Novel adenovirus vaccine therapy for treating recurrent respiratory papilloma disease

Abstract

Multi-antigenic HPV molecular vaccine constructs for use and treatment of diseases and pathologies associated with human papilloma virus (HPV), such as HPV molecular vaccines targeting recurrent respiratory papilloma disease (RRP) associated with HPV6 and HPV11.

Description

Background of the Invention

[0002] Recurrent respiratory papilloma (RRP) is a rare, difficult-to-treat, and sometimes fatal neoplastic disease of the upper and lower respiratory tracts. RRP is caused by infection with human papillomavirus (HPV) types 6 or 11. Mounts P, Shah KV, Kashima H: Viral etiology of juvenile- and adult-onset squamous papilloma of the larynx. Proc Natl AcadSci USA 1982, 79(17):5425-5429. Approximately 1,500 new cases of RRP are diagnosed annually in the United States. Derkay CS, Wiatrak B: Recurrent respiratory papillomatosis: a review. Laryngoscope 2008, 118(7):1236-1247.

[0003] RRP is classified into juvenile-onset and adult-onset types based on age of onset. The incidence of juvenile-onset RRP is 4 / 100,000, and it often has an aggressive clinical course. The incidence of adult-onset RRP is 2-3 / 100,000, and it often has a relatively indolent clinical course.

[0004] The morbidity and mortality of RRP stem from the mass effect of papillomatosis on the vocal cords, trachea, and lungs. This can lead to voice changes, wheezing, airway obstruction, lung volume loss, and / or post-obstructive pneumonia. Silver RD, Rimell FL, AdamsGL, Derkay CS, Hester R: Diagnosis and management of pulmonary metastasis from recurrent respiratory papillomatosis. Otolaryngol Head Neck Surg 2003, 129(6):622-629. Repeated surgeries are required to reduce tumor burden and monitor disease, exposing participants to anesthesia and surgical risks as well as emotional stress. The economic cost of RRP in the United States is estimated at $150 million annually. Derkay CS, Wiatrak B: Recurrent respiratory papillomatosis: a review. Laryngoscope 2008, 118(7):1236-1247. Although rare (accounting for 1% to 3% of cases), RRP can transform into invasive squamous cell carcinoma. DedoHH, Yu KC: CO(2) laser treatment in 244 patients with respiratory papillomas. Laryngoscope 2001, 111(9):1639-1644. Subsequent mortality depends on the clinical stage of the malignancy at diagnosis.

[0005] There is currently no cure for RRP, no approved drug treatment, and complete remission of RRP has not been observed through immunotherapy. The main treatment for RRP is repeated endoscopic debulking combined with ablation or excision of papillary lesions. Surgical principles require that, in order to minimize treatment-related morbidity, only papillary lesions should be removed, without excising the epithelium that appears normal. It is generally believed that latent HPV virus particles persist in an inactive state in clinically normal mucosa and are subsequently reactivated, leading to RRP recurrence. Armstrong LR, Derkay CS, Reeves WC: Initial results from the national registry for juvenile-onset recurrent respiratory papillomatosis. RRP Task Force. Arch Otolaryngol Head Neck Surg 1999, 125(7):743-748.

[0006] Individuals with adolescent-onset RRP require an average of 20 surgeries in their lifetime to control the disease. Ibid. Individuals with adult-onset RRP typically require fewer interventions; however, more than 50% still require five or more surgeries to control symptoms. Kashima HK, Shah F, Lyles A, Glackin R, Muhammad N, Turner L, Van Zandt S, Whitt S, Shah K: A comparison of risk factors in juvenile-onset and adult-onset recurrent respiratory papillomatosis. Laryngoscope 1992, 102(1):9-13. Some individuals with more aggressive disease may require hundreds of surgeries in their lifetime to maintain usable vocal capacity and a patent airway. Adjunctive systemic therapies, including systemic interferon-alpha and local injections of antiviral and anti-angiogenic drugs, have been tested in clinical trials. The research results are inconsistent, and no adjunctive therapy has been widely adopted or accepted as a standard treatment.

[0007] Local immunotherapy failed to eradicate RRP, presumably due to the long-term persistence of latent HPV in seemingly normal mucosa. This view is supported by a study that showed the presence of HPV DNA in clinically healthy mucosa of RRP patients. Smith EM, Pignatari SS, Gray SD, Haugen TH, Turek LP: Human papillomavirus infection in papillomas and nondiseased respiratory sites of patients with recurrent respiratory papillomatosis using the polymerase chain reaction. Arch Otolaryngol Head Neck Surg 1993, 119(5):554-557. Previous studies on systemic immunotherapy for RRP have been limited. Adjuvant IFN-α after papilloma treatment has been shown to prolong the short-term recurrence time, but no long-term benefit has been shown.

[0008] It has been confirmed that the expression of programmed death-ligand 1 (PD-L1) on tumor cells is closely associated with poor prognosis in various human cancers. Healy GB, Gelber RD, Trowbridge AL, Grundfast KM, Ruben RJ, Price KN: Results of a multicenter randomized clinical trial of treatment of recurrent respiratory papillomatosis with human leukocyte interferon. N Engl J Med 1988, 319(7):401-407. In recent years, a number of drugs targeting the programmed death receptor 1 (PD-1) / PD-L1 pathway have been approved by regulators and have shown remarkable duration of response in various tumor types, including head and neck cancer.Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP, Heath K, McClanahan T, Lunceford J, Gause C, et al.: Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol 2016, 17(7):956-965; Ferris RL, Blumenschein G Jr., Fayette J, Guigay J, Colevas AD, Licitra L, Harrington K, Kasper S, Vokes EE, Even C et al.: Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. NEngl J Med 2016, 375(19):1856-1867. It is noteworthy that atezolizumab, durvalumab, and avelumab are all regulatory-approved anti-PD-L1 antibodies with proven efficacy. However, none of these anti-PD-L1 antibodies have been proven to safely and effectively cure adult RRP.

[0009] Therefore, there remains a need in the field for relatively non-invasive methods to safely and effectively treat adult RRP, thereby providing cost and emotional benefits to patients, including methods that focus on inducing T-cell responses against HPV antigens.

[0010] Therapeutic vaccines are an immunotherapy strategy that enhances nascent T-cell responses in individuals with recurrent vulvar intraepithelial neoplasia (RRP). Therapeutic vaccination with a long peptide vaccine encoding the HPV-16 antigen induced complete remission of the disease in nearly 50% of individuals with precancerous vulvar intraepithelial neoplasia. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van derMeer DM, Vloon AP, Essahsah F, Fathers LM, Offringa R, Drijfhout JW et al.: Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med 2009, 361(19):1838-1847. In these participants, the intensity of the HPV-16-specific T-cell response correlated with the magnitude of the clinical response. van Poelgeest MI, Welters MJ, Vermeij R, Stynenbosch LF, Loof NM, Berends-van der Meer DM, Lowik MJ, Hamming IL, van Esch EM, Hellebrekers BW et al.: Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar / Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response. Clin Cancer Res 2016, 22(10):2342-2350. A study in Mexico City reported that a modified Ankara vaccinia virus encoding bovine papillomavirus E2 was injected directly into the papilloma via high-frequency intralesional injection (4 injections in total, once every two weeks) to achieve durable remission without papillary lesion recurrence in children and adults with recurrent lesions (RRP).Cabo Beltran OR, Rosales Ledezma R: MVA E2 therapeutic vaccine for marked reduction in likelihood of recurrence of respiratory papillomatosis. Head Neck 2019, 41(3):657-665. Although this study demonstrated clinical activity, further improvements are needed, and translational studies defining the mechanisms of papilloma clearance are still lacking. Summary of the Invention

[0011] This article provides non-naturally occurring polynucleotides (and polypeptides expressed therefrom) that encode non-naturally occurring polypeptides containing human papillomavirus (HPV) polypeptides that induce immune responses. This article also provides conformations of non-naturally occurring, variable-arrangement HPV immune response-inducing polypeptides linked by various polypeptide linker sequences; thereby comprising fusion proteins useful as vaccine antigens.

[0012] The present invention includes, but is not limited to, compositions (e.g., substances comprising polynucleotides, polypeptides, carriers, vaccines, or cells), methods for preparing and delivering compositions, and the use of non-naturally occurring polynucleotides encoding non-naturally occurring polypeptides comprising vaccine antigens for human papillomavirus (HPV) (especially for HPV6 and HPV11); and therapeutic methods for treating pathological conditions caused by these specific HPV pathogens.

[0013] Specifically, the present invention comprises polynucleotides and polypeptides encoded therefrom, which encode polyantigenic polypeptides derived from HPV6, HPV11 and HPV16 and other polypeptide sequences, for use as vaccine components and for the treatment of diseases associated with HPV infection.

[0014] For example, one embodiment of the invention relates to the use of the compositions described herein as a therapeutic vaccine for diseases induced or associated with HPV6 and / or HPV11 (HPV6 / 11); for example, but not limited to, for the treatment of recurrent respiratory papillomavirus (RRP), anogenital warts, and other HPV6 / 11-related diseases, such as lower genital tract tumors (e.g., cervical, vaginal, and vulvar intraepithelial neoplasia), cervical cancer, vulvar cancer, anal cancer, penile cancer, and head and neck cancer. In one embodiment, the compositions described herein are used as a therapeutic vaccine against RRP.

[0015] This invention includes a unique and innovative method for designing HPV peptide vaccines targeting HPV6 / 11-induced disease. The invention incorporates a combination of design strategies, such as using full-length protein sequences, using peptide “fragments,” hybrid peptide constructs, introducing amino acid substitutions, insertions, deletions, and rearrangements of peptides and peptide HPV gene products (proteins).

[0016] As shown above, the antigen described herein has been uniquely modified by introducing point mutations, substitution mutations, and / or reordering the viral polypeptide sequence, in particular to prevent oncogenic gene expression and / or to inactivate essential viral functions (e.g., viral replication).

[0017] In some implementations, the early HPV proteins E2 and E4 have been identified as novel antigens of HPV6 / 11 and incorporated into the vaccine design described herein.

[0018] In some embodiments, the present invention includes the innovative incorporation of four antigenic components from HPV into an HPV vaccine design.

[0019] In some embodiments, the present invention includes the innovative incorporation of four antigenic components from HPV into an HPV vaccine design.

[0020] In some embodiments, the present invention includes the novel integration and combination of high-carcinogenic and low-carcinogenic HPV genotype epitopes into an HPV antigen construct.

[0021] In some embodiments, the HPV antigen construct comprises a polynucleotide encoding a polypeptide that has at least 90% sequence identity with SEQ ID NO:68 or a functional variant thereof (e.g., with the polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity: SEQ ID NO:68, or a conserved substitution variant of SEQ ID NO:68).

[0022] In some embodiments, the vector is an adenovirus vector, such as a chimpanzee adenovirus vector. In some such embodiments, the vector is derived from GC44, GC45, or GC46.

[0023] In some embodiments, the vector is an adenovirus vector lacking all or part of the E1 and / or E4 regions of GC46.

[0024] In some embodiments, the vector comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:119 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:119, or a codon degenerate variant of SEQ ID NO:119).

[0025] In some embodiments, the expression cassette contains a promoter, and the expression of the transgene is controlled by the promoter. In some such embodiments, the promoter is a cytomegalovirus promoter or a synthetic promoter.

[0026] In some embodiments, the promoter is a synthetic promoter. In some such embodiments, the synthetic promoter comprises a blocking sequence, an enhancer, and / or a reaction element. In some embodiments, the enhancer comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:96 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:96, or a codon degenerate variant of SEQ ID NO:96). In some embodiments, the reaction element comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:98 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:98, or a codon degenerate variant of SEQ ID NO:98).

[0027] In some embodiments, the expression cassette comprises a 5' UTR. In some such embodiments, the 5' UTR comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:99 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:99, or a codon degenerate variant of SEQ ID NO:99).

[0028] In some embodiments, the expression cassette includes a termination sequence. In some such embodiments, the termination sequence includes a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:104 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:104, or a codon degenerate variant of SEQ ID NO:104).

[0029] In some embodiments, the vector is an adenovirus vector, and the expression cassette is located at the E1 region deletion junction.

[0030] In some implementations, the expression cassette is cloned relative to the adenovirus genome in a right-to-left direction.

[0031] In some embodiments, the vector is an adenovirus vector, such as a chimpanzee adenovirus vector. In some such embodiments, the vector is derived from GC44, GC45, or GC46.

[0032] In some embodiments, the vector is an adenovirus vector lacking all or part of the E1 and / or E4 regions of GC46.

[0033] This invention also relates in part to a pharmaceutical composition comprising the carriers described herein and pharmaceutically acceptable carriers.

[0034] In some embodiments, the composition further comprises other therapeutic agents. In some such embodiments, the other therapeutic agents are used to treat pathological conditions caused by HPV pathogens.

[0035] The present invention also relates in part to a kit comprising the pharmaceutical composition described herein. In some embodiments, the kit further comprises a label.

[0036] The present invention also relates in part to a method for treating a disease or ailment (including RRP) for treating a subject in need, the method comprising administering the carrier described herein to the subject.

[0037] In some embodiments of the method, the carrier is at approximately 0.1 × 10⁻⁶. 11 To approximately 1×10 12 A dose of one viral particle unit was administered.

[0038] In some embodiments, the method includes administering a composition comprising the carrier. In some such embodiments, the composition is administered subcutaneously, intramuscularly, intravenously, intracranially, intra-articularly, intradermally, percutaneously, intratumorally, or intralesionally. In some embodiments, the composition is administered to the limbs, buttocks, and / or abdomen. In some embodiments, the composition is administered to the lungs or upper respiratory tract via aerosol spray or nebulization.

[0039] In some embodiments, the method includes administering a composition comprising the carrier with a plurality of agents. In some embodiments, each agent may contain about 0.1 × 10⁻⁶. 11 Approximately 5×10 11One viral particle unit. In some embodiments, each dose is administered at least 11 days apart. In some embodiments, the second dose is administered two weeks after the first dose, the third dose six weeks after the second dose, and the fourth dose twelve weeks after the third dose. In some embodiments, at least one dose is administered at least one month apart from the previous dose.

[0040] In some embodiments, cytoreductive surgery is performed before administering the first dose. In some embodiments, one or more cytoreductive surgeries are performed 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, or 30 months prior to administering the first dose. In one embodiment, three cytoreductive surgeries are performed before administering the first dose. In another embodiment, one or more cytoreductive surgeries are performed 12 months prior to administering the first dose. In a further embodiment, three cytoreductive surgeries are performed 12 months prior to administering the first dose.

[0041] In some embodiments, one or more debulking procedures are performed after the first dose and before the final dose. In some embodiments, one or more debulking procedures are performed after the final dose. In some embodiments, one or more debulking procedures are performed during the administration of at least one dose of the composition.

[0042] In one embodiment, the patient may undergo at least one debulking surgery at least 6 months, 1 year, or 2 years after receiving one dose of the composition described herein, or within a timeframe determined by the patient's physician, after which the patient may continue to receive one or more additional doses of the composition. In some embodiments, the clinical efficacy of the treatment is evaluated after the final dose is administered.

[0043] In some implementations, providing the carrier described herein can reduce the need for surgical interventions (such as debulking surgery).

[0044] In some implementations, administration of the vector described herein results in an increased HPV6 / 11 antigen-specific T-cell response.

[0045] In some implementations, administering the carrier described herein results in an improvement in the Derkay score.

[0046] In some embodiments, the method further includes administering one or more other therapeutic agents. In some such embodiments, the additional therapeutic agents may be chemotherapeutic drugs, anti-inflammatory agents, analgesics, and / or bioresponse modifiers.

[0047] This document also provides a polynucleotide encoding a fusion protein comprising (a) an HPV6 protein and (b) an HPV11 protein. In some embodiments, the polynucleotide described herein encodes a fusion protein comprising (a) an HPV6 protein selected from HPV6 E2, HPV6 E4, HPV6 E6, and HPV6 E7 proteins; and (b) an HPV11 protein selected from HPV11 E6 and HPV11 E7 proteins. In some embodiments, the polynucleotide described herein comprises HPV6 E2, HPV6 E4, HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7 proteins. In some embodiments, the HPV6 E2 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the HPV6 E2 protein comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3 or 7. In some embodiments, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO: 3 or 7. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 or 40. In some embodiments, the HPV6 E6 protein comprises the amino acid sequence of SEQ ID NO: 11 or 40. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5 or 9. In some embodiments, the HPV6 E7 protein comprises the amino acid sequence of SEQ ID NO: 5 or 9. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42. In some embodiments, the HPV11 E6 protein comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45. In some embodiments, the HPV11 E7 protein comprises the amino acid sequence of SEQ ID NO: 45.

[0048] In some embodiments, the fusion protein comprises HPV6 E4 protein containing the amino acid sequence of SEQ ID NO:3 and HPV6 E4 protein containing the amino acid sequence of SEQ ID NO:7. In some embodiments, the fusion protein comprises HPV6 E6 protein containing the amino acid sequence of SEQ ID NO:11 and HPV6 E6 protein containing the amino acid sequence of SEQ ID NO:40. In some embodiments, the fusion protein comprises HPV6 E7 protein containing the amino acid sequence of SEQ ID NO:5 and HPV6 E7 protein containing the amino acid sequence of SEQ ID NO:9.

[0049] In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 95% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 97% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 98% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 99% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:68 or a conserved substitution variant thereof. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:68.

[0050] In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:70. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:72. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:74.

[0051] In some embodiments, the fusion protein further comprises a rigid linker polypeptide. In some embodiments, the fusion protein further comprises an HPV16 E6 agonist enhancer. In some embodiments, the fusion protein further comprises an HPV16E7 agonist enhancer.

[0052] In some embodiments, the fusion protein is operatively linked to at least one of the following: a promoter; a 5' untranslated region (UTR); a transcription start site (TSS); a 3' UTR; a tetracycline response element; and a Kozak region. In some embodiments, the promoter is operatively linked to a promoter enhancer region.

[0053] This document also provides vectors comprising any of the embodiments described herein. In some embodiments, the vector is a plasmid, a viral vector, or a non-viral vector. In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the adenovirus vector is defective in one or more elements selected from the E1-E4 and L1-L5 regions. In some embodiments, the adenovirus vector comprises one or more elements selected from E2B, L1, L2, L3, E2A, L4, E3, L5, inverted terminal repeat (ITR), poly(a) site, and spacer sequence. In some embodiments, the adenovirus vector is a chimpanzee adenovirus vector. In some embodiments, the adenovirus vector is a GC46 chimpanzee adenovirus vector.

[0054] This document also provides a method for inducing an anti-HPV immune response in subjects in need. In some embodiments, the method includes administering to the subject a therapeutically effective amount of any of the carriers described herein. In some embodiments, the therapeutically effective amount comprises about 1 × 10⁻⁶. 11 Approximately 5×10 11 Particle unit (PU).

[0055] This document also provides methods for treating HPV-related diseases or conditions in subjects who require treatment. In some embodiments, the method includes administering a therapeutically effective amount of any of the carriers described herein to the subject. In some embodiments, the HPV-related disease or condition is an HPV6-related disease or condition or an HPV11-related disease or condition. In some embodiments, the HPV-related disease or condition is an HPV-related cancer. In some embodiments, the HPV-related disease or condition is recurrent respiratory papillomavirus (RRP), anogenital warts, lower genital tract tumors, cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer. In some embodiments, the HPV-related disease or condition is RRP. In some embodiments, the therapeutically effective amount comprises about 1 × 10⁻⁶. 11 Approximately 5×10 11Individual particle units (PUs). In some embodiments, the method further includes administering additional therapy. In some embodiments, the additional therapy comprises administering at least one of the following: angiogenesis inhibitors, such as bevacizumab (AVASTIN®), and immune checkpoint inhibitors, such as PD-1 inhibitors (e.g., pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and cimipril (LIBTAYO®)) and / or PD-L1 inhibitors (e.g., atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®)). In some embodiments, the method further includes cytoreductive surgery.

[0056] This article also provides fusion proteins encoded by any of the polynucleotides described herein.

[0057] This document also provides compositions comprising any of the polynucleotides described herein. In some embodiments, any of the compositions described herein are used to treat a disease or condition in a subject of need.

[0058] This article also provides the use of any of the polynucleotides described herein in the preparation of medicaments for the treatment of diseases or conditions in subjects in need.

[0059] This article also provides kits containing any of the polynucleotides described herein.

[0060] This document also provides vaccines comprising any of the polynucleotides described herein. In some embodiments, the vaccine is used to treat diseases or conditions in subjects of need.

[0061] The present invention also relates in part to the use of the carrier described herein in the preparation of a medicament for treating a disease or condition (e.g., RRP) in a subject of need.

[0062] The features of this disclosure are specifically defined in the appended claims. The features and advantages of this disclosure can be better understood by referring to the following detailed description, which illustrates exemplary embodiments utilizing the principles of this disclosure, and the accompanying drawings.

[0063] Brief description of the attached figures

[0064] Figure 1A The engineered GC46 vector (AdV-HPV6 / 11) with deletions in the E1 and E4 regions and the inclusion of the CMV HPV6 / 11 expression cassette in the E1 region is shown.

[0065] Figure 1BHPV-6 / 11 proteins (HPV-E2, HPV-E4, HPV-E6, and HPV-E7) (AdV-HPV6 / 11) expressed by an engineered GC46 vector with deletions in the E1 and E4 regions and the inclusion of the CMV HPV6 / 11 expression cassette in the E1 region are shown.

[0066] Figure 1C The protein / antigen expressed by AdV-HPV6 / 11 is shown as a fusion of selected regions of HPV proteins (HPV-E2, HPV-E4, HPV-E6, and HPV-E7) expressed in HPV-6 and HPV-11 infected cells.

[0067] Figure 1D A schematic diagram of the vector backbone is shown, which includes a CMV promoter, an SV40 polyadenylation signal, a 3' untranslated region, and an antigen open reading frame flanked by an inverted terminal repeat (ITR), and contains the following genes arranged in 5' to 3' order: HPV6 E6, HPV11 E7, HPV6 E7, HPV11 E6, HPV6 E4, HPV6 E6, HPV6 E7, HPV11 E6, HPV6 E4, HPV11 E7, and HPV6 E2.

[0068] Figure 2 This demonstrates the use of a basic chimpanzee adenovirus construct encoding GFP (5 × 10⁻⁶) in autologous dendritic cells cultured from recurrent respiratory papillomavirus (RRP) subjects. 3 Fluorescent activity detected by flow cytometry 24 hours after MOI transduction.

[0069] Figure 3 The results of IFNγ ELISA on T lymphocytes from three RRP subjects, after three rounds of stimulation with dendritic cells transduced with AdV-HPV6 / 11 or a control, are shown. The X-axis represents the adenovirus constructs tested (differences between constructs include gene linkers and encoded antigens).

[0070] Figure 4A illustrates the experimental design for evaluating peripheral T lymphocytes from wild-type C57BL / 6 mice inoculated with Adv-HPV6 / 11 to detect HPV antigen-specific immune responses.

[0071] Figure 4B shows an optical micrograph of a representative ELISpot pore, which demonstrates the response to the HPV6 and HPV11 overlapping 15-mer peptide pool and the synthesized minimum peptide after in vivo inoculation with AdV-HPV6 / 11, while the empty GC46 does not show this response.

[0072] Figure 4C shows the quantification of IFNγ spots in inoculated male (n=3) and female (n=3) mice.

[0073] Figure 4D shows the quantification of IFNγ spots in female wild-type C57BL / 6 mice (n=5) in an independently validated in vivo inoculation experiment.

[0074] Figure 4E shows the quantitative (left) and representative flow cytometry dot plot (right) of CD8+ T lymphocyte IFNγ production after assessing HPV antigen-specific T lymphocyte responses in mice inoculated with Adv-HPV6 / 11 or empty GC46, as measured by intracellular flow cytometry (n=6 / group). The dot plot shows gated viable CD3+CD8+ T lymphocytes from Adv-HPV6 / 11-inoculated mice.

