Engineered human hookworms as a novel biodelivery system for vaccines and biologicals

Patent metadata

Field	Value
Publication number	US-2019069528-A1
Application number	US-201716087196-A
Country	US
Kind code	A1
Filing date	Mar 21, 2017
Priority date	Mar 21, 2016
Publication date	Mar 7, 2019
Grant date	—

Abstract

The present disclosure generally relates to genetic methods of manipulating helminths, e.g., hookworms, to act as a biological delivery vehicle for therapeutic polypeptides in mammals. Furthermore, the disclosure is drawn to compositions comprising genetically modified hookworms and methods of use, including the administration of the helminths to one or more mammals to provide a continuous supply of a synthetic or modified polypeptide (e.g., HIV neutralizing antibodies) which may mitigate infection and/or infection intensity, otherwise resulting in an increase in a desirable phenotypic trait.

First claim

What is claimed is:

1 . A transgenic helminth comprising: cells containing a polynucleotide sequence comprising one or more control sequences operably linked to at least one heterologous nucleic acid sequence, wherein the at least one heterologous nucleic acid sequence encodes a vaccine antigen and/or a therapeutic polypeptide.

2 . The transgenic helminth of claim 1 , wherein the helminth is a trematode, cestode, or nematode.

3 . The transgenic helminth of claim 2 , wherein the helminth is a nematode selected from the genera consisting of: Necator, Ancylostoma, Agriostomum, Bunostomum, Cyclodontostomum, Galonchus, Aonodontus, Uncinaria, Enterobius, Trichuris, Capillostrongyloides, Liniscus, Orthominx, Pearsonema, Sclerotrichum, Strongyloides , and Tenoranema.

4 . The transgenic helminth of claim 1 , wherein the transgenic helminth is a transgenic hookworm.

5 . The transgenic helminth of claim 3 , wherein the nematode is a hookworm selected from species of: Ancylostoma braziliense, Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma pluridentatum, Ancylostoma tubaeforme, Necator americanus , and Uncinaria stenocephala.

6 . The transgenic hookworm of claim 4 , wherein the vaccine antigen is an HIV polypeptide.

7 . The transgenic hookworm of claim 6 , wherein the HIV polypeptide is an envelope V2 region polypeptide.

8 . The transgenic hookworm of claim 4 , wherein the therapeutic polypeptide is selected from the group consisting of: insulin, gamma-interferon, beta-interferon, Factor VIII, Factor IX, tissue plasminogen activator, human growth hormone, bovine growth hormone, and a neutralizing antibody.

9 . The transgenic hookworm of claim 8 , wherein the therapeutic polypeptide is aneutralizing antibody, which neutralizes viral envelope proteins.

10 . The transgenic hookworm of claim 9 , wherein the neutralizing antibody neutralizes HIV envelope proteins.

11 . The transgenic hookworm of claim 8 , wherein the therapeutic polypeptide is a neutralizing antibody of VRC01.

12 . The transgenic hookworm of claim 4 , wherein the at least one heterologous nucleic acid sequence further encodes an adjuvant.

13 . The transgenic hookworm of claim 12 , wherein the adjuvant is selected from the group consisting of: flagellin, Escherichia coli heat labile toxin, cholera toxin, an AB5 toxin, a viral coat protein, a chemokine, a cytokine, and a defensin.

14 . The transgenic hookworm of claim 4 , wherein the one or more control sequences is selected from the group consisting of: a promoter, a terminator, a secretion signal, an enhancer, and an operator.

15 . The transgenic hookworm of claim 14 , wherein the one or more control sequences is a hookworm promoter.

16 . The transgenic hookworm of claim 15 , wherein the hookworm promoter is selected from asp-1, asp-2, asp-3, asp-4, asp-5, snr-3, lpp-1, tbg-1, myo-2, myo-3, ges-1, eft-3, ama-1, daf-11, daf-16, daf-21, daf-2, let-858, unc-119, vit-2, sur-5, hlh-13, pie-1, and spe-11.

17 . The transgenic hookworm of claim 15 , wherein the hookworm promoter is a stage-specific promoter that is parasitic L3 stage-specific, L4 stage-specific, or adult stage-specific.

18 . The transgenic hookworm of claim 4 , wherein the at least one heterologous nucleic acid sequence encodes an HIV envelope V2 region and an AB5 toxin.

