or functional portion thereof comprises non-canine complementarity determining regions (CDRs) in canine immunoglobulin constant framework regions.

14. The composition according to claim 13 , wherein sequences encoding the anti-GnRH antibody or functional portion thereof is optimized for canine delivery.

15. The composition according to claim 9 , wherein the antibody is an IgG protein.

16. The composition according to claim 9 , wherein the AAV has a capsid which is selected from the group consisting of AAV1, AAV8, and AAV9.

17. A lyophilized composition comprising an adeno-associated virus (AAV) vector having a composition comprising an adeno-associated virus (AAV) vector having an AAV capsid having packaged therein nucleic acid sequences comprising an AAV 5′ inverted terminal repeat (ITR), a sequence encoding a polypeptide which specifically binds gonadotropin releasing hormone (GnRH) under control of regulatory sequences which direct expression of the ligand, and an AAV 3′ ITR, wherein the functional portion of the antibody has a heavy chain variable amino acid sequence of:

(a) murine, SEQ ID NO: 12:

QIQLVQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGKGLKWMGW

INTYTGEPACADDFRGRFA(I)SLETSA(R)TAYLQINNL(I)NEDTATY

FCARTGGGRYNYGMDYWGQGTSVTVSS; or

(b) canine, SEQ ID NO: 14:

EVQLVESGGDLVKPAGSLRLSCVASGYPFTNYGMNWVRQAPGKGLQWVAW

INTYTGEPACADDFRGRFTISRDNAKRTLYLQMNSLIAEDTAVYYCAKTG

GGRYNYGMDYWGHGTSLFVSS; or

(c) feline, SEQ ID NO: 16:

DVQLMESGGDLVKPGGSLRLTCVASGYPFTNYGMNWVRQAPGKGLQWVAW

INTYTGEPACADDFRGRFTISRDNAKRTLYLQMNSLITEDTATYYCTRTG

GGRYNYGMDYWGQGALVTVSS,

or a sequence having about 97% to about 99% identity with one of (a) to (c).

18. A reconstituted composition comprising the lyophilized composition according to claim 17 comprising about 10 9 to about 5×10 13 vector particles per 1 mL aqueous suspension.

19. A lyophilized composition comprising an adeno-associated virus (AAV) vector having a composition comprising an adeno-associated virus (AAV) vector having an AAV capsid having packaged therein nucleic acid sequences comprising an AAV 5′ inverted terminal repeat (ITR), a sequence encoding a polypeptide which specifically binds gonadotropin releasing hormone (GnRH) under control of regulatory sequences which direct expression of the ligand, and an AAV 3′ ITR, wherein the polypeptide comprises a light chain variable amino acid sequence of:

(a) murine, SEQ ID NO: 6:

QIVLTQSPAIMSASPGEKVTITCSATSSVSYIHWFQQKPGTSPKLWIYST

SNLASGVP(V)RFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPPTFG

GGTKLEIKR;

(b) canine, SEQ ID NO: 8:

DIVMTQTPLSLSVSPGEPASISCSATSSVSYIHWYLQKAGQSPRLLPEST

SNLASGVPVRFSGSGSGTDFTLRIGRVEAEDAGIYYCQQRSSYPPTFGQG

TRLEVRR, or

(c) feline, SEQ ID NO: 10:

DIVMTQTPLSLSVTPGEPASISCSATSSVSYIHWYLQKPGQSPRRLIYST

SNLASGVPVRFSGSGSGTDFTLRISRVEADDVGVYYCQQRSSYPPTFGPG

TKLEIKR;

or a sequence having about 97% to about 99% identity with one of (a) to (c).

20. A reconstituted composition comprising the lyophilized composition according to claim 19 comprising about 10 9 to about 5×10 13 vector particles per 1 mL aqueous suspension.