[0075] Figure 5 The ELISpot is shown, which assesses the response of peripheral T lymphocytes in mice (N=4) treated with Adv-HPV6 / 11 (top row) or empty vector (bottom row) to the overlapping 15-mer peptide or minimal common epitope IYSYAYKHLK (SEQ ID NO:120) from HPV6 or HPV11 E6. PMA / Iono is a positive control. Figure 5 SEQ ID NO:120 has been disclosed.

[0076] Figure 6A shows a schematic diagram of retroviral transduction used to create MOC1 cells expressing HPV6 E6.

[0077] Figure 6B shows a flow cytometry plot, revealing E6 (NGFR) positivity in parental MOC1 or MOC1-E6 cells, and quantification is performed on the right.

[0078] Figure 6C shows a summary of tumor growth curves in mice carrying parental MOC1 or MOC1-E6 tumors (n=7 mice / group) after treatment with Adv-HPV6 / 11, empty C46, ​​or control (PBS). Red dots indicate treatments. Tumor volume on day 40 is quantified separately on the right.

[0079] Figure 6D shows a summary of growth curves of mice carrying MOC1-E6 tumors after treatment with AdV-HPV6 / 11 in the presence or absence of CD8 or CD4 depletion antibodies. Red dots represent treatments, and blue dots represent depletion. Tumor volume on day 40 is quantified separately on the right.

[0080] Figure 7A shows a representative dot plot of freshly digested parental pMOC1 or MOC1-E6 tumors obtained from mice treated with Adv-HPV6 / 11 or empty GC46, with T lymphocyte accumulation assessed by flow cytometry. Normalized quantification is shown on the right.

[0081] Figure 7B shows representative H&E and immunofluorescence optical micrographs showing the localization of T lymphocytes within MOC1-E6 tumors treated with Adv-HPV6 / 11 or empty GC46.

[0082] Figure 7C shows the quantification of total T lymphocytes per high-power field (HPF). mpIF represents multiplex immunofluorescence.

[0083] Figure 7D shows the quantitative localization of CD8+ and CD4+ T lymphocytes at the tumor margin and within the tumor parenchyma.

[0084] Figure 8A shows a schematic diagram of tumor-infiltrating lymphocytes (TILs) cultured from tumors of mice carrying MOC1-E6 tumors and treated with Adv-HPV6 / 11 or empty GC46 vector (n=5 mice / group).

[0085] Figure 8B shows representative impedance analysis of TILs cultured from MOC1-E6 tumors treated with Adv-HPV6 / 11, empty GC46, or control (PBS), followed by co-culture with parental MOC1 or MOC1-E6 target tumor cells (n=5 tumors per condition). TILs were added to target cells at experimental time 0. The percentage of cell killing (expressed as % loss of target cell index) was quantified 16 hours after TIL addition to target cells and is shown on the right.

[0086] Figure 8C shows an optical micrograph of a representative ELISpot well and quantification of IFNγ spots. The results demonstrate the response to HPV6 and HPV11 overlapping 15-mer peptide pools and the synthesized minimum peptides in TIL cultures of MOC1-E6 tumors treated with Adv-HPV6 / 11 or empty GC46.

[0087] Figure 9A shows a bar chart displaying the total number of clinically indicated interventions required for patients treated in DL1 (patients 1–3) and DL2 (patients 4–15) within 12 months before (left) and after (right) the study. Responders are shown in blue, and non-responders in gold. Patients requiring no intervention within 12 months after study treatment are considered full responders, while patients requiring 50% or more fewer interventions within 12 months after study treatment are considered partial responders. Responders include both full and partial responders.

[0088] Figure 9B shows a waterfall plot illustrating changes in clinically indicated surgical procedures one year after treatment compared to the year before treatment. Patients are sorted from left to right by the magnitude of the reduction in intervention frequency. The solid horizontal line represents the 50% reduction threshold, used to distinguish between responders and non-responders.

[0089] Figure 9C shows representative clinical endoscopic images, illustrating the larynx appearance of four out of six complete responders (CRs) without visible disease after AdV-HPV6 / 11 treatment. Derkay scores (top left) and time points after completion of treatment (top right) are shown in inset. Post-treatment images are from the most recent endoscopy performed at the data cutoff.

[0090] Figure 10A shows a dot plot illustrating the fold change in the logarithmic transformation of peripheral blood HPV-specific T-cell responses relative to pre-treatment levels at 6 weeks post-treatment. Each dot represents the fold change in IFNγ concentration following peptide stimulation with a specific HPV peptide pool encoded by Adv-HPV6 / 11. Peptide pools for which no IFNγ response was detected in pre- or post-treatment samples are not shown. N=14 patients; patient 5 had insufficient PBMCs pre-treatment. Responders are shown in blue, and non-responders in gold.

[0091] Figure 10B shows a scatter plot summarizing the changes in HPV-specific peripheral blood response. Significance was determined using the Mann-Whitney two-tailed test.

[0092] Figure 10C shows a bar chart displaying the proportion of the top 10 HPV-specific CDR3 frequencies identified in the HPV 6 / 11 peptide-stimulated FEST assay in the TCRβ repertoire after treatment. The Simpson clonality index is displayed above each bar. The top horizontal bar represents the treatment response. R, responder; NR, non-responder.

[0093] Figure 10D shows a dot plot illustrating the logarithmic fold change in the frequency of the top 10 HPV-specific CDR3 sequences (determined in FEST assays) in peripheral blood 6 weeks after completion of AdV-HPV6 / 11 treatment, relative to pre-treatment levels and under peptide-free conditions.

[0094] Figure 10E shows a scatter plot summing the changes in the top 10 HPV-specific CDR3 sequences. Significance was determined by an unpaired two-tailed t-test.

[0095] Figure 10F shows a bar chart summarizing the proportion of the top 10 peripheral blood HPV-specific CDR3 sequences (determined in FEST assays) detected after treatment, compared to those undetectable (newly appearing), detected at a lower frequency (amplified), or detected at a higher frequency (shrinkage) in peripheral blood before treatment. The top horizontal bar represents the treatment response.

[0096] Figure 10G shows a dot plot illustrating the log2 fold change in HPV-specific papilloma-infiltrating lymphocytes (PILs) after completion of the study treatment relative to pre-treatment levels. Each dot represents the logarithmic fold change in IFNγ spot counts after co-culturing PILs with antigen-presenting cells loaded with an HPV peptide pool encoded by Adv-HPV6 / 11. Peptide pools for which no IFNγ response was detected in pre- or post-treatment samples are not shown. N=9 patients; patients 7, 10, 11, and 13 had no post-treatment biopsy samples, and patients 2 and 3 were unable to establish pre-treatment PIL cultures.

[0097] Figure 10H shows a scatter plot summarizing the changes in HPV-specific PIL response. Significance was determined using a Mann-Whitney two-tailed test.

[0098] Figure 10I shows clinical endoscopic images of the larynx in patient 5 before treatment and at a 6-week time point. Red arrows indicate the biopsy sites used to generate the PIL and for the experiments shown in Figures 11J and 11K before and at 6 weeks.

[0099] Figure 10J shows representative IFNγ ELISpot wells, which show IFNγ spots after stimulation with Peptide Pool 2 peptide at pretreatment and 6-week time points (IFNγ spot counts are shown in inset), as well as negative control (DMSO only) and positive control (PMA / ionomycin).

[0100] Figure 10K shows a bar chart illustrating the IFNγ concentration after co-culturing 6-week PIL samples with antigen-presenting cells containing the individual peptides in peptide-loaded pool 2.

[0101] Figure 11A shows representative optical micrographs of T-cell immunofluorescence in baseline papilloma biopsy samples collected from responders (top row) and non-responders (bottom row).

[0102] Figure 11B shows a dot plot illustrating CD8 T cell density in papillomas and stroma in both responders and non-responders. Significance was determined using the Mann-Whitney two-tailed test.

[0103] Figure 11C shows a box plot displaying the HPV gene transcript counts normalized to papilloma cells in responders and non-responders, determined by single-cell RNA sequencing. Significance was determined by two-way ANOVA.

[0104] Figure 11D shows a box plot displaying cell-normalized Reactome IFNγ signal scores (X-axis) across different cell types (responders and non-responders), determined by single-cell RNA sequencing. Significance was determined using two-way ANOVA.

[0105] Figure 11E shows a heatmap displaying normalized chemokine transcript counts in different cell types (Y-axis). The bars on the right represent mean expression. The top horizontal bar represents treatment response. The significance of the difference between responders and non-responders was determined by two-way ANOVA.

[0106] Figure 11F shows a violin plot illustrating the CXCR3 transcript counts in CD8 and CD4 papilloma T cells, determined by single-cell RNA sequencing. Significance was determined using the Mann-Whitney two-tailed test.

[0107] Figure 11G shows a violin plot indicating the percentage of (total) CXCL9 or CXCL10 positive cells. Significance was determined using the Mann-Whitney two-tailed test. Representative optical micrographs of RNAscope immunofluorescence are also shown.

[0108] Figure 11H shows a dot plot illustrating the expression of selected T-cell-related genes in T lymphocyte clusters identified by single-cell RNA sequencing. The dot plot is sorted by the fold change in the number of cells detected in responders versus non-responders (responder / non-responder; bottom bar). T cells enriched in non-responders are shown in the left column, and T cells enriched in responders are shown in the right column. The color of the dots corresponds to the scaled average expression; the size of the dots indicates the proportion of cells with non-zero expression of the corresponding gene. The top bar represents the total number of cells.

[0109] Figure 12A shows a dot plot illustrating the expression of myeloid-related genes in selected myeloid cell clusters identified by single-cell RNA sequencing. The dot plot is sorted by the fold change in the number of cells detected in responders versus non-responders (responders / non-responders; bottom bar). Myeloid cells enriched in non-responders are located in the left column, and myeloid cells enriched in responders are located in the right column. The color of the dots corresponds to the scaled average expression; the size of the dots indicates the proportion of cells with non-zero expression of the corresponding gene. The top bar represents the total number of cells.

[0110] Figure 12B shows a heatmap displaying normalized chemokine transcript counts in different cell types (Y-axis). The bars on the right represent mean expression. The top horizontal bar represents treatment response. The significance of the difference between responders and non-responders was determined by two-way ANOVA.

[0111] Figure 12C shows a heatmap displaying normalized VEGF transcript counts in different cell types (Y-axis). The bars on the right represent mean expression. The top horizontal bar represents treatment response. The significance of the difference between responders and non-responders was determined by two-way ANOVA.

[0112] Figure 12D shows a violin plot illustrating the percentage of CXCL8-positive (total) cells. Significance was determined using the Mann-Whitney two-tailed test. Representative optical micrographs of RNAscope immunofluorescence are also shown.

[0113] Figure 12E shows representative optical micrographs of myeloid cell immunofluorescence in baseline papilloma biopsy samples collected from responders (top row) and non-responders (bottom row).

[0114] Figure 12F shows a dot plot illustrating the density of neutrophil-like cells (PMNs) and macrophages (Mθs) in papillomas and stroma in both responders and non-responders. Significance was determined using the Mann-Whitney two-tailed test.

[0115] Figure 13 Optical micrographs of H&E stained sections from each of the 15 patients included in this study are shown. The patient number is indicated by an inset in the upper left corner of each image.

[0116] Figure 14 A dot plot is shown, displaying the Derkay score (Y-axis) for each patient, obtained from available clinical endoscopic images at 12 months before the study, during the study, and 12 months after the study (X-axis). The lines are color-coded according to response status.

[0117] Figures 15A-15C show representative clinical endoscopic images of the larynx and (if applicable) trachea before and after treatment, where (A) represents patients with complete responses (CR) but with visible lesions remaining after treatment, (B) represents partial responses, and (C) represents non-responders. The inset in the upper left corner of each image shows the Derkay score, and the inset in the upper right corner shows the image time point. For complete responses, the post-treatment image is the image from the most recent endoscopy at the data cutoff. For partial and non-responders, the image is the image from the first clinically indicated surgical procedure after completion of study treatment.

[0118] Figure 16A shows a representative immunofluorescence optical micrograph of T cell staining.

[0119] Figure 16B shows a representative immunofluorescence optical micrograph of the T cell phenotype.

[0120] Figures 16C-16G show the densities of (C) Ki67+ CD8 T cells, (D) total CD4 T cells, (E) Ki67+ CD4 T cells, and (F) regulatory T cells (Tregs) in papillomas and stroma in both responders and non-responders. PD-L1 H scores for papilloma cells are shown in (G). Significance was determined using the Mann-Whitney two-tailed test.

[0121] Figure 17A shows a scatter plot displaying the UMAP embedding results for all sequenced cells from all 13 patients, annotated by cell type.

[0122] Figure 17B shows a bar chart illustrating the average chemokine expression in monocyte-like or neutrophil-like cells based on single-cell RNA sequencing.

[0123] Figure 17C shows a representative immunofluorescence optical micrograph of chemokine RNAscope staining.

[0124] Figure 17D shows a scatter plot illustrating the UMAP embedding results of CD8 T cells, with each cluster distinguished by color.

[0125] Figure 17E shows a scatter plot illustrating the UMAP embedding results of CD4 T cells, with each cluster distinguished by color.

[0126] Figure 17F shows a dot plot illustrating the expression of selected T-cell-related genes in T lymphocyte clusters identified by single-cell RNA sequencing. The dot plot is sorted by the fold change in the number of cells detected in responders versus non-responders (responders / non-responders; bars below). T cells enriched in non-responders are shown in the left column, and T cells enriched in responders are shown in the right column. The color of the dots corresponds to the scaled average expression; the size of the dots indicates the proportion of cells with non-zero expression of the corresponding gene. The top bars represent the total number of cells.

[0127] Figures 18A and 18B show scatter plots illustrating the items enriched in responders and non-responders after gene set enrichment analysis of (A) all papilloma mononuclear cell-like cells or (B) all papilloma neutrophil-like cells. P-values ​​were calculated based on a hypergeometric distribution and adjusted using the Benjamini-Hochberg method.

[0128] Figure 19A shows a bar chart illustrating the average chemokine expression in monocyte-like or neutrophil-like cells based on single-cell RNA sequencing.

[0129] Figure 19B shows a representative immunofluorescence optical micrograph of myeloid cell marker staining.

[0130] Figure 20 The results are shown in a bar chart, derived from the determination of neutralizing antibodies in serum samples from participants in Phase I of the clinical trial. These serum samples were collected from participants at baseline (before treatment) and at days 43 (6 weeks), 12 weeks, and 24 weeks after treatment with AdV-HPV6 / 11, and were further categorized by the participants’ overall clinical response (complete response (CR), partial response (PR), or no response (NR)).

[0131] Figure 21 A dot plot is shown, which illustrates the differences in DL1 (1×10⁻⁶) levels between participants in the Phase I clinical trial before treatment (baseline) and after treatment with Adv-HPV6 / 11. 11 (each particle unit) and DL2 (5×10) 11 Neutralizing HPV6 / 11 antibody titers induced after treatment with (each particle unit).

[0132] Figure 22 The Phase II study protocol is shown.

[0133] Figure 23 A graph comparing the number of surgeries performed in the past 12 months before AdV-HPV6 / 11 treatment with the number of surgeries performed in the 12 months after treatment is shown.

[0134] Figure 24 A bar chart is shown, displaying the number of months since the surgery and the number of pre-treatment surgeries in the 12 months prior to AdV-HPV6 / 11 treatment. Each bar represents an individual patient who has not undergone surgery since treatment. Detailed Implementation

[0135] It should be understood that this disclosure is not limited to the specific embodiments described herein, and therefore variations are possible. Although various features of this disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Those skilled in the art will recognize that variations and modifications of this disclosure are possible, all of which are included within the scope of this disclosure.

[0136] All terms are intended to be understood in the manner of those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0137] The chapter titles used in this document are for organizational purposes only and should not be construed as limiting the topics discussed.

[0138] definition

[0139] The following definitions are supplementary to those in the art and are specific to this application, and should not be attributed to any related or unrelated cases, such as any shared patents or applications. Therefore, the terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0140] In this application, unless otherwise expressly stated, the use of the singular form includes the plural form. It should be noted that, as used in the specification, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural form.

[0141] In this application, unless otherwise stated, “or” means “and / or”. The terms “and / or” and “any combination thereof” and their grammatical equivalents are used interchangeably throughout this application. These terms convey that any combination is explicitly considered. For illustrative purposes only, the phrases “A, B and / or C” or “A, B, C or any combination thereof” can be interpreted as “A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C”. The term “or” may be used in combination or separately unless the context explicitly indicates its use in combination.

[0142] The use of the term "including" and other forms such as "contains," "comprises," and "includes" is not restrictive; that is, "including" does not mean "limited to."

[0143] The use of terms such as "some embodiments," "one embodiment," "an embodiment," or "other embodiments" in the specification means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily in all embodiments of this disclosure.

[0144] The terms “comprising” (and any form of inclusion, such as “comprising” and “containing”), “having” (and any form of having, such as “containing” and “possessing”), “including” (and any form of inclusion, such as “comprising” and “containing”), or “containing” (and any form of containing, such as “containing” and “comprising”) as used in this specification and claims are closed or open-ended and do not exclude other possible components, elements, or method steps. Any embodiment described in this specification may be practiced in conjunction with any method or composition disclosed herein, and vice versa. Furthermore, the compositions of the present invention can be used to implement the methods of the present invention.

[0145] The term "about" or "approximately" means, with respect to a particular numerical value, within an acceptable range of error that can be determined by a person skilled in the art, which will depend in part on how the value is measured or determined, for example, the limits of the measurement system. For example, according to practice in the art, "about" may refer to one or more standard deviations. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. As another example, the quantity of "about 10" includes any quantity between 10 and 9-11. As another example, the term "about" in relation to a reference numerical value may also include a range of values ​​added to or subtracted from that value by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Alternatively, particularly with respect to biological systems or processes, the term "about" may mean within an order of magnitude, preferably within 5 times the numerical value, more preferably within 2 times. Where a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to indicate that the particular value is within an acceptable range of error.

[0146] In describing the ranges of numbers in this article, each interval number with the same precision is explicitly considered. For example, for the range of 6-9, 7 and 8 are considered in addition to 6 and 9; for the range of 6.0-7.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

[0147] As used herein, the term "isolated" and its grammatical equivalents refer to nucleic acids, proteins, peptides, cells, or other substances removed from their natural environment. The term "purified" and its grammatical equivalents refer to molecules or compositions whose purity has been increased, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, where "purity" is a relative term rather than "absolute purity." However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants and remain isolated for practical purposes. For example, when used for introduction into cells, nucleic acids are typically mixed with an acceptable carrier or diluent. The term "substantially purified" and its grammatical equivalents refer to nucleic acid sequences, peptides, proteins, or other compounds that are substantially free of, i.e., free of more than about 50%, more than about 70%, or more than about 90%, of polynucleotides, proteins, peptides, and other molecules naturally associated with that nucleic acid, peptide, protein, or other compound.

[0148] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” “polynucleotide construct,” “oligonucleotide,” and their grammatical equivalents refer to polymeric forms of nucleotides or nucleic acids of any length, which may be ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. It also includes modified forms, such as those modified by methylation and / or capping, as well as unmodified polynucleotide forms. The term is also intended to include molecules containing non-naturally occurring, synthetic, and semi-synthetic nucleotides and polynucleotides, as well as nucleotide analogs. When discussing the structure of a particular double-stranded DNA molecule, sequences in this document may be described, according to common practice, only the sequences in the 5' to 3' directions of the non-transcribed strand of DNA (i.e., the strand with a sequence homologous to mRNA). “Recombinant polynucleotide” refers to a polynucleotide that has undergone molecular biological manipulation. Polynucleotide sequences and vectors disclosed or envisioned herein may be introduced into cells by, for example, transfection, transformation, or transduction.

[0149] When used for polynucleotide or nucleic acid sequences, the term "fragment" refers to a nucleotide sequence shortened relative to a reference nucleic acid and containing the same nucleotide sequence as the reference nucleic acid in its common portion. Such nucleic acid fragments according to the invention may, where appropriate, be contained within a larger polynucleotide in which they are components. Such fragments comprise, or optionally consist of, oligonucleotides in the following length ranges: at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000 or more consecutive nucleotides derived from the nucleic acid according to the invention.

[0150] As used herein, "isolated polynucleotide" or "isolated nucleic acid fragment" refers to a single-stranded or double-stranded RNA or DNA polymer that optionally contains synthetic, non-natural, or modified nucleotide bases. An isolated nucleic acid fragment in the form of a DNA polymer may consist of one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0151] The term "gene" and its grammatical equivalents refer to a polynucleotide containing nucleotides encoding a functional molecule, including functional molecules produced solely through transcription (e.g., biologically active RNA species) or functional molecules produced through both transcription and translation (e.g., polypeptides). The term "gene" encompasses both cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment expressing a specific RNA, protein, or polypeptide, including regulatory sequences located before (5' non-coding) and after (3' non-coding) the coding sequence. "Natural gene" refers to a gene found in nature that possesses its own regulatory sequence. "Chimeric gene" refers to any non-natural gene containing regulatory and / or coding sequences that are not commonly found in nature. Therefore, a chimeric gene can contain regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged differently from their natural counterparts. A chimeric gene can contain coding sequences derived from different sources and / or regulatory sequences derived from different sources. "Endogenous gene" refers to a naturally occurring gene located in the genome of an organism. "Foreign" or "heterologous" genes are genes that are not normally present in a host organism but are introduced into the host organism through gene transfer. Foreign genes can include natural genes or chimeric genes inserted into non-natural organisms. "Transgenic" genes are genes that have been introduced into the genome through transformation methods.

[0152] The term "genome" includes chromosomes as well as mitochondria, chloroplasts, and viral DNA or RNA. The term "probe" refers to a single-stranded nucleic acid molecule that can pair with complementary single-stranded target nucleic acid bases to form a double-stranded molecule.

[0153] "Heterologous DNA" refers to DNA that is not naturally located in cells or at chromosomal loci within cells. Heterologous DNA can include foreign genes. "Foreign genes" refer to genes that are heterologous to the target organism, i.e., genes introduced into the target organism through transformation, unmutated forms of endogenous mutant genes, or mutated forms of unmutated endogenous genes. Foreign genes can be natural or synthetic genes, introduced into the target organism in the form of DNA or RNA, and can function through DNA intermediates such as reverse transcriptase. These genes can be introduced into target cells, directly into the target organism, or indirectly by transferring transformed cells into the target organism.

[0154] A primer is an oligonucleotide that hybridizes with a target nucleic acid sequence to form a double-stranded nucleic acid segment, which can serve as the starting point for DNA synthesis under appropriate conditions. These primers can be used in polymerase chain reactions or DNA sequencing.

[0155] A DNA “coding sequence” or “coding region” refers to a double-stranded DNA sequence that encodes a polypeptide. When placed under the control of appropriate regulatory sequences, it can be transcribed and translated into a polypeptide in cells, in vitro, or in vivo. An “appropriate regulatory sequence” refers to a nucleotide sequence located upstream (5' non-coding), inside, or downstream (3' non-coding) of the coding sequence, and that influences the transcription, RNA processing, or stability of the relevant coding sequence, or its translation. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of a coding sequence are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences can include, but are not limited to, prokaryotic sequences, cDNA derived from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the goal is to express the coding sequence in eukaryotic cells, then the polyadenylation signal and transcription termination sequence will typically be located at the 3' end of the coding sequence.

[0156] An "open reading frame" (ORF) is a nucleic acid sequence (DNA, cDNA, or RNA) that contains a translation initiation signal or start codon (e.g., ATG or AUG) and a stop codon, and can potentially be translated into a polypeptide sequence.