19 . A method of preparing a transgenic helminth, the method comprising introducing into cells of a helminth a polynucleotide comprising one or more control sequences operably linked to at least one heterologous nucleic acid sequence,
wherein the at least one heterologous nucleic acid sequence encodes a vaccine antigen and/or a therapeutic polypeptide.

20 . The method of claim 19 , wherein the helminth is a trematode, cestode, or nematode.

21 . The method of claim 20 , wherein the helminth is a nematode selected from the genera consisting of: Necator, Ancylostoma, Agriostomum, Bunostonmum, Cyckdontostomum, Galonchus, Monodonttus, Uncinaria, Enterobius, Trichuris, Capillostrongyloides, Liniscus, Orthominx, Pearsonema, Sclerotrichum, Sirongyloides , and Tenoranema.

22 . The method of claim 19 , wherein the transgenic helminth is a transgenic hookworm.

23 . The method of claim 22 , wherein the introducing a polynucleotide into cells of the helminth comprises biolistic bombardment or viral transfection.

24 . The method of claim 22 , wherein the vaccine antigen is an HIV polypeptide.

25 . The method of claim 24 , wherein the HIV polypeptide is an envelope V2 region polypeptide.

26 . The method of claim 19 , wherein the therapeutic polypeptide is selected from the group consisting of: insulin, gamma-interferon, beta-interferon, Factor VIII, Factor IX, tissue plasminogen activator, human growth hormone, bovine growth hormone, and a neutralizing antibody.

27 . The method of claim 26 , wherein the therapeutic polypeptide is a neutralizing antibody, which neutralizes viral envelope proteins.

28 . The method of claim 27 , wherein the neutralizing antibody neutralizes HIV envelope proteins.

29 . The method of claim 26 , wherein the therapeutic polypeptide is a neutralizing antibody of VRC01.

30 . The method of claim 25 , wherein the hookworm is selected from the group consisting of: Ancylostoma braziliense, Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma pluridentatum, Ancylostoma tubaeforme, Necator americanus , and Uncinaria stenocephala.

31 . The method of claim 25 , wherein the at least one heterologous nucleic acid sequence further encodes an adjuvant.

32 . The method of claim 31 , wherein the adjuvant is selected from the group consisting of: flagellin, Escherichia coli heat labile toxin, cholera toxin, an AB5 toxin, a viral coat protein, a chemokine, a cytokine, and a defensin.

33 . The method of claim 25 , wherein the one or more control sequences is selected from the group consisting of: a promoter, a terminator, a secretion signal, an enhancer, and an operator.

34 . The method of claim 33 , wherein the one or more control sequences is a hookworm promoter.

35 . The method of claim 34 , wherein the hookworm promoter is a stage-specific promoter that is parasitic L3 stage-specific, L4 stage-specific, or adult stage-specific.

36 . The method of claim 34 , wherein the hookworm promoter is selected from asp-1, asp-2, asp-3, asp-4, asp-5, snr-3, lpp-1, tbg-1, myo-2, myo-3, ges-1, eft-3, ama-1, daf-11, daf-16, daf-21, daf-2, let-858, unc-119, vit-2, sur-5, hlh-13, pie-1, and spe-11.

37 . The method of claim 25 , wherein the at least one heterologous nucleic acid sequence encodes an HIV envelope V2 region and an AB5 toxin.

38 . The method of claim 25 , wherein the method of introducing a polynucleotide into cells of a hookworm comprises viral transfection utilizing a lentivirus vector to introduce the polynucleotide, wherein the lentivirus vector has been pseudotyped with a VSV-G envelope protein.

39 . The method of claim 25 , wherein the method of introducing a polynucleotide into cells of a hookworm comprises viral transfection utilizing a retrovirus vector to introduce

Assignees

• Univ George Washington

Inventors

• Hawdon John M
• Nixon Douglas
• Han Suhao
• Brockmeyer Claudia
• Bethony Jeffrey
• Ratnappan Ramesh
• Diemert David

Classifications

• A61K39/12

  Viral antigens · CPC title

• A61K9/0053

  Mouth and digestive tract, i.e. intraoral and peroral administration · CPC title

• C12N2740/16021

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• A61P31/18

  for HIV · CPC title

• A61K9/0014

  Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00) · CPC title