[0157] The term "downstream" refers to the nucleotide sequence located at the 3' end relative to a reference nucleotide sequence. Specifically, downstream nucleotide sequences are typically associated with sequences following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0158] The term "upstream" refers to the nucleotide sequence located at the 5' end relative to a reference nucleotide sequence. Specifically, the upstream nucleotide sequence is typically associated with a sequence located 5' to the coding sequence or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0159] The term "response element" refers to one or more cis-acting DNA elements that confer promoter responsiveness by interacting with the DNA-binding domain of a transcription factor. This DNA element may be a palindromic sequence (complete or incomplete) in its sequence, or may comprise sequence motifs or half-sites separated by a variable number of nucleotides. Half-sites may be similar or identical, arranged as direct or inverted repeats, or as single half-sites or multimers of adjacent half-sites in tandem. Response elements may include minimal promoters isolated from different organisms, depending on the nature of the cell or organism in which the response element is introduced. In the presence or absence of a ligand, the DNA-binding domain of the transcription factor binds to the DNA sequence of the response element to initiate or repress transcription of downstream genes under the regulation of that response element. Examples of DNA sequences for the response elements of the natural ecdysone receptor include: RRGG / TTCANTGAC / ACYY (see Cherbas et al., Genes Dev. 1991); AGGTCAN(n)AGGTCA, where N(n) may be one or more spacer nucleotides (see D'Avino et al., Mol. Cell. Endocrinol. 113:1 1995); and GGGTTGAATGAATTT (see Antoniewski et al., Mol. CellBiol. 14:4465 1994).

[0160] As used herein, the term "operational linking" refers to the physical and / or functional connection of one DNA segment to another in a manner that allows each segment to function as intended. The DNA sequence encoding a gene product is operationally linked to a regulatory sequence (e.g., a promoter, enhancer, and / or silencer) in a manner that allows direct or indirect regulation of transcription of that DNA sequence. For example, the DNA sequence is operationally linked to a promoter when it is downstream of the transcription start site, in the correct reading frame relative to the transcription start site, and allows transcription to extend through the DNA sequence. An enhancer or silencer is operationally linked to the DNA sequence encoding a gene product when it is linked to the DNA sequence encoding the gene product in a manner that increases or decreases transcription of that DNA sequence, respectively. Enhancers and silencers may be located upstream, downstream, or embedded within the coding region of the DNA sequence. When the DNA of a signaling sequence is expressed as a precursor protein involved in polypeptide secretion, the DNA of the signaling sequence is operationally linked to the DNA encoding the polypeptide. The ligation of DNA sequences to regulatory sequences is typically achieved by ligation at appropriate restriction sites, or by using aptamers or adapters inserted into the sequence using restriction endonucleases known to those skilled in the art.

[0161] As used herein, the term "codon degenerate variant" refers to a modified nucleic acid sequence that encodes the same amino acid sequence as the original sequence, but differs in the specific nucleotides that make up the codons. The genetic code is degenerate, meaning that multiple codons can encode the same amino acid. For example, the amino acid leucine can be encoded by six different codons: CTG, CTT, CTC, CTA, TTG, and TTA. A codon degeneracy table (also called a genetic code table or codon table) is a diagram that provides information about the relationship between codons (a sequence of three nucleotides) and the corresponding amino acids they encode. This table lists 64 possible codons and indicates the amino acid each codon represents. Table 1 is an example of a codon degeneracy table: Table 1: Codon Degeneracy Table

[0162] (U stands for "uracil," which is incorporated into mRNA to replace T (thymine) present in DNA; both form complementary base pairs with "A" (adenine); "Indicates the stop codon."

[0163] The following definitions supplement those in the art and are specific to this application, and should not be attributed to any related or unrelated cases, such as any shared patents or applications. Therefore, the terminology used herein is for descriptive purposes only and is not intended to be limiting.

[0164] Furthermore, publicly available software resources are readily available for computer-generated “reverse translation,” also known as “back translation” of polypeptide sequences (i.e., converting a polypeptide sequence into a nucleotide sequence encoding that polypeptide sequence). See, for example, Madeira, F. et al., Nucleic Acids Res, 47(Wl), W636-W641 (2019); Madeira, F. et al., Curr Protoc in Bioinformatics, 66(1):e74 (2019); Chojnacki, S. et al., Nucleic Acids Res. 2017 Jul 3;45(Wl):W550-W553 (2017); Athey, J. et al., BMC Bioinformatics 18:391 (2017).

[0165] As used in this article, codon degenerate variants can be used to optimize gene expression or enhance protein production. By modifying codons in a nucleic acid sequence, codons that are used more frequently or preferred by the host organism's translation mechanisms can be utilized. This can lead to increased protein expression efficiency or improved compatibility with a specific host organism.

[0166] As used in this article, the term "expression" refers to the transcription and stable accumulation of the sense strand (mRNA) or antisense RNA derived from nucleic acids or polynucleotides. Expression can also refer to the translation of mRNA into proteins or polypeptides.

[0167] The terms "cassette," "expression cassette," and "gene expression cassette" refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at a specific restriction site or through homologous recombination. The DNA segment contains a polynucleotide encoding a target polypeptide, and the cassette and restriction site are designed to ensure that the cassette is inserted in the correct reading frame for transcription and translation. A "transformation cassette" refers to a specific vector containing a polynucleotide encoding a target polypeptide and, in addition to the polynucleotide, elements that promote the transformation of a specific host cell. The cassettes, expression cassettes, gene expression cassettes, and transformation cassettes of the present invention may also contain elements that allow for enhanced expression of the polynucleotide encoding the target polypeptide in host cells. These elements may include, but are not limited to: promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, etc. The total length of the expression cassette described herein may include approximately 500 to 10000 bp, approximately 1000 to 5000 bp, approximately 1500 to 4500 bp, approximately 1800 to 4400 bp, approximately 2000 to 4500 pp, approximately 2100 to 4400 bp, approximately 2200 to 4300 bp, approximately 2300 to 4200 bp, approximately 2400 to 4100 bp, approximately 2500 to 4000 bp, approximately 2600 to 3900 bp, approximately 2700 to 3800 bp, approximately 2800 to 3800 bp, approximately 2900 to 3700 bp, approximately 3000 to 3600 bp, approximately 3100 to 3500 bp, approximately 3150 to 3450 bp, approximately 3200 to 3400 bp, and approximately 3250 to 3350 bp. bp or approximately 3300 bp. Alternatively, the expression cassette can include any number of base pairs falling within these ranges.For example, expression boxes may include approximately 500 bp, approximately 750 bp, approximately 1000 bp, approximately 1250 bp, approximately 1500 bp, approximately 1750 bp, approximately 2000 bp, approximately 2250 bp, approximately 2500 bp, approximately 2550 bp, approximately 2600 bp, approximately 2650 bp, approximately 2700 bp, approximately 2750 bp, approximately 2800 bp, approximately 2850 bp, approximately 2900 bp, approximately 2950 bp, approximately 3000 bp, approximately 3050 bp, approximately 3100 bp, approximately 3150 bp, approximately 3200 bp, approximately 3250 bp, approximately 3300 bp, approximately 3350 bp, approximately 3400 bp, approximately 3450 bp, approximately 3500 bp, and approximately 3550 bp. The bp values ​​are approximately 3600 bp, 3650 bp, 3700 bp, 3750 bp, 3800 bp, 3850 bp, 3900 bp, 3950 bp, 4000 bp, 4050 bp, 4100 bp, 4150 bp, 4200 bp, 4250 bp, 4300 bp, 4350 bp, 4400 bp, 4450 bp, or 4500 bp. In one aspect, the expression box comprises approximately 2800 bp. In another aspect, the expression comprises 2825 bp.

[0168] As used herein, the term "vector" refers to any delivery vehicle used for cloning and / or transferring nucleic acids into host cells. A vector may be a replicon to which another DNA segment is attached to achieve replication of the attached segment. "Replicon" refers to any genetic element (e.g., plasmid, bacteriophage, granuloma, chromosome, virus) that functions in vivo as a unit of autonomous DNA replication, i.e., capable of replicating under its own control. The term "vector" includes viral and non-viral delivery vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. A large number of vectors known in the art can be used to manipulate nucleic acids, incorporate reactive elements and promoters into genes, etc. Available vectors include, for example, plasmids or modified viruses, including, for example, λ-derived bacteriophages, or plasmids such as pBR322 or pUC plasmid derivatives, or Bluescript vectors. Another example of a vector useful in this invention is the ULTRAVECTOR® production system (Intrexon Corp., Blacksburg, VA) described in WO2007 / 038276. For example, inserting a DNA fragment corresponding to the reaction element and promoter into a suitable vector can be achieved by inserting the DNA fragment corresponding to the reaction element and promoter into a selected vector with complementary sticky ends and ligating an appropriate DNA fragment. Alternatively, the ends of the DNA molecule can be enzymatically modified, or any site can be created by ligating a nucleotide sequence (adapter) to the DNA end. Such vectors can be engineered to contain a selective marker gene for selecting cells that have integrated the marker into their cellular genome. This marker allows for the identification and / or selection of host cells that contain and express a protein encoded by the marker.

[0169] As used herein, the term "plasmid" refers to an extrachromosomal element that typically carries genes not metabolized at the cellular center and is usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genome-integrated sequences, bacteriophages, or nucleotide sequences. They can be linear, circular, or supercoiled, and can be single-stranded or double-stranded DNA or RNA of any origin. Multiple nucleotide sequences are linked or recombined into a unique construct capable of introducing a promoter fragment and the DNA sequence of a selected gene product, along with an appropriate 3' untranslated sequence, into the cell.

[0170] As used herein, the terms "cloning vector" and "replicon" refer to a unit-length nucleic acid, preferably DNA, that replicates sequentially and contains an origin of replication, such as a plasmid, bacteriophage, or kinase, to which another nucleic acid segment can be attached to achieve replication of the attached segment. A cloning vector can replicate in one cell type and be expressed in another cell type ("shuttle vector"). A cloning vector may contain one or more sequences for selecting cells containing the vector and / or one or more multiple cloning sites for inserting a target sequence.

[0171] As used herein, the term "viral vector" refers to a virus, viral particle, or a derivative thereof, capable of transferring nucleic acids into cells, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. Viral vectors, especially retroviral vectors, have been used in a variety of gene delivery applications in cells and live animal subjects. Available viral vectors include, but are not limited to, retroviruses, adeno-associated viruses, poxviruses, baculoviruses, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus, geminiviruses, and cauliflower mosaic virus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cell transfection agents), DNA-protein complexes, and biopolymers. In addition to nucleic acids, vectors may also contain one or more regulatory regions and / or selectivity markers, which help in selecting, measuring, and monitoring nucleic acid transfer outcomes (which tissues to which, duration of expression, etc.).

[0172] As used herein, the terms “adenovirus” and “adenovirus vector” refer to adenoviruses that retain the ability to participate in the adenovirus life cycle and / or adenoviruses that are physically inactivated, for example, by disruption (e.g., sonication), denaturation (e.g., using heat or solvents), or cross-linking (e.g., formaldehyde cross-linking). The “adenovirus life cycle” includes (1) viral binding and entry into cells, (2) transcription of the adenovirus genome and translation of adenovirus proteins, (3) replication of the adenovirus genome, and (4) viral particle assembly (see, for example, Fields Virology, 5th ed., Knipe et al., Lippincott Williams & Wilkins, Philadelphia, PA (2006)). Adenoviruses as used herein and as described herein can also be made replication-deficient (i.e., not retaining the ability to participate in the adenovirus life cycle) by deleting one or more portions of the naturally occurring viral genome. As used herein and as described herein, "adenovirus" and "adenovirus vector" can include an adenovirus whose genome has been manipulated to accommodate a non-natural nucleic acid sequence relative to the adenovirus genome. Typically, an adenovirus vector is generated by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus's adenovirus genome to accommodate the insertion of a non-natural nucleic acid sequence (e.g., for gene transfer).

[0173] As used herein, the term “MOI” or “multiple of infection” refers to the average number of viral particles that infect a single cell in a particular experiment (e.g., recombinant virus or control virus).

[0174] As used herein, the term "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by cells. Cells are "transfected" when exogenous or heterologous RNA or DNA has been introduced into the cell. Cells are "transformed" when the transfected RNA or DNA causes a phenotypic change. The transformed RNA or DNA can be integrated (covalently linked) into the chromosomal DNA that constitutes the cell's genome.

[0175] As used herein, the term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. A host organism containing the transformed nucleic acid fragment is referred to as a "transgenic," "recombinant," or "transformed" organism.

[0176] As used herein, the term "electroporation" refers to the use of transmembrane electric field pulses to momentarily increase cell membrane permeability, thereby allowing the introduction of exogenous biological materials (e.g., DNA, RNA, peptides, polypeptides, proteins, enzymes, or ribonucleoproteins (RNPs)) into cells. The electric field pulses create transient pores in the cell membrane, thereby facilitating the uptake of biological materials. Electroporation can be performed using specialized buffers and devices that control pH, conductivity, osmotic pressure, and other parameters to optimize the process and improve transfection efficiency while minimizing cell damage. Electroporation can be used to introduce exogenous materials (e.g., biomolecules, plasmids, oligonucleotides, expression cassettes, siRNA, drugs, and ions) into various cell types, including primary human hemoglobin, immune cells, pluripotent progenitor cells, fibroblasts, and endothelial cells, for applications in gene therapy, cell therapy, and biotechnology research.

[0177] As used herein, the terms “induction,” “inducing effect,” and their grammatical equivalents refer to an increase in nucleic acid sequence transcription, promoter activity, and / or expression caused by transcriptional regulators relative to a given basal transcriptional level.

[0178] As used herein, the terms "promoter" and "promoter sequence" are used interchangeably to refer to a DNA sequence that controls the expression of a coding sequence or functional RNA. Generally, the coding sequence is located at the 3' end of the promoter sequence. A promoter may be derived entirely from a natural gene, or may consist of different elements derived from different promoters in nature, or may even contain synthetic DNA fragments. Those skilled in the art will understand that different promoters can guide gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters that express genes in most cell types and at most times are generally called "constitutive promoters." Promoters that express genes in specific cell types are generally called "cell-specific promoters" or "tissue-specific promoters." Promoters that express genes at specific developmental stages or cell differentiation stages are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced to express genes after cells are exposed to or treated with inducers such as drugs, biomolecules, chemicals, ligands, or light are generally called "inducible promoters" or "regulatory promoters." It should also be recognized that, since the exact boundaries of the regulatory sequence are not fully determined in most cases, DNA fragments of different lengths may have the same promoter activity.

[0179] Promoter sequences typically demarcate at their 3' end with the transcription start site and extend upstream (in the 5' direction) to include the minimum number of bases or elements necessary to initiate transcription beyond the background detectable level. Within the promoter sequence are the transcription start site (e.g., conveniently defined by nuclease S1 localization) and the protein-binding domain (a shared sequence) responsible for RNA polymerase binding.

[0180] The promoter may be natural or synthetic, and the source of the promoter should not limit the scope of the invention described herein. In other words, the promoter may be cloned directly from a cell, or the promoter may have been previously cloned from a different source, or the promoter may be obtained synthetically.

[0181] As used herein, the term “transcription regulator” refers to a biochemical element that functions under certain environmental conditions to prevent or inhibit the transcription of a promoter-driven DNA sequence (e.g., a repressor or nuclear repressor protein), or under certain environmental conditions to allow or stimulate the transcription of a promoter-driven DNA sequence (e.g., an inducer or enhancer).

[0182] As used herein, the term "enhancer" refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence operatively linked to it. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a wide variety of sources are well known in the art and are available as either cloned polynucleotides (e.g., from depositories such as ATCC and other commercial or personal sources) or contained within cloned polynucleotides. Many promoter-containing polynucleotides (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, inside, or downstream of the coding sequence. The term “Ig enhancer” refers to an enhancer element derived from an enhancer region mapped within an immunoglobulin (Ig) gene locus (such enhancers include, for example, heavy chain (μ) 5' enhancers, light chain (κ) 5' enhancers, κ and μ intrinsic enhancers and 3′ enhancers (see, generally, Paul WE, ed., Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pp. 353-363; and U.S. Patent No. 5,885,827).

[0183] As used herein, the term "therapeutic switch promoter" ("TSP") refers to a promoter that controls the expression of gene switch components. Gene switches and their various components are described in detail elsewhere herein. In some embodiments, the TSP is constitutive, i.e., persistently active. A constitutive TSP can be constitutive-ubiquitous (i.e., it typically functions in any tissue or cell without additional factors or regulators) or constitutive-tissue- or cell-specific (i.e., it typically functions in a specific tissue or cell type without additional factors or regulators). In some embodiments, the TSP of the present invention is activated under conditions associated with a disease, symptom, or condition. In some embodiments of the present invention involving two or more TSPs, the promoter may be a combination of a constitutive promoter and an activatable promoter. As used herein, "promoters activated under conditions associated with a disease, symptom, or condition" includes, but is not limited to, disease-specific promoters, promoters responding to specific physiological, developmental, differentiation, or pathological conditions, promoters responding to specific biomolecules, and tissue- or cell-type-specific promoters associated with a disease, symptom, or condition, such as tumor tissue or malignant cells. TSPs may contain sequences of naturally occurring promoters, modified sequences derived from naturally occurring promoters, or synthetic sequences (e.g., inserting a reactive element into a minimal promoter sequence to alter the promoter's responsiveness).

[0184] Therapeutic switch promoters useful in this invention may include any promoter useful for treating, improving, or preventing a specific disease, symptom, or condition. Examples include, but are not limited to, promoters of genes that exhibit increased expression only during a specific disease, symptom, or condition, and promoters of genes that exhibit increased expression under specific cellular conditions (e.g., proliferation, apoptosis, pH changes, oxidative state, oxygen level). In some embodiments, when the gene switch contains more than one transcription factor sequence, the specificity of the treatment method can be improved by binding a disease or condition-specific promoter to a tissue or cell type-specific promoter to restrict the tissue of expression of the therapeutic product. Therefore, tissue or cell type-specific promoters are included within the definition of therapeutic switch promoters.

[0185] Regarding gene switches, the term "ecdysone receptor-based" refers to a gene switch that includes at least a functional portion of a naturally occurring or synthetic ecdysone receptor ligand-binding domain and that regulates gene expression in response to a ligand binding to the ecdysone receptor ligand-binding domain. Examples of ecdysone response systems are described in U.S. Patent Nos. 7,091,038 and 6,258,603. Other examples of chimeric ecdysone receptor systems are described in U.S. Patent Nos. 7,091,038, U.S. Patent Publication Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457 and 2006 / 0100416, and International Publication Nos. WO 01 / 70816, WO 02 / 066612, WO 02 / 066613, WO 02 / 066614, WO 02 / 066615, WO 02 / 29075 and WO 2005 / 108617. In one implementation, the system is the RheoSwitch® Therapeutic System (RTS), which comprises two fusion proteins: a DEF domain of a mutagenized ecdysone receptor (EcR) fused with a Gal4 DNA-binding domain, and an EF domain of a chimeric RXR fused with a VP16 transcriptional activation domain, both expressed under a constitutive promoter.

[0186] When RNA polymerase transcribes a coding sequence into mRNA in a cell, and then the mRNA undergoes transRNA splicing (if the coding sequence contains introns) and is translated into a protein encoded by that coding sequence, the coding sequence is "under the control" of transcriptional and translational control sequences in the cell.

[0187] "Transcription and translation control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, and terminators, which enable the expression of coding sequences in host cells. In eukaryotic cells, polyadenylation signals are control sequences. Enhancers that can be used in embodiments of this invention include, but are not limited to: SV40 enhancer, cytomegalovirus (CMV) enhancer, elongation factor 1 (EF1) enhancer, yeast enhancer, viral gene enhancer, etc.

[0188] The terms “3' non-coding sequence” and “3' untranslated region (UTR)” refer to DNA sequences located downstream (3') of the coding sequence, and may contain polyadenylation [poly(A)] recognition sequences as well as other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by influencing the addition of polyadenylate chains to the 3' end of mRNA precursors.

[0189] As used herein, the term "regulatory region" refers to a nucleic acid sequence that regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences naturally responsible for expressing a specific nucleic acid (homologous regions) or sequences of different origins responsible for expressing different proteins or even synthesizing proteins (heterologous regions). Specifically, the sequences may be sequences or derived sequences of prokaryotic, eukaryotic, or viral genes that specifically or non-specifically stimulate or inhibit gene transcription and inducible or non-inducible ways. Regulatory regions include origins of replication, RNA splicing sites, promoters, enhancers, transcription termination sequences, and signaling sequences that introduce polypeptides into target cell secretion pathways.

[0190] As used in this article, the term “regulation” refers to the induction, reduction, or inhibition of nucleic acid or gene expression, which in turn leads to the corresponding induction, reduction, or inhibition of protein or polypeptide production.

[0191] As used herein, the term "inducible promoter" refers to a promoter that is induced to activate in the presence or absence of transcriptional regulators (e.g., biotic or abiotic factors). Inducible promoters are useful because the expression of genes operationally linked to them can be turned on or off at certain stages of an organism's development or in specific tissues. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenicity-regulated promoters, temperature-regulated promoters, and light-regulated promoters. The inducible promoter can be part of a gene switch or a genetic switch. The inducible promoter can be a gene switch ligand-induced promoter. In some cases, the inducible promoter can be a small molecule ligand-induced, two-peptide, ecdysone receptor-based gene switch. In some cases, the gene switch can be selected from the ecdysone receptor component, such as, but not limited to, any of the systems described below: International Patent Applications WO 2001 / 070816; WO 2002 / 029075; WO 2002 / 066613; WO 2002 / 066614; WO 2002 / 066612; WO 2002 / 066615; WO 2003 / 027266; ​​WO 2003 / 027289; WO2005 / 108617; WO 2009 / 045370; WO 2009 / 048560; WO 2010 / 042189; WO 2010 / 042189; WO2011 / 119773; and WO 2012 / 122025; and U.S. Patent Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; 8,105,825; 8,168,426; 7,531,326; 8,236,556; 8,598,409; 8,715,959; 7,601, 508; 7,829,676; 7,919,269; 8,030,067; 7,563,879; 8,021,878; 8,497,093; 7,935,510; 8,076,454; 9,402,919; 9,493,540; 9,249,207; and 9,492,482.

[0192] As used herein, two or more individually operable gene regulatory systems are referred to as "orthogonal" when the following conditions are met: a) at selected concentrations, regulation of each system by its respective ligand results in a measurable change in the magnitude of gene expression in that system; and b) this change is statistically significantly different from the expression changes of all other simultaneously operable systems in the same cell, tissue, or organism, regardless of whether the actual regulation occurs simultaneously or sequentially. Preferably, regulation of each individually operable gene regulatory system results in a gene expression change that is at least 2 times larger than that of all other operable systems in the cell, tissue, or organism, for example, at least 5, 10, 100, or 500 times larger. Ideally, at selected concentrations, regulation of each system by its respective ligand results in a measurable change in the magnitude of gene expression in that system, without resulting in a measurable change in the expression of all other operable systems in the cell, tissue, or organism. In this case, the multiple inducible gene regulatory system is referred to as "completely orthogonal". Useful orthogonal ligands and receptor-based orthogonal gene expression systems are described in US 2002 / 0110861 A1.

[0193] As used herein, the term "gene switch" refers to a combination of a promoter-associated response element and a ligand-dependent transcription factor system that regulates the expression of a gene incorporating both the response element and the promoter in the presence of one or more ligands. The term "polynucleotide encoding a gene switch" refers to a combination of a promoter-associated response element and a polynucleotide encoding a ligand-dependent transcription factor system that regulates the expression of a gene incorporating both the response element and the promoter in the presence of one or more ligands. Tightly regulated inducible gene expression systems or gene switches (e.g., EcR-based systems) are useful for a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, functional genomics, and trait regulation in transgenic plants and animals. Such inducible gene expression systems may include ligand-induced heterologous gene expression systems.

[0194] As used herein, the term “CAP” or “cap” refers to a modified nucleotide, typically 7-methylguanosine, attached to the 5' end of eukaryotic mRNA via a 3' to 5' linker (7meG-ppp-G), which serves as an essential element in the normal translation initiation pathway from which proteins are expressed on the mRNA.

[0195] As used herein, the term "Sleeping Beauty (SB) transposon system" refers to a synthetic DNA transposon system for introducing DNA sequences into the chromosomes of vertebrates. Exemplary embodiments of this system are described, for example, in U.S. Patent Nos. 6,489,458, 8,227,432, 9,228,180, and WO / 2016 / 145146. The Sleeping Beauty transposon system comprises a Sleeping Beauty (SB) transposon enzyme and an SB transposon. In embodiments, the Sleeping Beauty transposon system may include the SB11 transposon system, the SB100X transposon system, or the SB110 transposon system.

[0196] As used herein, the term "transposon" or "transposable element" (TE) refers to a vector DNA sequence that can change its position within the genome, sometimes producing or reversing mutations and altering the size of the cell's genome. Transposition typically results in the replication of the TE. Type I TEs are replicated in two stages: first, they are transcribed from DNA into RNA, and then the resulting RNA is reverse transcribed back into DNA. The replicated DNA is then inserted into a new location within the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the TE itself. Retroposons are similar in characteristics to retroviruses such as HIV. The cut-and-paste transposition mechanism of Type II TEs does not involve RNA intermediates. Transposition is catalyzed by several transposases. Some transposases bind to any target site in the DNA in a non-specific manner, while others bind to specific DNA sequence targets. Transposases create staggered cuts at the target site, forming single-stranded 5' or 3' DNA overhangs (sticky ends). This step involves the excision of the DNA transposon, which is then ligated to a new target site; this process involves DNA polymerase activity to fill the gap and DNA ligase activity to block the sugar-phosphate backbone. This leads to replication of the target site. The insertion site of the DNA transposon can be identified by short direct repeat sequences, which can be generated by staggered cutting of the target DNA and subsequent filling by DNA polymerase, followed by a series of inverted repeat sequences important for transposase excision of the TE. When the transposition of the cut-and-paste TE occurs during the S phase of the cell cycle (when the donor site has been replicated but the target site has not), the cut-and-paste TE can be replicated. In class I and class II TEs, transposition can be classified as autonomous or non-autonomous. Autonomous TEs can move on their own, while non-autonomous TEs require the presence of another TE to move. This is usually because non-autonomous TEs lack transposase (for class II) or reverse transcriptase (for class I).

[0197] As used in this article, the term "transposase" refers to an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome via a cut-and-paste mechanism or a replication-transposon mechanism.

[0198] As used herein, the terms “polypeptide,” “peptide construct,” and “peptide construct,” and their grammatical equivalents, refer to polymeric compounds consisting of covalently linked amino acid residues. “Mature protein” refers to a full-length protein, which optionally includes glycosylation or other modifications typical of that protein in a given cellular environment. Embodiments of the invention, as disclosed herein, include the HPV antigenic / antigenic polypeptides, peptides, and mature proteins described herein, and also include those encoding the same polynucleotides (DNA or RNA). The polypeptides and proteins disclosed herein (including functional fragments and functional variants thereof) may contain synthetic amino acids at the positions of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline-2-carboxylic acid. Acids, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0199] As used herein, the term "peptide fragment" refers to a polypeptide whose amino acid sequence is shorter than that of a reference polypeptide, and which contains the same amino acid sequence over its corresponding portion as those reference polypeptides. Where appropriate, such fragments may be contained within a larger polypeptide to which they belong. Such polypeptide fragments according to the invention may be at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 or more amino acids.

[0200] As used herein, the terms “isolated polypeptide,” “isolated peptide,” or “isolated protein” refer to a polypeptide or protein that is substantially free of those compounds (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids) that are typically associated with it in their natural state. “Isolated” is not intended to exclude the presence of artificial or synthetic mixtures with other compounds, or impurities that do not interfere with biological activity, and such impurities may arise, for example, due to incomplete purification, the addition of stabilizers, or formulation into pharmaceutically acceptable preparations.

[0201] As used herein, in the context of two nucleic acid sequences or two amino acid sequences of a polypeptide, the term "identical" or "sequence identity" refers to the same residues in two sequences when the two sequences are aligned for maximum correspondence within a specified comparison window. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically about 50 to about 200 positions, and more typically about 100 to about 150 positions, after optimal alignment of the two sequences, which can then be compared with a reference sequence having the same number of consecutive positions. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be performed by: Smith and Waterman, Adv. Appl. Math., The local homology algorithm of 2:482 (1981); through Needleman and Wunsch, J Mal. Biol., The alignment algorithm of 48:443 (1970); by Pearson and Lipman, Proc. Nat. Acad Sci US.A.,Similarity search methods described in 85:2444 (1988); computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the Intelligentics (Mountain View, CA) PC / Gene program, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Avenue, Madison, Wisconsin, USA); the CLUSTAL program is described in detail in Higgins and Sharp (Gene, 73:237-244 (1988)) and Higgins and Sharp (CABIOS, 5:151-153 (1989)); and Corpet et al. (Nucleic Acids Res., 16:10881-10890 (1988)); Huang et al. (Computer Applications in the Biosciences, 8:155-165). (1992)); and Pearson et al. (Methods in Molecular Biology, 24:307-331 (1994)). Comparisons are also often performed through visual inspection and manual comparison.

[0202] In one embodiment, the polypeptide described herein is at least 80%, 85%, 90%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to a reference polypeptide or fragment thereof, for example, as measured by BLASTP (or CLUSTAL or any other available alignment software) using default parameters. Similarly, nucleic acids may be described with reference to the starting nucleic acid, for example, being 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to a reference nucleic acid or fragment thereof, for example, as measured by BLASTN (or CLUSTAL or any other available alignment software) using default parameters. When a molecule is said to have a certain percentage of sequence identity with a larger molecule, this means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds matching residues in the larger molecule in the order of optimal alignment between the two molecules.

[0203] As used herein, the terms "percentage similarity" or "percentage identity" as known in the art refer to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences determined by comparing sequences. In the art, "similarity" or "identity" also means the degree of sequence correlation between polypeptide or polynucleotide sequences, determined by matching such sequence strings. "Identity" and "similarity" can be readily calculated using known methods, including but not limited to those mentioned above, or, for example, *Computational Molecular Biology* (edited by Lesk, AM), Oxford University Press, New York (1988); *Biocomputing: Informatics and Genome Projects* (edited by Smith, DW), Academic Press, New York (1993); *Computer Analysis of Sequence Data, Part I* (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); *Sequence Analysis in Molecular Biology* (edited by von Heinje, G.), Academic Press, San Diego (1987); and *Sequence Analysis Primer* (edited by Gribskov, M and Devereux). The methods described in J. (ed.), Stockton Press, New York (1991) are used to determine identity and similarity. Methods for determining identity and similarity are incorporated into publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using sequence analysis software, such as the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc., Madison, Wisconsin) with MegAlign (or the newer MegAlign Pro). Multiple alignment of sequences can be performed using the Clustal alignment method (Higgins et al., CABIOS. 5:151 1989) with default parameters (gap penalty = 10, gap length penalty = 10).The default parameters for the Clustal method used for pairing are: KTUPLE = 1, gap penalty = 3, window size = 5, and number of diagonals saved (DIAGONALS SAVED) = 5.

[0204] As used herein, the term “substantially similar” and its grammatical equivalents, when applied to nucleic acid or amino acid sequences, mean that a nucleic acid or amino acid sequence contains a sequence that, when compared using the comparison procedures described above (e.g., BLAST with standard parameters), has at least 90% or higher sequence similarity to a reference sequence, such as at least 95%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and at least 99.99% sequence similarity. The term "substantially identical" and its grammatical equivalents, when applied to nucleic acid or amino acid sequences, mean that a nucleic acid or amino acid sequence contains a sequence that, when compared using the aforementioned comparison procedures (e.g., BLAST with standard parameters), has at least 99% sequence identity relative to a reference sequence, such as at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and at least 99.99% sequence identity. For example, the default parameters for the BLASTN procedure (for nucleotide sequences) are word length (W) 11, expected value (E) 10, M=5, N=-4, and it compares two strands. For amino acid sequences, the default parameters for the BLASTP procedure are word length (W) 3, expected value (E) 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The sequence identity percentage is determined by comparing two best-aligned sequences within a comparison window, wherein the polynucleotide sequence portion within the comparison window may contain additions or deletions (i.e., gaps) relative to a reference sequence (which does not contain insertions or deletions) to achieve optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. In an embodiment, substantial identity exists in sequence regions of at least about 50 residues in length, in regions of at least about 100 residues, and in an embodiment, the sequences are substantially identical at at least about 150 residues. In an embodiment, the sequences are substantially identical over the entire length of the coding region.

[0205] As used herein, the term "functional fragment" and its grammatical equivalents refer to a portion, segment, or region of a biomolecule that retains the essential functional characteristics or activity of the original biomolecule. As used herein, the term "functional variant" and its grammatical equivalents refer to a modified form of a biomolecule that exhibits a degree of variation while retaining the essential functional characteristics or activity of the original molecule. This includes altered biomolecules, for example, by introducing specific changes through genetic engineering or mutagenesis techniques, while retaining the overall functionality of the biomolecule. Compared to the original molecule, a functional variant may have one or more amino acid substitutions, insertions, or deletions while still maintaining the desired biological activity or function. Techniques for obtaining these variants, including genetic techniques (inhibition, deletion, mutation, etc.), chemical techniques, and enzymatic techniques, are known to those skilled in the art. In one embodiment, the variant biomolecule comprises at least about 14 monomers (e.g., nucleotides or amino acids).

[0206] As used herein, the term "homology," and all its grammatical forms and spelling variations, refers to the percentage of similarity between two polynucleotide or polypeptide segments. The correspondence between the sequences of one segment and the sequence of another segment can be determined using techniques known in the art. For example, homology can be determined by aligning the sequence information of two polypeptide molecules and performing a direct comparison using readily available computer programs. Alternatively, homology can be determined by polynucleotide hybridization under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease and determination of the size of the digested fragments.

[0207] The term "substitution," in the context of amino acid sequences, refers to a variation in which one amino acid in the sequence is replaced by another. The nomenclature used to indicate amino acid substitution follows a standardized format. For example, in "L50G," "L" indicates that the original amino acid is leucine (abbreviated as "L"), "50" indicates the position of this amino acid relative to its N-terminus in the amino acid sequence (in this example, the 50th amino acid from the N-terminus), and "G" indicates the substituted amino acid, in this example, glycine (abbreviated as "G"). Therefore, "L50G" indicates a substitution in which leucine at position 50 (relative to its N-terminus) in the amino acid sequence has been replaced by glycine.

[0208] As used herein, the term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid with another that shares a common property. One functional way to define the common property among amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins from homologous organisms (see Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analysis, groups of amino acids can be defined such that amino acids within the same group preferentially exchange with each other, and are therefore most similar in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, ibid.). Examples of conserved mutations include amino acid substitutions between amino acids within the aforementioned subgroups, such as lysine replacing arginine and vice versa to maintain a positive charge; glutamic acid replacing aspartic acid and vice versa to maintain a negative charge; serine replacing threonine to maintain free -OH; and glutamine replacing asparagine to maintain free -NH2. Exemplary conserved amino acid substitutions are shown in the table below.

[0209]

[0210] As used herein, an amino acid sequence that differs from a reference amino acid sequence solely through conserved amino acid substitutions is referred to herein as a “conserved substitution variant” of the reference sequence. Given the established knowledge and well-known techniques in the field of protein science, those skilled in the art are fully capable of determining the functional impact of a “conserved substitution variant” relative to the reference amino acid sequence.

[0211] In some embodiments, the functional variant may be a conserved substitution variant of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 100 conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 90 amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 80 amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 70 conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 60 conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 50 conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may differ from the amino acid sequence of the reference protein by 40 or fewer conserved amino acid substitutions. In some embodiments, the conserved substitution variant may have no more than 30 conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 20 conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the conserved substitution variant may have no more than 10 conserved amino acid substitutions compared to the reference sequence. In some embodiments, the conserved substitution variant may differ from the reference sequence by 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conserved amino acid substitution. In some embodiments, the conserved substitution variant may have at least 100 and no more than 150 conserved amino acid substitutions compared to the reference sequence. In some embodiments, the conserved substitution variant may have at least 150 conserved amino acid substitutions compared to the reference sequence.

[0212] An amino acid sequence having at least one nonconservative amino acid substitution compared to a reference amino acid sequence is referred to herein as a “nonconservative substitution variant” of the reference sequence. As used herein, the term “nonconservative amino acid substitution” refers to an amino acid substitution between different groups of amino acids, such as lysine replacing tryptophan, or phenylalanine replacing serine, etc. In this case, it is preferable that the nonconservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The nonconservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased relative to the homologous parent protein. A more detailed discussion of amino acid substitutability can be found, for example, LY Yampolsky and A. Stoltzfus, “The Exchangeability of Amino Acids in Proteins,” Genetics, August 2005, 170(4):1459-1472. Given the established knowledge and well-known techniques in the field of protein science, those skilled in the art are fully capable of determining the functional effects of nonconservative amino acid substitutions in functional variants relative to the reference amino acid sequence.

[0213] In some embodiments, the functional variant has at least one non-conserved amino acid substitution compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant has at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 10 to 20 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 21 to 30 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 31 to 40 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 41 to 50 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 51 to 60 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 61 to 70 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 71 to 80 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 81 to 90 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have 91 to 100 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence. In some embodiments, the functional variant may have at least 100 non-conserved amino acid substitutions compared to the amino acid sequence of the reference sequence.

[0214] As used herein, the term "antibody" refers to either a monoclonal antibody or a polyclonal antibody. Specifically, a "monoclonal antibody" is an antibody produced by a single B cell clone and binding to the same epitope. In contrast, a "polyclonal antibody" is a group of antibodies produced by different B cells and binding to different epitopes of the same antigen. A complete antibody typically consists of four polypeptides: two identical heavy (H) chain polypeptides and two identical light (L) chain polypeptides. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, while each light chain contains an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains together form the antigen-binding site of the antibody. The VH and VL regions have similar general structures, each containing four framework regions whose sequences are relatively conserved. These framework regions are connected by three complementarity-determining regions (CDRs). These three CDRs, referred to as CDR1, CDR2, and CDR3, form the "hypervariate region" of the antibody, which is responsible for antigen binding.

[0215] As used herein, the terms “functional antibody fragment” and “functional fragment of an antibody” and their grammatical equivalents are used interchangeably to refer to an antibody portion, fragment, or segment that retains the essential functional characteristics or activity of the original antibody. In one embodiment, this activity is the ability to specifically bind to an antigen. (See, for example, Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). The functional antibody fragment may, for example, comprise one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Non-limiting examples of functional antibody fragments include: (i) antigen-binding fragments (Fab), which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bonds in a stem region; (iii) variable fragments (“Fv”), which consist of the VL and VH domains of an antibody single arm; and (iv) single-chain Fv (scFv), which are monovalent molecules consisting of two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that enables the synthesis of the two domains into a single polypeptide chain (see, for example, Bird et al.). Science, 242 : 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85 : 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16 (v) and 778 (1998) are biantibodies, which are dimers of polypeptide chains, wherein each polypeptide chain contains a VH linked to a VL via a peptide linker, the peptide linker being too short to pair between VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimer molecule having two functional antigen-binding sites. Functional antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent 8,603,950.

[0216] As used herein, the term "antibody-like molecule" can, for example, be a member of the immunoglobulin (Ig) superfamily of proteins capable of selectively binding ligands. MHC molecules and T-cell receptors are examples of such molecules. In one embodiment, the antibody-like molecule is a TCR. In one embodiment, the TCR has been modified to enhance its MHC binding affinity.

[0217] As used herein, the terms "antigen recognition unit" or "antigen recognition domain" refer to a molecule or portion thereof that specifically binds to an antigen. In one embodiment, the antigen recognition unit is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0218] As used herein, the term "immune cells" includes dendritic cells, macrophages, neutrophils, mast cells, eosinophils, basophils, natural killer cells, and lymphocytes (such as B cells and T cells).

[0219] As used herein, the term "T cell" or "T lymphocyte" refers to a class of lymphocytes that play a central role in cell-mediated immunity. They are distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of the T cell receptor (TCR) on their cell surface. The TCR is a protein molecule present on the surface of T cells, a class of white blood cells involved in adaptive immune responses. The variable domain of the TCR contains a highly polymorphic loop called the complementarity-determining region (CDR), which is responsible for binding to the major histocompatibility complex (MHC) that presents the peptide. There are two main forms of TCR: αβ TCR and γδ TCR. Both forms consist of two protein chains: an α chain and a β chain for αβ TCR, and a γ chain and a δ chain for γδ TCR. These chains together form a heterodimeric structure. In the human immune system, most T cells express αβ TCR. The α chain and β chain of the αβ TCR are encoded by different gene segments that recombine during T cell development to produce diverse TCR specificities. The α and β chains each contain variable (V), diverse (D), and linked (J) gene segments, similar to antibody gene rearrangement processes. The combination of V, D, and J gene segments confers unique antigen-binding specificity to the αβ TCR. The αβ TCR recognizes antigenic peptides presented against the background of the major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Compared to the αβ TCR, the γδ TCR accounts for a smaller proportion of the immune system but still plays an important role. The γ and δ chains of the γδ TCR are also encoded by different gene segments and undergo recombination during T cell development. The gene rearrangement process of the γδ TCR differs from that of the αβ TCR. γδ T cells typically exhibit tissue-specific distribution and can be found in epithelial tissues such as the skin and intestines. The γδ TCR can recognize a variety of antigens, including certain peptide and non-peptide molecules, independently of MHC presentation. Both the αβ and γδ TCRs are involved in immune surveillance and response, but their functions and specificities differ. αβ TCRs are primarily involved in recognizing peptides presented by major histocompatibility complex (MHC) molecules, while γδ TCRs can have a more diverse range of antigen recognition capabilities.

[0220] TCRs capable of recognizing MHC-antigen complexes and constructs encoding TCRs can be generated and introduced into T cells (called TCR T cells), and can trigger an immune response by utilizing the subsequent TCR-peptide-MHC interaction. Greenbaum et al., Cancer Immunol Res, 1 November 2021; 9(11):1252-1261. There is interest in using TCRs with an affinity for peptide-MHC antigens (type I) above the normal range, termed high-affinity TCRs, to: 1) drive the activity of CD4 helper T cells (lacking CD8 co-receptors), or 2) develop soluble TCRs that directly target cells by attaching “effective” molecules (e.g., antibody Fc regions, toxic drugs, or antibody scFvs, such as anti-CD3 antibodies, to form bispecific proteins) (Ashfield and Jakobsen, IDRugs, 9, 554-9 (2006); Foote and Eisen, Proc Natl Acad Sci USA, 97:10679-81 (2000); Holler et al., Proc Natl Acad Sci USA, 97:5387-92 (2000); Molloy et al., Curr OpinPharmacol, 5:438-43 (2005); Richman and Kranz, Biomol Eng, 24:361-73 (2007)). This method also overcomes a problem faced by some cancer patients whose T cells do not express TCRs with sufficient specificity and binding affinity for potential tumor antigens. For example, more than 300 MHC-restricted, T cell-defined tumor antigens have been identified (Cheever et al., Clin Cancer Res. 2009; 15(17):5323-5337). These tumor antigens include mutant peptides, differentiation antigens, and overexpressed antigens, all of which can serve as therapeutic targets. Since most cancer antigens described to date are derived from intracellular proteins and can only be targeted on the cell surface in the context of MHC molecules, TCRs are ideal therapeutic candidates because they have evolved to recognize such antigens. Similarly, TCRs can detect peptides derived from viral proteins that have been naturally processed in infected cells and displayed on the cell surface by MHC molecules. However, patients with these diseases may not have optimized TCRs to bind to and destroy infected cells. Finally, in highly specific approaches, TCRs can be used as receptor antagonists targeting autoimmune targets, or as delivery vectors to suppress local immune cell responses, thereby avoiding systemic immunosuppression.

[0221] As used herein, the term "helper T cells" (TH or Th cells) assist other white blood cells in the immune process, including promoting the maturation of B cells into plasma cells and memory B cells, and activating cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when MHC class II molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. Once activated, they rapidly divide and secrete small proteins called cytokines, which regulate or assist active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH9, TH17, TH22, or TFH (follicular helper T cells), which secrete different cytokines to promote different types of immune responses. Signals from APCs direct T cells to specific subtypes.

[0222] As used in this article, the terms "cytotoxic T cells" (TC cells or CTLs) or "cytotoxic T lymphocytes" destroy virus-infected and tumor cells and are also associated with transplant rejection. These cells are also called CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. CD8+ cells can be inactivated into an unresponsive state through IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases.

[0223] As used herein, the term "memory T cells" refers to a subset of antigen-specific T cells that persist long after infection has resolved. Upon re-exposure to their corresponding antigens, they rapidly proliferate into a large number of effector T cells, thus providing the immune system with a memory of the previous infection. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells typically express the cell surface proteins CD45RO, CD45RA, and / or CCR7.

[0224] As used herein, the term "regulatory T cells" (Treg cells), formerly known as suppressor T cells, refers to T cells that play a role in maintaining immune tolerance. Their main function is to shut down T cell-mediated immunity at the end of the immune response and to suppress autoreactive T cells that escape during thymic negative selection.

[0225] As used in this article, the term "natural killer T cells" (NKT cells—not to be confused with the natural killer cells of the innate immune system) refers to cells that bridge the adaptive and innate immune systems. Unlike regular T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CD1d. Once activated, these cells can perform functions attributable to T helper (TH) cells and cytotoxic T (TC) cells (i.e., cytokine production and the release of cytolytic / cytokilling molecules). They are also capable of recognizing and eliminating certain tumor cells and cells infected with herpesviruses.

[0226] As used in this article, the term “proliferative disease” refers to a unified concept in which excessive cell proliferation and / or cell matrix renewal make a significant contribution to the pathogenesis of diseases, including cancer.

[0227] As used herein, "patient" or "object" refers to a mammalian object diagnosed or suspected of having or developing a disease or condition (e.g., cancer). In some embodiments, the term "patient" refers to a mammalian object with a higher-than-average likelihood of developing a proliferative condition such as cancer. Exemplary patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents, and other mammals that may benefit from the treatments disclosed herein. Exemplary human patients may be male and / or female. "Patient in need of" or "object in need of" as used herein refers to a patient diagnosed or suspected of having a disease or condition, such as, but not limited to, human papillomavirus (HPV) infection.

[0228] As used herein, “giving” or “administering” refers to providing a patient or subject with one or more of the compositions described herein. For example, and without limitation, the composition may be administered (e.g., by injection) via intravenous, subcutaneous, intradermal, intraperitoneal, or intramuscular injection. One or more of these routes may be used. Parenteral administration may be performed, for example, by bolus injection or gradual perfusion over time. Alternatively or concurrently, administration may be performed via oral route. Furthermore, administration may be performed via surgical deposition or placement / positioning of a medical device. Pharmaceutical compositions may comprise the compositions of the invention described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers (e.g., neutral buffered saline, phosphate buffered saline, etc.); carbohydrates (e.g., glucose, mannose, sucrose, or dextran, mannitol); proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA or glutathione); adjuvants (e.g., aluminum hydroxide); and preservatives.

[0229] As used herein, the term "therapeutic product" refers to a therapeutic peptide or therapeutic polynucleotide that confers beneficial function to a host cell expressing the product. Therapeutic peptides may include, but are not limited to, peptides, single-chain or multi-chain proteins, and fusion proteins with a length as short as three amino acids. Therapeutic polynucleotides may include, but are not limited to, antisense oligonucleotides, small interfering RNA, ribozymes, and RNA external guide sequences. Therapeutic products may comprise naturally occurring sequences, synthetic sequences, or combinations of natural and synthetic sequences.

[0230] As used herein, the terms “treatment,” “under treatment,” or their grammatical equivalents refer to achieving the desired pharmacological and / or physiological effect. In some embodiments, this effect is therapeutic, meaning that it partially or completely cures the disease and / or adverse symptoms or pathological manifestations caused by the disease. To this end, the method of the present invention includes administering a therapeutically effective amount of the composition of the present invention (which expresses the nucleic acid sequence of the present invention) or a vector containing the nucleic acid sequence of the present invention.

[0231] As used herein, a “treatment interval” refers to a treatment cycle, such as a period of administration of a therapeutic agent, which may be repeated, for example, according to a regular schedule. In some embodiments, the dosing regimen may include one or more periods between treatment intervals during which no therapeutic agent is administered.

[0232] As used herein, a “dosing regimen” or “administration regimen” includes a treatment regimen based on a set of determined doses. As used herein, the terms “dosage” and “administration” refer to the administration of a substance to achieve a therapeutic goal (e.g., the treatment of cancer).

[0233] As used herein, the terms "co-administration," "co-dosing," or "co-delivery" refer to the delivery of two (or more) different treatments to a subject during the course of a disease or condition, such as after the subject has been diagnosed with a disease or condition and before the disease or condition is cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, the first treatment is still in progress when the second treatment is started, resulting in overlap in dosing. This is sometimes referred to herein as "simultaneous" or "parallel delivery." In other embodiments, the delivery of the first treatment ends before the delivery of the second treatment begins. In some embodiments of either case, the treatment is more effective due to co-administration. For example, the second treatment is more effective than the second treatment alone in the absence of the first treatment, for example, an equivalent effect can be observed with less of the second treatment, or the second treatment is more significant in symptom reduction, or a similar situation exists for the first treatment. In some embodiments, the delivery method results in a reduction of a symptom or other parameters related to the condition that is greater than the effect observed by delivering one of the treatments alone. The effects of the two treatments can be partially additive, completely additive, or greater than additive. The delivery method can also ensure that the effect of the first treatment can still be detected when the second treatment is delivered.

[0234] In some embodiments of the invention, the first and second treatments may be administered simultaneously (e.g., at the same time), with the same or different compositions, or sequentially. Sequential administration means administering one treatment before administering an additional (e.g., a secondary) treatment (e.g., immediately before; less than 5, 10, 15, 30, 45, or 60 minutes before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96, or more hours before; 4, 5, 6, 7, 8, 9, or more days before; or 1, 2, 3, 4, 5, 6, 7, 8, or more weeks before). The order of administration of the first and secondary treatments may also be reversed.

[0235] The terms “therapeutic effective dose,” “therapeutic dose,” “immunologic effective dose,” “antitumor effective dose,” “tumor-suppressive effective dose,” or their grammatical equivalents, refer to the amount that effectively achieves the desired therapeutic outcome at the required dose and within the required time. Therapeutic effective doses can vary depending on factors such as disease state, individual age, sex, weight, and the ability of the composition described herein to induce the desired response in one or more subjects.

[0236] Alternatively, the pharmacological and / or physiological effects produced when one or more of the compositions described herein are administered to a patient or subject may be "preventive," meaning that the effect completely or partially prevents the disease or its symptoms. "Preventive effective amount" refers to the amount that effectively achieves the desired preventive outcome (e.g., prevention of disease or prevention of the occurrence of a target pathology) at the required dose and within the required time.

[0237] The term "Derkay score" refers to a scoring system used to assess the severity of recurrent respiratory papillomatosis (RRP) in children. The Derkay score is based on factors such as age of onset, frequency of surgery, location of the papilloma, and tracheotomy dependence. It helps clinicians assess the severity of the disease and guide treatment decisions. Higher scores indicate more severe cases requiring more aggressive management. See Hester RP, Derkay CS, Burke BL, Lawson ML: Reliability of a staging assessment system for recurrent respiratory papillomatosis. Int J Pediatr Otorhinolaryngol. 2003;67(5):505-9; Derkay CS: Recurrent respiratory papillomatosis. Laryngoscope. 2001;111(1):57-69.

[0238] I. carrier

[0239] Gene therapy, which involves introducing a transgene (e.g., through vaccination) into a subject to express a foreign protein, has proven useful for treating diseases and conditions in subjects who require it. For such introductions, vectors containing the transgene encoding the protein, such as viral vectors, are typically used.

[0240] This invention relates in part to a vector comprising an expression cassette containing a transgene encoding an HPV antigen.

[0241] In some embodiments, the vector is a plasmid.

[0242] Another suitable vector is the integrative expression vector. These vectors can randomly integrate into the host cell's DNA, or may contain recombination sites to achieve specific recombination between the expression vector and the host cell's chromosome. Such integrative expression vectors can utilize the endogenous expression control sequences of the host cell's chromosome to achieve the expression of the desired protein. Examples of site-specific integration vectors include components of Invitrogen's (Carlsbad, California) flp-in system (e.g., pcDNA). TM5 / FRT), or cre-lox systems, such as the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that randomly integrate into the host cell chromosome include pcDNA3.1 from Ingenie (Carlsbad, CA) (when introduced in the absence of T antigen), and pCI or pFN10A(ACT)FLEXITM from Promega (Madison, Wisconsin).

[0243] A. Methods for introducing nucleic acids into cells

[0244] Methods of introducing and expressing genes in cells are well known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0245] 1. physical methods

[0246] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory, New York (2001). In some embodiments, the method for introducing polynucleotides into host cells is calcium phosphate transfection or polyethyleneimine (PEI) transfection.

[0247] In some embodiments, the method of introducing polynucleotides into host cells is electroporation. Electroporation is a technique that uses electrical pulses to temporarily increase cell membrane permeability, thereby allowing nucleic acid molecules to be taken up by the cells. This process enhances the delivery and expression of biological materials (such as peptides or nucleic acids) in the target cells, potentially improving the immune response against HPV. In some embodiments, the biological material is an HPV antigen. In other embodiments, the biological material is a nucleic acid encoding an HPV antigen.

[0248] Electroporation buffers may contain water, sugars, sugar alcohols, chloride salts, and buffers. The pH, conductivity, and osmotic pressure of the buffer are carefully controlled. The buffer is compatible with the UltraPorator™ electroporation device and cartridge. The UltraPorator™ electroporation device is designed for the rapid fabrication of gene and cell therapies and serves as a scale-up and commercialization solution for dispersed cell fabrication. See, for example, PCT / US20 / 59984 (filed November 11, 2020) and U.S. Patent Application Serial No. 17 / 095,028 (filed November 11, 2020).

[0249] In some embodiments, a suspension is formed by combining human-derived cells with exogenous biological material in a buffer solution, and then an electric current is applied to the suspension to facilitate the delivery of the biological material into the cells. The voltage pulse may have a field strength of 1-10 kV / cm, a duration of 5-250 μs, and at least 2 A / cm. 2 The current density. This method can be used to introduce biological materials (e.g., nucleic acids, peptides, polypeptides, proteins, enzymes, or RNPs) into primary human blood cells, pluripotent precursor cells, fibroblasts, and endothelial cells. In some embodiments, the method is used to introduce bioactive materials into primary human blood cells, human blood pluripotent precursor cells, and primary human fibroblasts and endothelial cells. In some embodiments, the cells are human blood cells, such as immune cells. In some embodiments, the immune cells are neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells), or some combination thereof. In some embodiments, the lymphocytes are T cells. In some embodiments, the cells are taken from a patient.

[0250] In some embodiments, the transfection yield and transfected cell recovery yield using the electroporation buffer are significantly higher than those obtained using the control buffer. In some embodiments, the transfection yield using the buffer of the present invention is at least approximately 1.1 times that of the transfection yield using the control buffer, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 times higher.

[0251] In some of the methods described herein, HPV antigens are administered to a subject. In some aspects, these methods include introducing the nucleic acid molecules of the present invention into the subject, followed by electroporation. In one specific embodiment, a nucleic acid molecule (e.g., a plasmid encoding an antigen of interest or a therapeutic protein) is injected into a target tissue of the subject, such as skin or muscle, using a conventional needle or needle-free injection device. Shortly after injection, a handheld electroporation device is applied to the injection site, for example, by contact with the skin or tissue. The device delivers brief, controlled electrical pulses to the tissue, thereby creating transient pores in the cell membrane, making the cell membrane more permeable to the nucleic acid molecule, which then enters the cell through the pores created by electroporation. Once inside the cell, the nucleic acid molecule is translated into the desired antigen or therapeutic protein. The resulting antigen stimulates an immune response, thereby providing protection against the targeted pathogen. In the case of a therapeutic protein, it can exert its intended effect within the cell or tissue.

[0252] In some embodiments, the method may involve administering multiple copies of a single nucleic acid molecule, such as a single plasmid, or administering multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. The number of different nucleic acid molecules administered may vary depending on the specific application and may include two, three, four, five, six, seven, eight, nine, ten, or more different nucleic acid sequences. This method allows for the delivery of multiple HPV antigens or the co-delivery of additional immunostimulatory factors to enhance the immune response.

[0253] Genetic constructs containing nucleic acids encoding HPV antigens can be administered using a variety of methods, including electroporation devices, conventional syringes, standard needles, side-hole needles (as described in U.S. Publication No. 2023 / 0017972), needle-free injection devices, or "microparticle bombardment gene guns." Each of these methods has its advantages and can be selected based on factors such as target tissue, desired gene expression levels, and specific applications.

[0254] Several minimally invasive electroporation devices and methods have been described in the literature. These include devices and methods disclosed in U.S. Patent Application No. 20080234655; U.S. Patent Nos. 6,520,950; 7,171,264; 6,208,893; 6,009,347; 6,120,493; 7,245,963; 7,328,064; 6,763,264; and US200240123052. These devices and methods are designed to efficiently deliver nucleic acid molecules into cells while minimizing tissue damage and patient discomfort. By using these minimally invasive electroporation techniques, nucleic acids encoding HPV antigens can be effectively introduced into the patient's cells, leading to the production of HPV antigens and stimulating an immune response.

[0255] 2. Chemical methods

[0256] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0257] B. Virus-based delivery systems

[0258] This article also provides virus-based delivery systems, such as viral vectors for delivering nucleic acids. Exemplary viral vectors include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, and herpesvirus-based vectors. Viral vectors can be used as delivery vehicles for nucleic acids encoding therapeutic molecules (e.g., anti-inflammatory agents) while avoiding immune surveillance by host cells. Retroviruses, adenoviruses, adeno-associated viruses (AAVs), and herpes simplex viruses have all been engineered for viral vector applications. (Robins et al., Pharmacology & Therapeutics, 80:35-47 (1998)). Specifically, recombinant adenovirus vectors provide high levels of transgenic expression, where the vector exists as free DNA without integrating into the host genome. High transduction efficiency and high levels of short-term gene expression make adenovirus vectors ideal for gene therapy and vaccine applications. Furthermore, these viral vectors can be made replication-deficient by deleting essential viral genes and replacing them with expression cassettes containing exogenous therapeutic genes.

[0259] When used in gene therapy, it is desirable to use viral vectors that are non-infectious to humans or engineered to remove or inactivate their infectious characteristics, because they are highly efficient in delivering transgenes and can deliver high loads of nucleic acids to dendritic cells.

[0260] However, the effectiveness of treatment using viral vectors is limited by their immunogenicity. For example, human adenovirus vectors are commonly used in gene therapy; however, because a large portion of the US population has been exposed to the wild-type form of such viruses, a significant portion of the population has existing immunity to them. Therefore, these vectors and the transgenes they carry are rapidly cleared from the bloodstream. Furthermore, the immunogenicity of these vectors limits their effectiveness in repeated administration scenarios.

[0261] 1. Retroviral vector

[0262] In some embodiments, the viral vector is a retroviral vector, such as a lentiviral vector. Vectors derived from retroviruses are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of transgenes and their delivery in daughter cells. Compared to vectors derived from oncogenic retroviruses (e.g., murine leukemia virus), lentiviral vectors have the added advantage of being able to transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0263] 2. Adeno-associated virus vector

[0264] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. Such vectors are derived from adeno-associated viruses. One advantage of using such vectors is their low immunogenicity in humans. Another advantage is their small and compact size, making them efficient for gene delivery to cells. However, their size also limits their payload capacity compared to adenovirus vectors. They are also more difficult to produce than adenovirus vectors. Furthermore, they exhibit narrow tissue tropism.

[0265] 3. Vectors based on herpes simplex virus

[0266] In some implementations, the viral vector is a herpesvirus-based vector. Such vectors are derived from herpes simplex virus (HSV). These vectors are known to infect multiple cell types and persist long-term in the host. However, they are more difficult to engineer than adenovirus vectors.

[0267] 4. Adenovirus-based vectors

[0268] In some embodiments, the viral vector is an adenovirus vector. Such vectors are derived from adenoviruses, such as human adenoviruses (e.g., human Ad5 adenovirus), avian adenoviruses, or chimpanzee adenoviruses. Adenoviruses are commonly associated with benign pathologies in humans, and adenovirus genomes isolated from multiple species, including humans, have been extensively studied. Adenovirus vectors are advantageous because they can infect a variety of cell types. They are also relatively easy to engineer and can carry high payloads.

[0269] Adenovirus vectors can be produced at high titers and efficiently transfer DNA to replicating and non-replicating cells. Adenovirus vector genomes can be generated using any species, strain, subtype, mixture of species / strain / subtype, or chimeric adenovirus as the vector DNA source. Adenovirus libraries that can be used as a source of adenovirus can be obtained by amplification of adenoviruses of serotypes 1 through 51, currently available from the American Type Culture Collection (ATCC, Manassas, Virginia), or from any other adenovirus serotype available from any other source. For example, adenoviruses can belong to subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenovirus serotype.

[0270] The adenovirus vector can be any adenovirus vector capable of growing in cells, which is derived to some extent (though not necessarily substantially) from or based on the genome of an adenovirus. The adenovirus vector can be based on the genome of any suitable wild-type adenovirus. In some embodiments, the adenovirus vector is derived from the genome of a group C wild-type adenovirus, particularly serotype 2 or 5. Adenoviral vectors are well known in the art, for example, as described in U.S. Patent Nos. 5,559,099, 5,712,136, 5,731,190, 5,837,511, 5,846,782, 5,851,806, 5,962,311, 5,965,541, 5,981,225, 5,994,106, 6,020,191, and 6,113,913; International Patent Applications WO 95 / 34671, WO 97 / 21826, and WO 00 / 00628; Thomas Shenk, “Adenoviridae and their Replication”; and M.S. Horwitz, Adenoviruses, Chapters 67 and 68, respectively, in Virology, BN Fields et al., eds., 3rd edition, Raven Press Ltd. Press, Ltd., New York (1996).

[0271] Adenoviruses are medium-sized (90-100 nm), non-enveloped icosahedral viruses containing approximately 36 kb of double-stranded DNA. The adenoviral capsid mediates key interactions in the early stages of viral infection of cells and is essential for packaging the adenoviral genome at the end of the adenoviral life cycle. The capsid contains 252 capsid particles, comprising 240 hexons, 12 penton-based proteins, and 12 fibers. (Ginsberg et al., Virology, 28: 782-783 (1966)). The hexons contain three identical proteins, namely polypeptide II. (Roberts et al., Science, 232: 1148-1151 (1986)). The penton-based proteins contain five identical proteins, and the fibers contain three identical proteins. Proteins IIIa, VI, and IX are present in the adenoviral capsid and are believed to stabilize the viral capsid. Stewart et al., Cell, 67: 145-54 (1991) and Stewart et al., EMBO J., 12(7): 2589-99 (1993). Except for pIX, the expression of capsid proteins depends on adenovirus polymerase proteins. Therefore, the major components of adenovirus particles are expressed by the genome only when the polymerase protein gene is present and expressed.

[0272] Several characteristics of adenoviruses make them an ideal delivery vehicle for transferring genetic material into cells for therapeutic applications. For example, adenoviruses can be produced at high titers (e.g., about 10). 13 Adenoviruses can transfer genetic material into both non-replicating and replicating cells. Furthermore, the adenovirus genome can be manipulated to carry large amounts of exogenous DNA (up to approximately 8 kb), and the adenovirus capsid can facilitate the transfer of even longer sequences. (Curiel et al., Hum. Gene Ther., 3: 147-154 (1992)). Additionally, adenoviruses typically do not integrate into the host cell chromosome but are maintained as linear, free-floating entities, thus minimizing the possibility of recombinant adenoviruses interfering with normal cellular function.

[0273] The adenovirus may be modified, for example, using methods known in the art, to be used as an adenovirus vector, such as a gene delivery vehicle. The adenovirus and adenovirus vector may be replication-competent, conditionally replication-competent, or replication-deficient.

[0274] Replicating adenoviruses or adenovirus vectors can replicate in typical host cells, i.e., cells that are normally susceptible to adenovirus infection. Replicating adenoviruses or adenovirus vectors may have one or more mutations relative to wild-type adenoviruses (e.g., one or more deletions, insertions, and / or substitutions), said mutations not inhibiting viral replication in host cells. For example, the adenovirus or adenovirus vector may have partial or complete deletions in an early adenovirus region called the E3 region, which is not essential for the replication of the adenovirus or adenovirus genome.

[0275] Conditionally replicating adenoviruses or adenovirus vectors are adenoviruses or adenovirus vectors engineered to replicate under predetermined conditions. For example, gene functions essential for replication, such as those encoded by the early region of the adenovirus, may be operatively linked to inducible, repressible, or tissue-specific transcriptional control sequences (e.g., promoters). In this implementation, replication requires the presence or absence of specific factors that interact with said transcriptional control sequences. Conditionally replicating adenovirus vectors are further described in U.S. Patent No. 5,998,205.

[0276] A replication-defective adenovirus or adenovirus vector is an adenovirus or adenovirus vector that requires complementation of one or more gene functions or regions of the adenovirus genome necessary for replication, such that the adenovirus or adenovirus vector does not replicate in typical host cells, particularly in human cells that can be infected by the adenovirus or adenovirus vector, for example due to defects in one or more gene functions or regions necessary for replication.

[0277] As used herein, a “defect” in a gene function or genomic region is defined as a disruption (e.g., deletion) of sufficient genetic material in the adenovirus genome to eliminate or weaken the function of the gene (e.g., reducing the function of the gene product by at least about 2, 5, 10, 20, 30, or 50-fold), wherein the nucleic acid sequence of the gene is completely or partially disrupted (e.g., deleted). Disruption of replication-essential gene function typically does not require the deletion of an entire gene region. However, to provide sufficient space in the adenovirus genome for one or more transgenes, it may be desirable to remove a large portion of one or more gene regions. While deletion of genetic material is preferred, mutations in the genetic material through addition or substitution are also suitable for disrupting gene function. Replication-essential gene functions refer to those gene functions necessary for adenovirus replication (e.g., proliferation) and are encoded, for example, by early adenovirus regions (e.g., E1, E2, and E4 regions), late regions (e.g., L1, L2, L3, L4, and L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA-1 and / or VA-RNA-2).

[0278] Regardless of whether the adenovirus or adenovirus vector is replicative or replication-deficient, it typically retains at least a portion of the adenovirus genome. The adenovirus or adenovirus vector may contain any portion of the adenovirus genome, including protein-coding and / or non-protein-coding regions. For example, the adenovirus or adenovirus vector may contain at least one nucleic acid sequence encoding an adenovirus protein. The adenovirus or adenovirus vector may contain a nucleic acid sequence encoding any suitable adenovirus protein, such as a nucleic acid sequence encoding a protein encoded by any early region gene (i.e., regions E1A, E1B, E2A, E2B, E3, and / or E4), or a nucleic acid sequence encoding a protein encoded by any late region gene (which encodes viral structural proteins, i.e., regions L1, L2, L3, L4, and L5).

[0279] It should be understood that the deletion of different regions of an adenovirus vector can alter the immune response in mammals. Specifically, the deletion of different regions can reduce the inflammatory response induced by the adenovirus vector. Furthermore, as described in International Patent Application WO 98 / 40509, the capsid protein of the adenovirus vector can be modified to reduce the ability or susceptibility of the adenovirus vector to be recognized by neutralizing antibodies against the wild-type capsid protein.

[0280] In some embodiments, the adenovirus or adenovirus vector comprises one or more nucleic acid sequences encoding a pIX protein, a DNA polymerase protein, a pentazocine protein, a hexazocine protein, and / or fibrin. The adenovirus or adenovirus vector may comprise a full-length nucleic acid sequence encoding the full-length amino acid sequence of an adenovirus protein. Alternatively, the adenovirus or adenovirus vector may comprise a portion of a full-length nucleic acid sequence encoding a fraction of the full-length amino acid sequence of an adenovirus protein. The “fraction” of the amino acid sequence comprises at least three amino acids (e.g., about 3 to about 1,200 amino acids). Preferably, the “part” of the amino acid sequence contains 3 or more (e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more) amino acids, but less than 1,200 (e.g., 1,000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less) amino acids. Preferably, a portion of the amino acid sequence comprises about 3 to about 500 amino acids (e.g., about 10, 100, 200, 300, 400, or 500 amino acids), about 3 to about 300 amino acids (e.g., about 20, 50, 75, 95, 150, 175, or 200 amino acids), or about 3 to about 100 amino acids (e.g., about 15, 25, 35, 40, 45, 60, 65, 70, 80, 85, 90, 95, or 99 amino acids), or a range defined by any two of the foregoing values. More preferably, a “portion” of the amino acid sequence comprises no more than about 500 amino acids (e.g., about 3 to about 400 amino acids, about 10 to about 250 amino acids, or about 50 to about 100 amino acids, or a range defined by any two of the foregoing values).

[0281] The adenoviral pIX protein, located in the adenoviral capsid, has been shown to enhance hexa-nenomilleric interactions and is essential for packaging the full-length genome. See, for example, Boulanger et al., J Gen. Virol., 44: 783-800 (1979); Horwitz MS, “Adenoviridae and their replication”, Virology, 2nd ed., BN Fields et al., Raven Press, Ltd., New York, pp. 1679-1721 (1990); Ghosh-Choudhury et al., EMBO J., 6: 1733-1739 (1987); and van Oostrum et al., J. Virol., 56: 439-448 (1985). In addition to its contribution to adenoviral structure, pIX has also been shown to possess transcriptional properties, such as stimulating the activity of the major late promoter (MLP) of adenovirus. For example, see Lutz et al., J.Virol., 71(7): 5102-5109 (1997). For example, nucleic acid sequences encoding all or part of the adenovirus pIX protein are described in WO 2019 / 173465 and WO 2022 / 115470.

[0282] Adenoviral DNA polymerase protein is essential for viral DNA replication both in vitro and in vivo. This polymerase is co-purified in a complex with a precursor of terminal protein (TP) (pTP), which is covalently linked to the 5′ end of adenoviral DNA. (Field et al., J. Biol. Chem., 259: 9487-9495 (1984)). Both adenoviral DNA polymerase and pTP are encoded by the E2 region. Except for pIX, the polymerase protein is essential for the expression of all structural proteins. Without the gene sequence for the polymerase protein, it is not produced. As a result, the viral genome does not replicate, the major late promoter is not activated, and the capsid protein is not expressed. Nucleic acid sequences encoding all or part of the adenoviral DNA polymerase protein are described, for example, in WO 2019 / 173465 and WO 2022 / 115470.

[0283] Hexagonal proteins are the largest and most abundant proteins in the adenovirus capsid. They are essential for capsid assembly, defining the icosahedral symmetry of the capsid (which in turn defines the limits of capsid volume and DNA packaging size), and capsid integrity. Furthermore, hexagonal proteins are a major target for modification to reduce the neutralization of adenovirus vectors. See, for example, Gall et al., J. Virol., 72: 10260-264 (1998), and Rux et al., J. Virol., 77(17): 9553-9566 (2003). The main structural features of hexagonal proteins are shared among different serotypes of adenovirus, but differences exist in size and immunological properties between serotypes. Jornvall et al., J. Biol. Chem., 256(12): 6181-6186 (1981). A comparison of 15 adenovirus hexagonal proteins revealed that the major antigenic and serotype-specific regions of the hexagonal proteins appear to be located in loops 1 and 2 (LI or l1, and LII or l2, respectively), containing seven discrete hypervariable regions (HVR1 to HVR7) whose length and sequence vary between adenovirus serotypes. (Crawford-Miksza et al., J. Virol., 70(3): 1836-1844 (1996)). Nucleic acid sequences encoding all or part of the adenovirus hexagonal proteins are described, for example, in WO 2019 / 173465 and WO 2022 / 115470.

[0284] Adenoviral fibrin is a homotrimer of adenoviral polypeptide IV, possessing three domains: tail, shaft, and knot. Devaux et al., J. Molec. Biol., 215: 567-88 (1990); Yeh et al., Virus Res., 33: 179-98 (1991). Fibrin mediates the initial binding of the virus to cell surface receptors via the knot and shaft domains. Henry et al., J. Virol., 68(8): 5239-46 (1994). The amino acid sequence for trimerization is located in the knot, which appears to be essential for the proper binding of the fiber's amino terminus (tail) to the penton base. Novelli et al., Virology, 185: 365-76 (1991). In addition to recognizing cell receptors and binding to the penton base, the fiber also contributes to serotype specificity. The fibrin proteins of different adenovirus serotypes vary considerably. See, for example, Green et al., EMBO J., 2: 1357-65 (1983), Chroboczek et al., Virology, 186: 280-85 (1992), and Signas et al., J. Virol., 53: 672-78 (1985). Therefore, fibrin proteins play several key roles in the adenovirus life cycle. For instance, nucleic acid sequences encoding all or part of the adenovirus fibrin proteins are described in WO 2019 / 173465 and WO 2022 / 115470.

[0285] The adenoviral pentagonal base protein is located at the vertices of the icosahedral capsid and comprises five identical monomers. The pentagonal base protein provides a structure for bridging hexagonal proteins on multiple faces of the icosahedral capsid and provides a crucial interface necessary for the incorporation of fibrin into the capsid. Each monomer of the pentagonal base protein contains an RGD tripeptide motif. Neumann et al., Gene, 69: 153-157 (1988). The RGD tripeptide mediates binding to αv integrins, and adenoviruses with point mutations in the RGD sequence of the pentagonal base protein are limited in their ability to infect cells. Bai et al., J. Virol., 67:5198-5205 (1993). Therefore, the pentagonal base protein is essential for the capsid architecture and for maximizing the efficiency of virus-cell interactions. Nucleic acid sequences encoding all or part of the adenoviral pentagonal base protein are described, for example, in WO 2019 / 173465 and WO 2022 / 115470.

[0286] The adenovirus or adenovirus vector may contain one, two, three, four, or all five of the above sequences, either alone or in any combination. In this respect, the adenovirus or adenovirus vector may contain any combination of any two of the above sequences, any combination of any three of the above sequences, any combination of any four of the above sequences, or all five of the above sequences.

[0287] In some embodiments, the adenovirus or adenovirus vector is replication-deficient, such that the replication-deficient adenovirus or adenovirus vector requires complementation of at least one replication-essential gene function in one or more regions of the adenovirus genome to achieve proliferation (e.g., formation of adenovirus vector particles).

[0288] The replication-defective adenovirus or adenovirus vector may be modified in any suitable manner to create a defect for replication in the function of one or more replication-essential genes in one or more regions of the adenovirus genome. Complementation of defects in the function of one or more replication-essential genes in one or more regions of the adenovirus genome refers to providing the missing replication-essential gene function exogenously. Such complementation may be achieved in any suitable manner, for example by using exogenous DNA that complements the cell and / or encodes the disrupted replication-essential gene function (e.g., helper adenovirus).

[0289] In some embodiments, the adenovirus or adenovirus vector is defective in the function of one or more replication-essential genes only in the early region (i.e., E1-E4 region) of the adenovirus genome, in the function of one or more replication-essential genes only in the late region (i.e., L1-L5 region) of the adenovirus genome, in both the early and late regions of the adenovirus genome, or in all adenovirus genes (i.e., high-load adenovirus vector (HC-Ad)). See Morsy et al., Proc. Natl. Acad. Sci. USA, 95: 965-976 (1998); Chen et al., Proc. Natl. Acad. Sci. USA, 94: 1645-1650 (1997); and Kochanek et al., Hum. Gene Ther., 10: 2451-2459 (1999). The adenoviral vector can also remove substantially the entire adenoviral genome, in which case at least viral inverted terminal repeats (ITRs) and one or more promoters are preserved, or viral ITRs and packaging signals (i.e., adenoviral amplicon) are preserved intact. The larger the region of the adenoviral genome removed, the larger the foreign nucleic acid sequence fragment that can be inserted into the genome. For example, given that the adenoviral genome is 36 kb, by preserving the viral ITRs and one or more promoters intact, the adenoviral foreign insertion capacity is approximately 35 kb. Alternatively, a multi-deficient adenoviral vector containing only ITRs and packaging signals can effectively allow the insertion of foreign nucleic acid sequences of approximately 37-38 kb. Of course, including spacer elements in any or all of the deficient adenoviral regions will reduce the adenoviral vector's capacity for large inserts.

[0290] In some embodiments, the adenovirus vector is "multiple defective," meaning that the adenovirus vector is defective in one or more regions of the adenovirus genome in terms of the function of one or more genes essential for viral replication. For example, the aforementioned E1-deficient or E1 / E3-deficient adenovirus vector may further be defective in at least one replication-essential gene function in the E4 region (referred to as an E1 / E4-deficient adenovirus vector). Adenovirus vectors lacking the entire E4 region may elicit a lower host immune response.

[0291] Examples of replication-defective adenovirus vectors are disclosed in U.S. Patent Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, as well as international patent applications WO 1994 / 028152, WO1995 / 002697, WO 1995 / 016772, WO 1995 / 034671, WO 1996 / 022378, WO 1997 / 012986, WO1997 / 021826, and WO 2003 / 022311.

[0292] The early regions of the adenovirus genome include regions E1, E2, E3, and E4. Region E1 contains subregions E1A and E1B, and defects in the function of one or more replication-essential genes within region E1 may include defects in the function of one or more replication-essential genes in either or both of the E1A and E1B subregions, thus requiring complementation of the E1A and / or E1B subregions of the adenovirus genome to enable the adenovirus or adenovirus vector to proliferate (e.g., form adenovirus vector particles). Region E2 contains subregions E2A and E2B, and defects in the function of one or more replication-essential genes within region E2 may include defects in the function of one or more replication-essential genes in either or both of the E2A and E2B subregions, thus requiring complementation of the E2A and / or E2B subregions of the adenovirus genome to enable the adenovirus or adenovirus vector to proliferate (e.g., form adenovirus vector particles).

[0293] The E3 region does not contain any gene functions essential for replication; therefore, partial or complete deletion of the E3 region does not require complementation of any gene function within the E3 region to enable the adenovirus or adenovirus vector to proliferate (e.g., form adenovirus vector particles). In the context of this disclosure, the E3 region is defined as an open reading frame (OPF) starting at a protein encoding a 12.5K protein highly homologous to the E3 region of human adenovirus 5 (NCBI reference sequence AP_000218) and terminating at an OPF encoding a 14.7K protein highly homologous to the E3 region of human adenovirus 5 (NCBI reference sequence AP_000224.1). The E3 region may be completely or partially deleted, or completely or partially retained. The size of the deletion can be customized to preserve the genome of the adenovirus or adenovirus vector that closely matches the optimal genome packaging size. Larger deletions will accommodate larger heterologous nucleic acid sequences inserted into the adenovirus or adenovirus genome. In some embodiments of this disclosure, the L4 polyadenylation signal sequence located within the E3 region is retained.

[0294] The E4 region contains multiple open reading frames (ORFs). Adenoviruses or adenovirus vectors lacking all open reading frames in the E4 region except ORF6, and in some cases except ORF3, do not require complementation of any gene function in the E4 region to enable the adenovirus or adenovirus vector to proliferate (e.g., form adenovirus vector particles). Conversely, adenoviruses or adenovirus vectors with ORF6 in the E4 region, and in some cases with ORF3 disrupted or missing (e.g., defects in replication-essential gene function in ORF6 and / or ORF3 in the E4 region), regardless of whether accompanied by disruption or loss of any other open reading frames in the E4 region or the native E4 promoter, polyadenylated sequences, and / or right inverted terminal repeat (ITR) sequences, require complementation of the E4 region (specifically ORF6 and / or ORF3 in the E4 region) to enable the adenovirus or adenovirus vector to proliferate (e.g., form adenovirus vector particles).

[0295] The late regions of the adenovirus genome include L1, L2, L3, L4, and L5 regions. The adenovirus or adenovirus vector may also have mutations in the major late promoter (MLP), as discussed in International Patent Application Publication WO 2000 / 000628, which can make the adenovirus or adenovirus vector replication-defective if needed.

[0296] The adenovirus genome contains one or more regions with defective functions of one or more replication-essential genes, preferably one or more early regions of the adenovirus genome, namely E1, E2, and / or E4 regions. Therefore, in some embodiments, the adenovirus vector lacks all or part of such regions.

[0297] The replication-deficient adenovirus or adenovirus vector may also have one or more mutations in the adenovirus genome that do not inhibit viral replication in the host cell (e.g., one or more deletions, insertions, and / or substitutions) relative to the wild-type adenovirus. Therefore, in addition to defects in one or more replication-essential genes, the adenovirus or adenovirus vector may also be defective in other non-replication-essential aspects. For example, the adenovirus or adenovirus vector may have a partial or complete deletion of the E3 region, an early region of the adenovirus that is not essential for the replication of the adenovirus or adenovirus genome.

[0298] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most complementation of the E1 or E4 region of the adenovirus genome for propagation (e.g., formation of adenovirus vector particles). In some such embodiments, the adenovirus vector may lack all or part of the E1 and / or E4 regions. Therefore, the replication-deficient adenovirus or adenovirus vector requires complementation of at least one replication-essential gene function in the E1A subregion and / or E1B region of the adenovirus genome (referred to as an E1-deficient adenovirus vector), or complementation of the E4 region of the adenovirus genome (referred to as an E4-deficient adenovirus vector) to achieve propagation (e.g., formation of adenovirus vector particles). The adenovirus or adenovirus vector may be defective in at least one replication-essential gene function (preferably all replication-essential gene functions) in the E1 region of the adenovirus genome, and defective in at least one gene function in the non-essential E3 region of the adenovirus genome (referred to as an E1 / E3-deficient adenovirus vector). The adenovirus or adenovirus vector may be defective in at least one replication-essential gene function (preferably all replication-essential gene functions) in the E4 region of the adenovirus genome, and defective in at least one gene function in the non-essential E3 region of the adenovirus genome (referred to as an E3 / E4 defective adenovirus vector).

[0299] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most complementation of the E2 region, preferably the E2A subregion, of the adenovirus genome for propagation (e.g., formation of adenovirus vector particles). Therefore, the replication-deficient adenovirus or adenovirus vector requires complementation of at least one replication-essential gene function in the E2A subregion of the adenovirus genome (referred to as an E2A-deficient adenovirus vector) to achieve propagation (e.g., formation of adenovirus vector particles). The adenovirus or adenovirus vector may be defective in at least one replication-essential gene function (preferably all replication-essential gene functions) in the E2A region of the adenovirus genome and defective in at least one gene function in the non-essential E3 region of the adenovirus genome (referred to as an E2A / E3-deficient adenovirus vector).

[0300] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most complementation of the E1 and E4 regions of the adenovirus genome for propagation (e.g., formation of adenovirus vector particles). In some such embodiments, the adenovirus vector may lack all or part of the E1 and / or E4 regions. Therefore, the replication-deficient adenovirus or adenovirus vector requires complementation of at least one replication-essential gene function in both the E1 and E4 regions of the adenovirus genome (referred to as an E1 / E4-deficient adenovirus vector) to achieve propagation (e.g., formation of adenovirus vector particles). The adenovirus or adenovirus vector may be deficient in at least one replication-essential gene function (preferably all replication-essential gene functions) in the E1 region of the adenovirus genome, deficient in at least one replication-essential gene function in the E4 region of the adenovirus genome, and deficient in at least one gene function in the non-essential E3 region of the adenovirus genome (referred to as an E1 / E3 / E4-deficient adenovirus vector). Preferably, the adenovirus or adenovirus vector requires at most complementation of the E1 region of the adenovirus genome for propagation and does not require complementation of any other defects in the adenovirus genome for propagation. More preferably, the adenovirus or adenovirus vector requires at most the complementation of the E1 and E4 regions of the adenovirus genome for propagation, and does not require the complementation of any other defects in the adenovirus genome for propagation.

[0301] When the adenovirus or adenovirus vector is defective in the function of multiple replication-essential genes in the adenovirus genome (e.g., E1 / E4-deficient adenovirus vector), it may include a spacer sequence to provide similar viral growth in complement cell lines as achieved by adenoviruses or adenovirus vectors defective in the function of a single replication-essential gene (e.g., E1-deficient adenovirus vector). The spacer sequence may comprise a nucleotide sequence of any desired length, such as a sequence of at least about 15 base pairs (e.g., between about 15 and about 12,000 nucleotides), preferably about 100 to about 10,000 nucleotides, more preferably about 500 to about 8,000 nucleotides, even more preferably about 1,500 to about 6,000 nucleotides, and most preferably about 2,000 to about 3,000 nucleotides, or a range defined by any two of the foregoing values. The spacer sequence may be coding or non-coding, and may be natural or non-natural relative to the adenovirus genome, but it does not restore the replication-essential function of the defective region. The spacer may also include an expression cassette. More preferably, the spacer includes a polyadenylated sequence and / or a gene that is non-natural relative to the adenovirus or adenovirus vector. Further description of the use of spacers in adenovirus vectors can be found, for example, in U.S. Patent No. 5,851,806 and International Patent Application Publication WO 1997 / 021826.

[0302] By removing all or part of the adenovirus genome, such as the E1, E3, and E4 regions of the adenovirus genome, the resulting adenovirus or adenovirus vector can accept the insertion of exogenous nucleic acid sequences while retaining its ability to be packaged into the adenovirus capsid. The exogenous nucleic acid sequence can be inserted at any location in the adenovirus genome, provided that the insertion allows for the formation of adenovirus or adenovirus vector particles. The exogenous nucleic acid sequence is preferably located in the E1, E3, or E4 regions of the adenovirus genome.

[0303] The replication-defective adenovirus or adenovirus vector disclosed herein can be prepared in complement cell lines that provide at appropriate levels the genetic functions absent in the replication-defective adenovirus or adenovirus vector but necessary for viral replication, thereby producing a high-titer stock solution of viral vector. Such complement cell lines are known and include human embryonic kidney (HEK) 293 cells (e.g., described in Graham et al., J. Gen. Virol., 36: 59-72 (1977)), PER.C6 cells (e.g., described in International Patent Application Publication WO 1997 / 000326 and U.S. Patent Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (e.g., described in International Patent Application Publication WO 95 / 34671 and Brough et al., J. Virol., 71:9206-9213 (1997)). Other suitable complement cell lines for preparing the replication-defective adenovirus or adenovirus vector of this disclosure include complement cells that have been generated for propagating adenovirus vectors encoding transgenes that express an inhibitory effect on viral growth in host cells (see, for example, U.S. Patent Application Publication 2008 / 0233650). Other suitable complement cells are described, for example, in U.S. Patent Nos. 6,677,156 and 6,682,929 and International Patent Application Publication WO 2003 / 020879.

[0304] In some cases, the cell genome need not contain nucleic acid sequences whose gene products can complement all the defects of the replication-defective adenovirus vector. The missing functions of one or more replication-essential genes in the replication-defective adenovirus vector can be provided by a helper virus, for example, by an adenovirus vector that provides the functions of one or more essential genes necessary for replication of the replication-defective adenovirus or adenovirus vector in a trans-trans manner. Alternatively, the adenovirus or adenovirus vector of the present invention may contain a non-natural replication-essential gene that complements the functions of one or more replication-essential genes missing in the replication-defective adenovirus or adenovirus vector of the present invention. For example, an E1 / E4-deficient adenovirus vector may be engineered to contain a nucleic acid sequence encoding E4 ORF 6, said nucleic acid sequence being obtained from or derived from a different adenovirus (e.g., an adenovirus of a different serotype than the adenovirus or adenovirus vector of the present invention, or an adenovirus of a different species than the adenovirus or adenovirus vector of the present invention).

[0305] a. Adenovirus vector based on chimpanzees

[0306] In some embodiments, the adenovirus described herein is isolated from gorillas. Western gorilla species include the western lowland gorilla (… Gorilla gorilla gorilla ) and Cross River gorillas ( Gorilla gorilla diehli These two subspecies, while the eastern gorilla species include the mountain gorilla ( Gorilla beringei beringei ) and eastern lowland gorillas ( Gorilla beringei graueri These two subspecies. See, for example, Wilson and Reeder (eds.), *Mammalian Species of the World*, 3rd ed., Johns Hopkins University Press, Baltimore, Maryland (2005). In some embodiments, the adenovirus of this disclosure is derived from mountain gorillas ( Gorilla beringei beringei Isolation from ) . Previous studies have characterized numerous chimpanzee adenoviruses and their genome sequences (see, for example, WO 2013 / 052832, WO 2013 / 052811, WO 2013 / 052799; WO 2019 / 173465, WO 2022 / 115470).

[0307] In terms of vector design and safety, chimpanzee adenoviruses are similar to human adenoviruses, offering benefits such as highly efficient transgene delivery and replication incompetence through targeted deletion. Importantly, humans have very little prior immunity to chimpanzee adenoviruses compared to human adenoviruses. This characteristic of not being recognized by the human immune system minimizes potential prior immune barriers in gene therapy and vaccine applications.

[0308] In some embodiments, the adenovirus vector is derived from chimpanzee adenovirus type 40 (GAd40), such as GC44, GC45, or GC46. In some embodiments, the adenovirus vector represents a functional adaptation of the aforementioned vectors. Such adaptations may include sequences encoding functional variants of their components, such as the E2B, E2A, E3, and L1-L5 regions, and inverted terminal repeat sequences. Functional adaptations of such vectors having codon degenerate variants encoding sequences of the E2B, E2A, E3, and L1-L5 regions are also contemplated.

[0309] In a particularly preferred embodiment, the adenovirus vector is derived from GC46, a newly isolated and unique strain of chimpanzee adenovirus, isolated from fecal samples of healthy African gorillas. Based on hexagonal, DNA polymerase, and exon 4 ORF6 protein sequence comparisons, this adenovirus is highly related to and phylogenetically clustered with human adenovirus type C. (Duncan et al., Virology, 444:119-123 (2013)). In the United States, the seroprevalence of chimpanzee adenovirus GC46 is less than approximately 6%. In contrast, the seroprevalence of Ad5 is approximately 57%, and most seropositive individuals have high titers (above 200 IC90). (Johnson et al., Molecular Therapy, 22:196-205 (2014)). Therefore, prior neutralizing activity against chimpanzee adenovirus GC46 has been rare and weak in the United States compared to conventional adenovirus treatment based on the Ad5 serotype. Furthermore, comparative studies of human serum samples from sub-Saharan Africa confirmed the presence of rare and weak prior neutralizing activity in the human population. These data suggest that prior neutralizing activity against GC46 would not significantly interfere with molecular vaccines and therapeutics constructed based on this platform, making gorilla adenovirus GC46 highly suitable as a backbone viral vector.

[0310] In a preferred embodiment of the invention, the goat adenovirus vaccine encodes a fusion protein (e.g., HPV-E2, HPV-E4, HPV-E6, and HPV-E7) of selected regions of HPV proteins expressed in HPV-6 and HPV-11 infected cells.

[0311] In one specific embodiment of the present invention, the chimpanzee adenovirus vaccine encodes an HPV protein of 791 amino acids, of which 731 amino acids (92.4%) are derived from HPV-6 and 60 amino acids (7.6%) are derived from HPV-11.

[0312] In some embodiments, the adenovirus vector is an engineered chimpanzee adenovirus vector that has partially or completely deleted the E1 and / or E4 regions. Deletion of the E1 region, for example, may render the adenovirus vector replication-defective and may contain bases 459 to 3411, resulting in the deletion of the E1A and E1B promoters and open reading frames. Deletion of the E4 region, for example, may contain bases 34144 to 36824 and remove all E4 open reading frames (ORFs), thereby eliminating key elements necessary for chimpanzee adenovirus replication. (The chimpanzee adenovirus coordinates provided herein are based on a wild-type adenovirus genome size of 37,213 base pairs.)

[0313] Modified chimpanzee adenovirus vectors with deletions and / or omissions of the E1 and / or E4 regions can offer one or more advantages over unmodified vector backbones. For example, deletions of extended adenovirus genomes can provide enhanced payload capacity for adenovirus vectors. A second potential advantage is the reduced risk of generating replicative adenoviruses (RCA) during adenovirus vector preparation. A third advantage is that removal of E1 and E4 expression products can further silence other regions of the viral genome.

[0314] Therefore, in one aspect, the chimpanzee adenovirus vector described herein has an E1 region or a portion thereof deleted. In another aspect, the chimpanzee adenovirus vector described herein has an E4 region or a portion thereof deleted. In yet another aspect, the chimpanzee adenovirus vector described herein has both the E1 region and the E4 region or a portion thereof deleted. In one aspect, the deletion in the E1 and / or E4 regions is about 100 to about 5,000 base pairs (bp) in length compared to the wild type. For example, the deletion in the E1 and / or E4 regions may be about 100 bp, about 500 bp, about 1,000 bp, about 1,500 bp, about 2,000 bp, about 2,500 bp, about 3,000 bp, about 3,500 bp, about 4,000 bp, about 4,500 bp, or about 5,000 bp compared to the wild type. In some implementations, the deletions in regions E1 and / or E4 comprise approximately 100 bp to approximately 5,000 bp, or approximately 500 bp to approximately 4,500 bp, approximately 750 bp to approximately 4,000 bp, or approximately 1,000 bp to approximately 3,750 bp, or approximately 1,250 bp to approximately 3,500 bp, or approximately 1,500 bp to approximately 3,500 bp, or approximately 1,750 bp to approximately 3,500 bp, or approximately 2,000 bp to approximately 3,500 bp, or approximately 2,000 bp to approximately 3,000 bp. In some specific implementations, the deletions in regions E1 and / or E4 are approximately 3,000 bp in length compared to the wild type.

[0315] In some embodiments, the deletion of the E4 region removes all predicted open reading frames (ORFs) within it. To avoid potentially low yields when preparing adenoviral vectors with an E4 deletion, a spacer sequence can be inserted within the E4 deletion region, as shown in Figure 1, to terminate any potential transcription initiated from the retained E4 promoter. In one aspect, the chimpanzee adenoviral vector described herein includes a spacer sequence inserted to replace the deleted portion of the E4 region. In one aspect, the spacer sequence includes a bovine growth hormone polyadenylation (BGH polyA) signal sequence inserted to replace the deleted E4 ORF, but any suitable spacer sequence may also be used. In some aspects, the spacer sequence is approximately 10 to approximately 500 base pairs (bp) in length. For example, the length of the interval sequence may be approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, approximately 2 50, approximately 260, approximately 270, approximately 280, approximately 290, approximately 300, approximately 310, approximately 320, approximately 330, approximately 340, approximately 350, approximately 360, approximately 370, approximately 380, approximately 390, approximately 400, approximately 410, approximately 420, approximately 430, approximately 440, approximately 450, approximately 460, approximately 470, approximately 480, approximately 490, or approximately 500 bp. Alternatively, compared to the wild type, the spacer sequence may be about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, or about 450 bp to about 500 bp. The spacer may also be of any length within these ranges. For example, the spacer length may be about 250 bp to about 350 bp, about 260 bp to about 340 bp, about 270 bp to about 330 bp, about 280 bp to about 320 bp, or about 290 bp to about 310 bp. In one aspect, the spacer sequence is about 300 base pairs long. In yet another aspect, the spacer sequence is 278 bp.

[0316] On the other hand, compared to the wild type, the spacer sequence is located at approximately 34,700 to 35,000 base pairs in the vector genome. On yet another hand, compared to the wild type, the spacer sequence is located at 34,692 to 34,969 base pairs in the vector genome. On yet another hand, the spacer sequence comprises the nucleic acid sequence of SEQ ID NO:95.

[0317] In some embodiments, both the E1 and E4 regions of the vector are deleted. In some such embodiments, the E1 region or the E4 region is replaced by an expression cassette containing a transgene or spacer. In some such embodiments, the E1 region is replaced by the expression cassette and the E4 region is replaced by a spacer.

[0318] In one embodiment, the E1 / E4-deficient GC46 adenovirus vector can be prepared in any complementary cell line providing E1 and E4 ORF6 functionality, such as engineered 293 cells. For example, such cells can be cultured in shake flasks in a serum-free suspension and infected with the master viral library at a multiplicity of infection (MOI) of 100 PU per cell. The culture harvest can be downstream processed and purified by three rounds of cesium chloride density gradient ultracentrifugation to obtain a highly purified material. This material can then be frozen, thawed, aseptically filtered, and aliquoted into vials that can be stored in a freezer at approximately -60 to approximately -90°C.

[0319] In some embodiments, the vector of the present invention can be prepared by isolating a GC46 chimpanzee adenovirus vector from a non-human primate source, cloning the isolated GC46 genome, deleting the E1 and E4 regions of GC46, and inserting an expression cassette into the E1 region, the expression cassette expressing a human papillomavirus (HPV) 6 / 11 antigen design controlled by the cytomegalovirus (CMV) immediate early promoter. In one embodiment described below, the CMV-HPV 6 / 11 antigen design includes epitopes of HPV 6 and 11—namely, key immunogenic peptides derived from E2 (HPV6), E4 (HPV6), E6 (HPV6 / 11), and E7 (HPV6 / 11), wherein the HPV6-derived peptide has high sequence similarity to HPV11.

[0320] In some embodiments, the vector of the present invention encodes any of the HPV antigen regions described herein or variants thereof. For example, the vector may contain a nucleic acid sequence having at least 80% identity with SEQ ID NO:68.

[0321] C. Non-virus-based delivery systems

[0322] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used in vitro and in vivo as a delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0323] Imagine using lipid formulations to introduce the nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acids can be associated with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous phase of liposomes, sandwiched within the lipid bilayer of liposomes, attached to liposomes via linkers associated with both liposomes and oligonucleotides, trapped within liposomes, forming complexes with liposomes, dispersed in solutions containing lipids, mixed with lipids, bound to lipids, present as suspensions in lipids, contained in micelles or forming complexes with micelles, or otherwise associated with lipids. Compositions associated with lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, they can be bilayers, micelles, or “collapsed” structures. They can also simply be dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances that can be naturally occurring or synthesized. For example, lipids include naturally occurring fat droplets in the cytoplasm and classes of compounds comprising long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0324] The lipids suitable for use are available from commercial sources. For example, dimyristoylphosphatidylcholine (“DMPC”) is available from Sigma (St. Louis, Missouri); hexadecyl phosphate (“DCP”) is available from K&K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; and dimyristoylphosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids dissolved in chloroform or chloroform / methanol can be stored at approximately -200°C. Chloroform is used only as a solvent because it evaporates more readily than methanol.

[0325] "Liposome" is a general term encompassing various monolayer and multilayer lipid delivery vehicles formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicle structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in excess aqueous solution. The lipid components undergo rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, it also encompasses compositions that have structures in solution different from normal vesicle structures. For example, lipids may exist as micelles or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes have also been envisioned.

[0326] In some cases, polynucleotides encoding polypeptides can also be introduced into cells using non-viral delivery systems, such as the Sleeping Beauty (SB) transposon system. In embodiments, the SB11, SB100X, SB110, and piggyBac transposon systems are used (see, for example, Wilson et al., "PiggyBac Transposon-mediated Gene Transfer in Human Cells"). Molecular Therapy 15 (139-145 (2007)) and / or the piggyBat transposon system (see, for example, Mitra et al., “Functional characterization of piggyBac from the bat Myotis lucifugus unveils an active mammalian DNA transposon)). Proc. Natl. Acad. Sci. USA 110 :234-239 (2013)), the modified effector cells and other genetic elements described herein are delivered to cells. Other transposases and transposon systems are provided in the following disclosures: U.S. Patent Nos. 6,489,458, 6,613,752, 7,148,203, 7,985,739, 8,227,432, 9,228,180; U.S. Patent Publication 2011 / 0117072; Mates et al., Nat Genet, 41(6): 753-61 (2009). doi: 10.1038 / ng.343. Electronic publication May 3, 2009; Gene Ther., 18(9):849-56 (2011). doi: 10.1038 / gt.2011.40 Electronic publication March 31, 2011; and Ivies et al., Cell, 91(4):501-10, (1997).

[0327] Other suitable non-viral systems may include integrative expression vectors, which can be randomly integrated into the host cell's DNA, or may contain recombination sites to achieve specific recombination between the expression vector and the host cell's chromosome. Directed integration of transgenes into predefined genetic sites is a desired goal for many applications. First, a first recombination site of a site-specific recombinase is inserted into the genome, the location of which can be random or predetermined. Subsequently, the cells are transfected with a plasmid carrying the gene or DNA of interest, a second recombination site, and the source of the recombinase (expression plasmid, RNA, protein, or a virus expressing the recombinase). Recombination between the first and second recombination sites results in the integration of the plasmid DNA. Such integrative expression vectors can utilize endogenous expression regulatory sequences of the host cell's chromosome to achieve the expression of the desired protein.

[0328] In some embodiments, the presence of sequences homologous to the sequences flanking the integration site on the donor polynucleotide can promote directed integration. For example, directed integration using the donor polynucleotide described herein can be achieved after conventional transfection techniques, such as those used to create gene knockout or knock-in genes through homologous recombination. In other embodiments, directed integration is promoted both by the presence of sequences homologous to the sequences flanking the integration site on the donor polynucleotide and by contacting the cell with the donor polynucleotide in the presence of a site-specific recombinase. A site-specific recombinase, or simply recombinase, is a polypeptide that catalyzes conserved site-specific recombination between its compatible recombination sites. As used herein, site-specific recombinases include native polypeptides and their active derivatives, variants, and / or fragments, as well as native polynucleotides, derivatives, variants, and / or fragments encoding active recombinases.

[0329] This document also provides a system for integrating a heterologous gene into a host cell, the system comprising one or more gene expression cassettes. In some cases, the system includes a first gene expression cassette containing a first polynucleotide encoding a first polypeptide construct. In other cases, the system may include a second gene expression cassette containing a second polynucleotide encoding a second polypeptide construct. In still other cases, the system may include a third gene expression cassette. In one embodiment, one of the gene expression cassettes may include a gene-switching polynucleotide encoding one or more of the following: (i) a transactivation domain; (ii) a nuclear receptor ligand-binding domain; (iii) a DNA-binding domain; and (iv) a ecdysone receptor-binding domain. In another embodiment, the system further includes a recombination attachment site and a serine recombinase; thereby, in the presence of the serine recombinase, when the host cell is in contact with at least the first gene expression cassette, the heterologous gene is integrated into the host cell.

[0330] In some cases, the system further comprises a ligand; thereby, upon contact with the host cell in the presence of the ligand, the heterologous gene is expressed in the host cell. In one case, the system further comprises a recombination attachment site. In some cases, a recombination attachment site is a phage genome recombination attachment site (attP) or a bacterial genome recombination attachment site (attB). In one case, the host cell is a eukaryotic cell. In another case, the host cell is a human cell. In a further case, the host cell is a T cell or an NK cell.

[0331] II. Expression Box

[0332] A. Genetically modified

[0333] The vector of the present invention may contain an expression cassette for expressing a transgene. The “transgene” comprises a non-natural nucleic acid sequence operatively linked to a suitable regulatory element (e.g., a promoter), such that the non-natural nucleic acid sequence can be expressed to produce a protein (e.g., a peptide or polypeptide). The regulatory element (e.g., the promoter) may be natural or non-natural with respect to an adenovirus or adenovirus vector.

[0334] A “non-natural” nucleic acid sequence refers to any nucleic acid sequence (e.g., DNA, RNA, or cDNA sequence) that is not located in a natural position within an adenovirus. Therefore, the non-natural nucleic acid sequence may be naturally present in adenoviruses, but located in a non-natural position within the adenovirus genome and / or operatively linked to a non-natural promoter. The terms “non-natural nucleic acid sequence,” “heterologous nucleic acid sequence,” and “exogenous nucleic acid sequence” are synonymous and are used interchangeably in the context of this disclosure. The non-natural nucleic acid sequence is preferably DNA and preferably encodes a protein (i.e., one or more nucleic acid sequences encoding one or more proteins).

[0335] The non-natural nucleic acid sequence may encode a therapeutic protein that can be used for preventative or therapeutic treatment of diseases in mammals. Examples of suitable therapeutic proteins include anti-inflammatory agents (e.g., cytokines), toxins, tumor suppressor proteins, growth factors, hormones, receptors, mitogens, immunoglobulins, neuropeptides, neurotransmitters, and enzymes. Alternatively, the non-natural nucleic acid sequence may encode an antigen of a pathogen (e.g., bacteria or virus), and the adenovirus or adenovirus vector may be used as a vaccine.

[0336] B. Promoter

[0337] The genetic regulatory components of therapeutic expression cassettes are selected to confer high levels of transgene expression. Therefore, another aspect of this disclosure is an expression cassette that also includes a promoter. A promoter is a region of a polynucleotide that initiates transcription of a coding sequence. The promoter is located near the transcription start site of the gene, on the same strand of DNA as the coding sequence, and upstream (towards the 5' region of the sense strand). Some promoters are constitutive because they are active under all conditions in the cell, while others are regulated and become active in response to specific stimuli, such as inducible promoters. Other promoters are tissue-specific or activating promoters, including but not limited to T cell-specific promoters.

[0338] As used in this article, “promoter activity” and its grammatical equivalents refer to the degree of expression of a nucleotide sequence operatively linked to a promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcripts produced, for example by Northern blotting analysis, or indirectly by determining the amount of product encoded by the linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0339] 1. Inducible promoters

[0340] In some embodiments, the promoter is an inducible promoter. An inducible promoter is a promoter whose activity is induced by the presence or absence of transcriptional regulatory factors, such as biological or abiotic factors. Inducible promoters are useful because the expression of the gene operationally linked to them can be turned on or off at specific stages of an organism's development or in specific tissues. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenicity-regulated promoters, temperature-regulated promoters, and light-regulated promoters. In some embodiments, the inducible promoter is part of a genetic switch. This inducible promoter can be a gene switch ligand-induced promoter. In some cases, the inducible promoter can be a small molecule ligand-induced dual-peptide ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch, as described in WO2018 / 132494. Other examples of gene switch systems include, but are not limited to, the systems described in U.S. Patent Nos. 6,258,603 and 7,045,315, U.S. Patent Application Publication Nos. 2006 / 0014711 and 2007 / 0161086, and International Publication No. WO01 / 70816.

[0341] In some cases, the gene switch can be selected from ecdysone-based receptor components, such as, but not limited to, any system described in the following documents: PCT / US2001 / 009050 (WO2001 / 070816); US Patent Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT / US2001 / 030608 (WO2002 / 029075); US Patent Nos. 8,105,825; 8,168,426; PCT / US2002 / 005235 (WO2002 / 066613); US Application Serial No. 10 / 468,200 (US Patent Publication No. 201201672). 39); PCT / US2002 / 005706 (WO2002 / 066614); US Patent Nos. 7,531,326; 8,236,556; 8,598,409; PCT / US2002 / 005090 (WO2002 / 066612); US Patent No. 8,715,959 (US Publication No. 20060100416); PCT / US2002 / 005234 (WO2003 / 027266); US Patent Nos. 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT / US2002 / 005708 (WO2002 / 066615) US Application Serial No. 10 / 468,192 (US Publication No. 20110212528); PCT / US2002 / 005026 (WO2003 / 027289); US Patent Nos. 7,563,879; 8,021,878; 8,497,093; PCT / US2005 / 015089 (WO2005 / 108617); US Patent Nos. 7,935,510; 8,076,454; PCT / US2008 / 011270 (WO2009 / 045370); US Application Serial No. 12 / 241,018 (US Publication No. 20090136465); PCT / US2008 / 01 1563 (WO2009 / 048560); U.S. Application Serial No. 12 / 247,738 (U.S. Publication No. 20090123441); PCT / US2009 / 005510 (WO2010 / 042189); U.S. Application Serial No. 13 / 123,129 (U.S. Publication No. 20110268766); PCT / US2011 / 029682 (WO2011 / 119773); U.S. Application Serial No. 13 / 636,473 (U.S. Publication No. 20130195800); PCT / US2012 / 027515 (WO2012 / 122025); and U.S. Patent No. 9,402,919.

[0342] Inducible promoters typically utilize ligands to dose-regulate the expression of at least two genes. In some cases, the ligands may be selected from the group consisting of: ecdysone, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazine, oxadiazoline, dibenzoylalkylcyanazide, N-alkyl-N,N'-diaroylhydrazine, N-acyl-N-alkylcarbonylhydrazine, N-aroyl-N-alkyl-N'-aroylhydrazine, arnidoketone, 3,5-di-tert-butyl-4-hydroxy-N-isobutylbenzamide, 8-O-acetylharbazoside, oxosterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-α-6-α-epoxycholesterol-3-sulfate (ECH). S), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-bisphosphonates, larval hormone III, RG-115819 (N-(1-ethyl-2,2-dimethylpropyl)-N'-(2-methyl-3-methoxybenzoyl)-hydrazine of 3,5-dimethylbenzoic acid), RG-115932 ((R)-3,5-dimethylbenzoic acid N-(1-tert-butylbutyl)-N'-(2-ethyl-3-methoxybenzoyl)-hydrazine), and RG-115830 (N-(1-tert-butylbutyl)-N'-(2-ethyl-3-methoxybenzoyl)-hydrazine of 3,5-dimethylbenzoic acid), and any combination thereof. In one embodiment, a gene switch is a system in which gene expression levels depend on the presence of ligands. Examples of ligand-dependent transcription factor complexes that can be used in the gene switch of this invention include, but are not limited to, members of the nuclear receptor superfamily, which can be activated by their respective ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysone, and their analogues and mimics), as well as rTTA activated by tetracycline. In one aspect of this invention, the gene switch is an EcR-based gene switch.

[0343] 2. Non-inducible promoters

[0344] In some embodiments, the promoter is a non-inducible promoter, including, for example, tissue-specific promoters, strongly constitutive promoters, or minimal promoters known to those skilled in the art. Suitable non-inducible promoters may include, for example, CMV promoters, SV40 promoters, CAG promoters, or other promoters. In some embodiments, the promoter is a CMV promoter.

[0345] 3. Tissue-specific promoters

[0346] In some embodiments, the promoter may be a tissue-specific promoter. Herein, “tissue-specific” means the regulated expression of a gene within a subset of tissue or cell types. In some cases, tissue-specific promoters may be spatially regulated such that the promoter drives expression only in certain tissues or cell types of an organism. In other cases, tissue-specific promoters may be temporally regulated such that the promoter drives expression differently in a particular cell type or tissue at different times (including during the organism’s development). In some cases, tissue-specific promoters are regulated both spatially and temporally. In some embodiments, tissue-specific promoters are constitutively activated in a particular cell type or intermittently at a particular time or stage. For example, a tissue-specific promoter may be a promoter that is activated when a particular cell (such as a T cell or NK cell) is activated. T cells can be activated in a variety of ways, such as when they present peptide antigens via MHC class II molecules.

[0347] 4. Synthesis and Engineering of Promoters

[0348] The use of synthetic promoters in the expression cassettes described herein is also envisioned, and these promoters can be engineered to improve expression properties. Synthetic promoters may include various subcomponents, including but not limited to blocking sequences, enhancers, and various reaction elements.

[0349] In some embodiments, the promoter is an engineered promoter or a variant thereof. As described herein, the promoter may combine a minimal promoter sequence from IL-2 with one or more of the following: activating T cell nuclear factor (NFAT) response element; NFIL2D response element, NF-κB / TCF response element, NFAT / NFIL2B response element, or NFIL2A / OCT response element. NFAT transcription factors are key regulators of effector T cell status. NFAT is a stepwise driving early transcriptional checkpoint. Upon TCR stimulation, NFAT is rapidly activated in T cells and forms a protein complex with AP-1 induced by appropriate co-stimulatory signals, thereby regulating effector genes and T cell function. NFAT response elements may be fused with other minimal promoter sequences (e.g., the IL2 minimal promoter) to drive transgene expression in response to T cell activation. Further examples of response elements are described in Mattila et al., EMBO J., 9(13):4425-33 (1990).

[0350] 5. Activation of specific promoters

[0351] In some embodiments, the promoter is an activation-specific promoter, such as the interleukin-2 (IL2) promoter and the programmed cell death (PD)-1 (CD279) promoter. Alternatively, the gene switch component can be conditionally expressed upon activation of immune cells by fusing the binding sites of other nuclear factors (e.g., NF-κB from pro-inflammatory signaling pathways) to a minimal promoter sequence (e.g., IL2).

[0352] In some embodiments, the promoter comprises an IL-2 core promoter. In some embodiments, at least one promoter comprises an IL-2 minimal promoter. In another embodiment, at least one promoter comprises an IL-2 enhancer and a promoter variant. In yet another embodiment, at least one promoter comprises an NF-κB binding site. In some embodiments, at least one promoter comprises a (NF-κB)1-IL2 promoter variant. In some embodiments, at least one promoter comprises a (NF-κB)3-IL2 promoter variant. In some embodiments, at least one promoter comprises a (NF-κB)6-IL2 promoter variant. In some embodiments, at least one promoter comprises a 1X Activated T Cell Nuclear Factor (NFAT) Response Element-IL2 promoter variant. In another embodiment, at least one promoter comprises a 3X NFAT Response Element. In yet another embodiment, at least one promoter comprises a 6X NFAT Response Element-IL2 promoter variant. In some embodiments, at least one promoter comprises a human EF1A1 promoter variant. In some embodiments, at least one promoter comprises a human EF1A1 promoter and an enhancer. In some embodiments, at least one promoter comprises a human UBC promoter. In some embodiments, at least one promoter comprises a 6-site GAL4-induced proximal factor-binding element (PFB). In some embodiments, at least one promoter comprises a synthetic minimal promoter 1 (inducible promoter). Sequences of such promoters are described, for example, in WO2019 / 173465 and WO 2022 / 115470.

[0353] In some embodiments, the promoter may be any one or more of the following: IL-2 core promoter, IL-2 minimal promoter, IL-2 enhancer and promoter variant, (NF-κB)1-IL2 promoter variant, (NF-κB)3-IL2 promoter variant, (NF-κB)6-IL2 promoter variant, 1X NFAT reaction element-IL2 promoter variant, 3X NFAT reaction element-IL2 promoter variant, 6X NFAT reaction element-IL2 promoter variant, human EEF1A1 promoter variant, human EEF1A1 promoter and enhancer, human UBC promoter, and synthetic minimal promoter 1.

[0354] 6. Constitutive promoters

[0355] In some embodiments, the promoter is a constitutive promoter. Examples of such promoters include the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0356] 7. Virus promoter

[0357] Exemplary promoters used in the vectors described herein include viral promoters that are operatively combined with heterologous nucleic acid sequences encoding the cytokines. Exemplary viral promoters may be derived from a variety of known viruses, including but not limited to retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), alphavirus vectors, etc. Non-limiting examples of potentially useful viral vectors include human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), simian virus 40, herpes simplex virus (HSV), adenovirus (AV), adeno-associated virus (AAV), or lentivirus (LV). For example, specific viral promoters contemplated herein include the cytomegalovirus (CMV) immediate early promoter, the CAG promoter (which is a combination of a CMV early enhancer element and a chicken β-actin promoter), the simian virus 40 (SV40) promoter, the 35S RNA and 19S RNA promoters of cauliflower mosaic virus (CaMV), the capsid protein promoter of tobacco mosaic virus (TMV), and any variants thereof. Examples of mammalian promoters include the human elongation factor 1α subunit (EF1-1α) promoter, the human ubiquitin C (UCB) promoter, the mouse phosphoglycerate kinase-1 (PGK) promoter, and any variant thereof.

[0358] 8. Other starter elements

[0359] Additional promoter elements, such as enhancers (e.g., promoter enhancers), regulate the frequency of transcription initiation. These elements are typically located in a region 30–110 bp upstream of the start site, although recent studies have shown that some promoters also contain functional elements downstream of the start site. The spacers between promoter elements are usually flexible, so promoter function can be maintained even when the elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function synergistically or independently to activate transcription.

[0360] The synthetic promoters useful in this invention may include enhancer sequences. In one aspect, the enhancer may be an mCMV enhancer sequence. In another aspect, the mCMV enhancer sequence is about 500 to about 1,000 bp in length. In yet another aspect, the enhancer sequence is about 700 bp in length. In still another aspect, the enhancer sequence comprises a nucleic acid sequence of SEQ ID NO:96 or a functional variant thereof, for example, a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with: SEQ ID NO:96, or a conserved substitution variant of SEQ ID NO:96, or a non-conserved substitution variant of SEQ ID NO:96.

[0361] In another aspect, the mCMV enhancer includes transcription factor binding sites. In yet another aspect, the transcription factor binding sites include Sp1, Ebox, ETS, TRE, CREB, and GATA binding sites.

[0362] The promoters described herein may also contain response elements. Various response elements are known in the art. For example, to reduce transgene expression during adenovirus production (which can sometimes negatively impact overall production titers), a tetracycline response element (TRE, 2X TetO) can be placed within a promoter element between the TATA box and the transcription start site in the promoter. Thus, when the vector is produced in a cell line expressing a tetracycline (Tet) repressor protein, transgene expression driven by a promoter containing a TRE is reduced. Gall et al., Molecular Biotechnology , 35:263–273 (2007). In the absence of tetracycline, the Tet repressor protein interacts with the TRE element and blocks transcription initiation. Normal expression levels can be observed when production cells that do not express the Tet repressor protein are infected. Therefore, in one aspect described herein, the synthetic promoter comprises a TRE. The TRE is about 10 to about 100 bp in length. In another aspect, the TRE is in the range of 10 bp to 100 bp in length. In one aspect, the TRE is about 50 bp in length. In yet another aspect, the TRE comprises a nucleic acid sequence of SEQ ID NO:98 or a functional variant thereof, for example, a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with: SEQ ID NO:98, or a conserved substitution variant of SEQ ID NO:98, or a non-conserved substitution variant of SEQ ID NO:98.

[0363] In some embodiments, the promoter comprises a transcriptional blocker, an enhancer sequence, and a response element. In some such embodiments, the promoter comprises an mCMV enhancer and a TRE.

[0364] C. Non-translation area

[0365] In some embodiments, the expression cassette may include an untranslated region (UTR) to regulate or enhance transgene expression. In one aspect, the expression cassette may include an artificially created untranslated region. A 5' UTR with a splicing unit has been shown to enhance transgene expression. Therefore, in one embodiment, the cassette includes a 5' UTR with a splicing unit. In some embodiments, the 5' UTR is engineered to include a synthetic splicing site sequence spanning intron 2 of the canine ATP2A2 gene, followed by the 5' UTR of the bovine CSN2 gene. In another aspect, the 5' UTR includes a nucleic acid sequence of SEQ ID NO:99 or a functional variant thereof, for example, a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with: SEQ ID NO:99, or a conserved substitution variant of SEQ ID NO:99, or a non-conserved substitution variant of SEQ ID NO:99.

[0366] D. Termination Sequence

[0367] In another aspect, the expression cassette also includes a termination sequence. The termination sequence may follow the open reading frame of the cytokine transgene. Similar to the promoter sequence, the termination sequence may also contain various regulatory elements to ensure proper 3' transcript end processing. In one aspect, the termination sequence includes a portion of the human growth hormone (HGH) 3' untranslated region. In another aspect, the termination sequence includes a polyadenylation signal, including but not limited to SV40 polyadenylation and / or LTR polyadenylation signals. In yet another aspect, the termination sequence includes a human β-actin (ACTb) transcription termination signal sequence. In yet another aspect, the termination sequence includes the HGH 3' untranslated region, a polyadenylation signal, and a human β-actin transcription termination sequence. In another aspect, the termination sequence comprises a nucleic acid sequence of SEQ ID NO:104 or a functional variant thereof, such as a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with: SEQ ID NO:104, or a conserved substitution variant of SEQ ID NO:104, or a non-conserved substitution variant of SEQ ID NO:104.

[0368] E. Polynucleotide linkers

[0369] This paper also envisions expression cassettes and constructs containing polynucleotide linkers to facilitate the expression of the polynucleotides described herein and the function of the peptides.

[0370] In some cases, the adapter may be a cleavable adapter. The polynucleotide adapter may be an oligomer. The polynucleotide adapter may be a double-stranded, single-stranded, or combination thereof DNA. In some cases, the adapter may be RNA. The polynucleotide adapter may be a double-stranded DNA fragment containing desired restriction sites, which may be added to form an end structure compatible with a vector containing the polynucleotides described herein.

[0371] In some cases, polynucleotide adapters can be used to modify vectors containing the polynucleotides described herein. For example, vector modifications containing polynucleotide adapters may include alterations to the multiple cloning site or the addition of a polyhistidine tail. Polynucleotide adapters can also be used to adapt blunt-ended insertions into the ends of DNA for cloning into vectors that have been cleaved by restriction endonucleases that produce sticky end groups. Using polynucleotide adapters is more efficient than directly attaching blunt ends to the vector and provides a method for releasing the insert from the vector in downstream applications. The insert may be a polynucleotide sequence encoding a polypeptide for therapeutic applications.

[0372] In some embodiments, the polynucleotide adapter may be ligated to a vector containing the polynucleotide described herein using a T4 ligase. To facilitate ligation, an excess of the polynucleotide adapter may be added to the composition containing the insert and the vector. In some cases, the insert and vector are pretreated prior to the introduction of the adapter. For example, pretreatment with a methyltransferase can prevent unwanted cleavage of the inserted DNA.

[0373] In some cases, the polynucleotides or genes described herein may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 linkers.

[0374] 1. IRES sequence

[0375] In some embodiments, the polynucleotides described herein can be linked via an "internal ribosome entry site" or "IRES" element. IRES can allow for the simultaneous expression of multiple genes. For example, an IRES sequence can allow for the production of multiple proteins from a single mRNA transcript. Ribosomes can bind to the IRES in a non-5'-cap-dependent manner and initiate translation.

[0376] In expression cassettes containing IRES sequences, the first gene can be translated via a cap-dependent ribosome scanning mechanism with its own 5'-UTR, while the translation of subsequent genes can be achieved by directly recruiting ribosomes to the IRES in a cap-independent manner. IRES sequences allow eukaryotic ribosomes to bind and initiate translation without binding the 5' cap. IRES sequences can allow the expression of multiple genes from a single transcript (Mountford and Smith, Trends Genet. 11(5):179-84(1995)).

[0377] In some cases, the IRES region may be derived from viruses, such as pituitary viruses, encephalocarditis viruses, and hepatitis C virus IRES sequences. In other cases, the IRES sequence may be derived from encephalocarditis viruses. As used herein, the term "EMCV" or "encephalocarditis virus" refers to isolates or strains of any member of the genus encephalocarditis virus in the family Picornaviridae. Examples include EMCV-R (Rueckert) and Columbia-SK viruses. In some cases, cellular IRES elements may be used, such as eukaryotic initiation factor 4G, immunoglobulin heavy chain binding protein, c-myc proto-oncogene, vascular endothelial growth factor, fibroblast growth factor-I IRES, or any combination or modification thereof. In some cases, cellular IRES may exhibit enhanced gene expression compared to viral IRES.

[0378] The vector may utilize IRES sequences from viruses, cells, or combinations thereof. IRES may originate from myocardial encephalitis virus (EMCV) or poliovirus (PV). In some cases, IRES elements are selected from poliovirus (PV), encephalomyelitis virus (EMCV), foot-and-mouth disease virus (FMDV), porcine TESE virus-1 (PTV-1), AiV, Seneca Valley virus (SVV), hepatitis C virus (HCV), classical swine fever virus (CSFV), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-1 (HIV-1), Moloney mouse leukemia virus (MoMLV), feline immunodeficiency virus (FIV), mouse mammary tumor virus (MMTV), human cytomegalovirus latent (pUL138), Epstein-Barr virus (EBNA-1), herpesvirus Marek's disease (MDV RLORF9), and SV40 polycistronic 19S (SV40). 19S), RhPV (RhV), CrPV (CrV), EoPV (EoPV), PSIV (PSIV), TrV (TrV), IAPV (IAPV, KBV), BRV (BRV), PFBV (PFBV), HCRSV (HCRSV), CrTMV (CrTMV), PLRV (PLRV), TEV (TEV), Giadiflavivirus (GLV), LRV (LRV-1), and combinations or modifications thereof.

[0379] In some cases, IRES are selected from Apaf-1, XIAP, HIAP2 / cIAP1, DAP5, Bcl-2, c-myc, CAT-I, INR, differentiation LEF-1, PDGF2, HIF-1a, VEGF, FGF2, BiP, BAG-I, CIRP, p53, SHIMTI, PITSLEP58, CDKI, Rpr, hid, hsp70, grim, skl, Antennapedia, dFoxO, dinR, Adh-Adhr, HSPI0I, Adh, URE-2, GPRI, NCE102, YMR18la, MSNI, BOil, FLO8, GICI, and any combination or modification thereof. When an IRES element is sandwiched between two open reading frames (ORFs), translation initiation can occur in the first ORF via a classic 5'-m7GpppN cap-dependent mechanism, and in the second ORF downstream of the IRES element via a cap-free mechanism.

[0380] In some cases, the IRES sequence can be about 9 to about 1,000 base pairs. For example, the IRES sequence can be about 9 to about 150 base pairs, or about 150 to about 400 base pairs, about 400 to about 600 base pairs, or about 600 to 1,000 base pairs. In some embodiments, the IRES sequence is about 9, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 275, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 750, about 800, about 850, about 900, about 950, or about 1,000 base pairs.

[0381] In some cases, downstream gene expression in a vector containing the IRES sequence may be reduced. For example, a gene following the IRES sequence may have reduced expression relative to a gene preceding the IRES sequence. Reduced expression can range from 1% to 99.9% relative to the preceding gene, including, for example, reductions of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% relative to the preceding gene.

[0382] 2. Virus 2A sequence

[0383] In some embodiments, the polynucleotides described herein can be linked via viral 2A elements or sequences. 2A elements can be shorter than IRES, ranging in length from 5 to 100 base pairs. In some cases, the 2A sequence can contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 base pairs. Genes linked by 2A can be expressed in a single open reading frame, and “self-cleavage” can occur during translational co-transfer between the last two amino acids (GP) at the C-terminus of the 2A polypeptide, resulting in equal amounts of co-expressed protein.

[0384] The viral 2A sequence can be approximately 20 amino acids long. In some cases, the viral 2A sequence may contain the shared motif Asp-Val / Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO:122). This shared motif sequence can play a role in translational co-transfer. For example, it can prevent the formation of a normal peptide bond between glycine and proline residues, leading to ribosome skipping and cleavage of the nascent polypeptide. This effect can generate multiple genes at an equimolar level.

[0385] 2A peptides can allow the translation of multiple proteins into polypeptides within a single open reading frame that can subsequently be cleaved into individual polypeptides via ribosomal skipping mechanisms (Funston et al., J Gen. Viral. 89(Pt 2):389-96(2008)). In some embodiments, the 2A sequence may include: F / T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, and any combination thereof.

[0386] In some cases, the vector may contain an IRES sequence and a 2A adapter sequence. In others, the expression of multiple genes linked by a 2A peptide can be promoted by placing a spacer sequence (GSG) preceding the 2A peptide. In some cases, the construct may combine spacers, adapters, aptamers, promoters, or combinations thereof. For example, the adapter may have a spacer (SGSG (SEQ ID NO:121) or GSG or Whitlow adapter) and a furin protease adapter (RAKR (SEQ ID NO:123)) cleavage site and be combined with different 2A peptides. The spacer may be I-Ceui. In some cases, the adapter may be engineered. For example, the adapter may be designed to include chemical properties such as hydrophobicity. In some cases, at least two adapter sequences may produce the same protein. In others, multiple adapters may be used in the vector. For example, the gene of interest may be separated by at least two adapters.

[0387] 3. A polypeptide linker encoded by one or more polynucleotides.

[0388] In some embodiments, the polynucleotides described herein may encode two or more polypeptides. In some of these embodiments, the polynucleotides may be separated by an intervention sequence encoding an intervention connector polypeptide. As used herein, the term "intervention connector polypeptide" refers to the amino acid sequence separating two or more polypeptides encoded by the polynucleotide and is distinguished from the term "peptide connector," which refers to the amino acid sequence optionally included in the polypeptide constructs disclosed herein for linking transmembrane domains to cell surface polypeptides (e.g., truncated variants of native polypeptides).

[0389] In some cases, the interventional connector peptide is a cleavable interventional connector peptide. In some embodiments, the interventional connector peptide is a cleavable connector or a ribosomal skipping connector. In some embodiments, the cleavable connector or ribosomal skipping connector sequence is selected from 2A, GSG-2A, GSG connector, SGSG connector (SEQ ID NO: 121), furin protease connector variants and their derivatives. In some embodiments, the 2A connector is a p2A connector, T2A connector, F2A connector, or E2A connector. In some embodiments, the peptide of interest is expressed as a fusion protein linked by a cleavable interventional connector peptide. In some embodiments, the cleavable interventional connector peptide may be one or more of the following: F / T2A, T2A, p2A, 2A, GSG-p2A, GSG connector, and furin protease connector variants. Connectors (polynucleotide and peptide sequences) are disclosed, for example, in PCT / US2016 / 061668 (WO2017083750), which was published on May 18, 2017.

[0390] In some embodiments, the adapter polypeptide comprises the sequences disclosed in Table 3.

[0391] Table 3: Linker amino acid sequences

[0392] In some cases, the adaptor peptide may contain the amino acid sequence “RAKR” (SEQ ID NO:123). In other cases, the furin-intervention adaptor peptide may be encoded by a polynucleotide sequence containing “CGTGCAAAGCGT” (SEQ ID NO:125) or “AGAGCTAAGAGG” (SEQ ID NO:126).

[0393] In some embodiments, the interventional linker polypeptide comprises a furin protease polypeptide and a 2A polypeptide linked by a polypeptide linker comprising at least three hydrophobic amino acids. In some cases, the at least three hydrophobic amino acids are selected from the list consisting of glycine (Gly) (G), alanine (Ala) (A), valine (V) (V), leucine (Leu) (L), isoleucine (Ile) (I), proline (Pro) (P), phenylalanine (Phe) (F), methionine (Met) (M), and tryptophan (Trp) (W). In some cases, the polypeptide linker may also comprise one or more GS linker sequences, such as (GS)n (SEQ ID NO:129), (SG)n (SEQ ID NO:130), (GSG)n (SEQ ID NO:131), and (SGSG)n (SEQ ID NO:132), where n can be any number from zero to fifteen.

[0394] The linkers described herein can, in certain circumstances, enhance biological activity, increase expression yield, and achieve desired pharmacokinetic profiles. Linkers may also contain hydrazones, peptides, disulfide bonds, or thioesters.

[0395] Flexible linkers can be applied when the connected domains require a certain degree of movement or interaction. Flexible linkers can be composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Flexible linkers can have sequences primarily composed of fragments of Gly and Ser residues (“GS” linkers). An example of a flexible linker may have a sequence of (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO:127). The length of this exemplary GS linker can be optimized by adjusting the copy number “n” to achieve proper separation of functional domains or to maintain necessary interdomain interactions. Besides GS linkers, other flexible linkers can also be used in recombinant fusion proteins. In some cases, flexible linkers may also be enriched with small or polar amino acids, such as Gly and Ser, but may contain additional amino acids, such as Thr and Ala, to maintain flexibility. In other cases, polar amino acids such as Lys and Glu may be used to improve solubility.

[0396] The flexible linkers useful in this invention can be enriched with small or polar amino acids, such as Gly and Ser, to provide good flexibility and solubility. Flexible linkers are a suitable choice when fusion protein domains are expected to have certain movements or interactions. Furthermore, although flexible linkers cannot have rigid structures, they can act as passive linkers to maintain distances between functional domains. The length of the flexible linker can be adjusted to allow for proper folding or to achieve optimal bioactivity of the fusion protein.

[0397] In some cases, the interventional linker peptides described herein are rigid linkers. Rigid linkers can be used to maintain a fixed distance between the domains of a peptide. Examples of rigid linkers may include: linkers that form α-helices, Pro-rich sequences, (XP)n, X-Pro backbones, A(EAAAK)nA (n=2-5) (SEQ ID NO:128), etc. Rigid linkers can exhibit a relatively rigid structure in some cases by employing an α-helical structure or containing multiple Pro residues.

[0398] In some embodiments, the interventional connector peptide may be non-cleavable. A non-cleavable connector can covalently link functional domains together to function as a single molecule throughout in vivo or in vitro processes.

[0399] In other embodiments, the interventional linker polypeptide may be cleavable. A cleavable linker may be introduced to release the free functional domain in vivo. The cleavable linker may be cleaved in the presence of reducing agents, proteases, etc. For example, the reduction of disulfide bonds may be used to generate a cleavable linker. In the case of disulfide linker, cleavage can occur through a cleavage event that involves disulfide bond exchange with a thiol group (e.g., glutathione). In other cases, in vivo cleavage of the linker in the recombinant fusion protein may also be accomplished by proteases that can be expressed in vivo under pathological conditions (e.g., cancer or inflammation), expressed in specific cells or tissues, or confined to certain cellular compartments. In some cases, the cleavable linker may allow targeted cleavage. For example, the specificity of many proteases can provide slower linker cleavage in confined compartments. The cleavable linker may also contain hydrazones, peptides, disulfide bonds, or thioesters. For example, hydrazones can confer serum stability. In other cases, hydrazones may allow cleavage in acidic compartments. The pH of the acidic compartment can be as high as 7. The linker may also contain thioethers. Thioethers may be non-reducible. Thioethers can be designed for the hydrolytic degradation of intracellular proteins.

[0400] A method for obtaining improved expression of a polypeptide construct is provided, comprising: providing a polynucleotide encoding the polypeptide construct, wherein the polypeptide construct comprises a first functional polypeptide and a second functional polypeptide, and the first functional polypeptide and the second functional polypeptide are linked by a linker polypeptide comprising a sequence having at least 60% identity with the sequence APVKQ (SEQ ID NO: 133); and expressing the polynucleotide in a host cell, wherein the expression results in improved expression of the polypeptide construct relative to a corresponding polypeptide construct that does not have a linker polypeptide comprising a sequence having at least 60% identity with the sequence APVKQ (SEQ ID NO: 133).

[0401] 4. Engineered and designed connectors

[0402] In some implementations, the polynucleotide linker may be engineered or designed. The method for designing the linker may be computational. In some cases, the computational method may include graphical techniques. The computational method can be used to search for suitable peptides from a three-dimensional peptide structure library derived from a database. For example, the Brookhaven Protein Database (PDB) can be used to measure the spatial distances of selected amino acids in the linker.

[0403] D. Packaging sequence

[0404] In addition to the expression cassette, the vector may also contain a packaging sequence. As used herein, the term "packaging sequence" refers to a sequence located within the chimpanzee adenovirus genome that is essential for loading viral DNA into the viral capsid or particle. See Ostapchuk et al., Curr. Topics in Microbiology and Immunology, 272:165-185 (1995), and Ahi et al., Frontiers in Microbiology, 7:150 (2016).

[0405] E. HPV early zone protein

[0406] Constitutive or induced expression of HPV early (E) region proteins provides targets for effective HPV vaccines.

[0407] HPV genes (E1-E8) regulate viral expression and replication, while late (L) genes control viral protein coding (8-10). The functions of early HPV proteins include: E1 and E2 function in viral replication / transcription (e.g., E2 regulates the expression of E6 and E7; and the E1 / E2 interaction is essential for viral replication); E4 and E5 have increased expression in the late stages of the viral replication cycle; and E6 and E7 work synergistically during replication (E6 is essential for epithelial genome maintenance, and E7 amplifies the epithelial cell compartments active in DNA replication).

[0408] An exemplary embodiment of the present invention is an HPV6 / 11 vaccine that delivers a multi-epitope antigen design containing HPV 6 and 11 epitopes—namely, key immunogenic peptides derived from E2 (HPV6), E4 (HPV6), E6 (HPV6 / 11), and E7 (HPV6 / 11), wherein the HPV6-derived peptides have high sequence similarity to HPV11.

[0409] F. Adenovirus expression cassette

[0410] In some cases using adenovirus vectors, the expression cassette may be located at an E1 region deletion junction or an E4 region deletion junction. In some embodiments, the expression cassette is located at an E1 region deletion junction.

[0411] In some embodiments, the expression cassette is cloned relative to the adenovirus genome in a right-to-left direction.

[0412] In some embodiments, when the expression cassette is cloned within the adenovirus genome in a right-to-left direction, it contains a nucleic acid sequence of SEQ ID NO:116 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with: SEQ ID NO:116 or a codon degenerate variant of SEQ ID NO:116, or a conserved substitution variant of SEQ ID NO:116, or a non-conserved substitution variant of SEQ ID NO:116).

[0413] III. Antigenic Bioinformatics Workflow for HPV Vaccine Design

[0414] A. Identification of new HPV antigenic components

[0415] Based on the important roles of HPV E2 and E4 genetic components in essential HPV functions, the locations of corresponding proteins, and computer simulation predictions, antigens derived from E2 and E4 were identified for use in HPV therapeutic vaccines. Non-carcinogenic and virally inactivating genetic modifications were also applied to eliminate the viral and carcinogenic biological activities of HPV proteins (e.g., HPV E2 and E6 proteins).

[0416] Genetic manipulation was also employed to produce protein sequences rearranged in such a way that the immunogenicity of the peptides was preserved, but the oncogenic and viral amplification functions of E7 and E4 were eliminated, respectively.

[0417] Therefore, some innovative aspects of the designs illustrated in this specification include: (1) using gene constructs encoding fusion proteins containing four or more different HPV proteins; (2) combining amino acid point mutations and overlapping polypeptide sequence shuffling techniques to inactivate oncogenic and essential viral functions; (3) incorporating HPV proteins containing multiple antigenic components from HPV proteins highly expressed in host infected cells; (4) the first known hybrid antigen design; (5) combining epitopes from high- and low-cancer-risk HPV strains; (6) using mixed and regularly repeating linkers; (7) using rigid linkers to stabilize polypeptide subunits and prevent unwanted intramolecular interactions; (8) using cleavable linkers between epitopes; and / or (9) dual use of linker sequences to provide protein-protein linker (-) function as well as antigen and epitope themselves (i.e., antigenicity conferred by the linker sequence).

[0418] Antigenicity is the ability to stimulate antibody production or cell-mediated immune responses. The antigenicity of the final designed sequences was predicted using Vaxjen software, an antigen recognition model based on the major chemical properties of amino acid sequences and independent of alignment. The results showed that the five antigen sequences were antigenic. See Table 4 (Antigenic Viruses and Tumors). (SEQ ID NO:121)(SEQ ID NO:123) In some embodiments, two or more polypeptides encoded by the polynucleotides described herein may be separated by an intervening sequence encoding a linker polypeptide. In some cases, the linker is a cleavable linker. In some embodiments, the polypeptide of interest is expressed as a fusion protein linked by a cleavable linker polypeptide. In some embodiments, the cleavable linker polypeptide may be any one or two of the furin protease linker, fmdv, p2a, GSG-p2a and / or fp2a described below. In some cases, the linker is APVKQGSG (SEQ ID NO:124).

[0419] Allergens are small antigens that typically trigger an antibody response. Allergenicity (i.e., whether an antigen is an allergen or not) was predicted using ALLERTOP, a bioinformatics-based software that uses machine learning methods for allergen prediction. ALLERTOP includes logistic regression (LR), decision tree (DT), Naive Bayes (NB), random forest (RF), multilayer perceptron (MLP), and k-nearest neighbors (kNN). The results showed that these five antigen sequences were non-allergens. See Table 4 (Allergenicity).

[0420] In adaptive immunotherapy, cross-reactivity in different tissues or the induction of autoimmune side effects have important safety implications. Sequence homology analysis was performed to assess whether these neoantigens cross-react with the human proteome using BLAST (a basic local alignment search tool). No host cross-reactivity was identified in these five antigen sequences. See Table 4 (Host Cross-Reactivity).

[0421] B. Software / Tools

[0422] Software tools used to implement the designs described herein include, but are not limited to: ALLERTOP (See: AllerTOP v.2—a server for in silico prediction of allergens; J Mol Model, June 2014, 20(6):2278, doi: 10.1007 / s00894-014-2278-5, published online May 31, 2014.) ANN (See: Reliable prediction of T-cell epitopes using neural networks with novel sequence representations; Protein Sci., May 2003, 12(5):1007–1017.) BLAST (Basic Local Alignment Search Tool; NCBI, National Center for Biotechnology Information, US National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA.) CLUSTALW2 (European Laboratory for Molecular Biology – European Institute for Bioinformatics (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK, Tel: +44 (0)1223 49 44 44.) GENEIOUS VI 1.1.5 (See: geneious.com / biopharma / ) IEDB CONSENSUS (see: A Common Epitope Prediction Method Reveals Mouse CD8) + The breadth of T cell responses to vaccinia virus (A consensus epitope prediction approach identifies the breadth of murine T(CD8+)-cell responses to vaccinia virus); Nat Biotechnol., July 2006, 24(7):817–819, published online June 11, 2006.) NETMHCPAN 4.0 (See: Gapped sequence alignment using artificial neural networks: application to the MHC class I system; Bioinformatics, Feb. 15, 2016, 32(4):511–517, doi: 10.1093 / bioinformatics / btv639, published online on Oct. 29, 2015.) PHYRE2 (See: The Phyre2 webportal for protein modeling, prediction and analysis; May 7, 2015; nature.com / articles / nprot.2015.053; doi: 10.1038 / nprot.2015.053.) PYMOL MOLECULAR GRAPHICS SYSTEM V2.1.1 (PyMOL Molecular Graphics System version 2.1.1; see: pymol.org / 2 / ; sourceforge.net / projects / pymol / support) VAXJEN (See: VaxiJen: a server for prediction of protective antigens, tumor antigens and subunit vaccines; BMC Bioinformatics, January 5, 2007, 8:4.) III. HPV Antigen Design and Variants In one exemplary embodiment, the polynucleotide encoding the fusion protein (e.g., an HPV antigen) comprises two or more HPV proteins. For example, the polynucleotide encoding the fusion protein comprises one or more HPV6 proteins, one or more HPV11 proteins, and one or more HPV45 proteins (e.g., one HPV6 protein and one HPV11 protein).

[0423] Exemplary HPV6 proteins include, but are not limited to, one or more HPV6 E2 proteins, one or more HPV6 E4 proteins, one or more HPV6 E6 proteins, one or more HPV6 E7 proteins, and combinations thereof, including, but not limited to, combinations of HPV6 and / or HPV11 protein types, and combinations of multiple copies or variants of a single HPV6 and / or HPV11 protein.

[0424] In some embodiments, the HPV6 E2 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:1. In some embodiments, the HPV6 E2 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the HPV6 E2 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1. In some embodiments, the HPV6 E2 protein comprises an amino acid sequence having 1-36 amino acid substitutions (e.g., 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 amino acid substitutions) relative to SEQ ID NO:1. In some embodiments, the HPV6 E2 protein comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the HPV E2 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:105. In some embodiments, the HPV E2 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:105.

[0425] In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:3 or 7. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3 or 7. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:3 or 7. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:3 or 7. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:3. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:3. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:3. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:7 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:7. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:7. In some embodiments, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO:3 or 7. In some embodiments, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the HPV E4 protein comprises an amino acid sequence selected from SEQ ID NO:3, 7, 107, 111, and 172-179.In some embodiments, the HPV E4 protein comprises an amino acid sequence selected from SEQ ID NO:3 and 7.

[0426] In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:11 or 40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:11 or 40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:11 or 40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:11 or 40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:11. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:11. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:11. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:11. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:40. In some embodiments, the HPV6 E6 protein comprises the amino acid sequence of SEQ ID NO:11 or 40. In some embodiments, the HPV6 E6 protein comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the HPV6 E6 protein comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:110 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:110.In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:110. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:110. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence selected from SEQ ID NO:11, 40, 110, and 197-204. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence selected from SEQ ID NO:11, 40, and 110.

[0427] In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:5 or 9. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5 or 9. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5 or 9. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having 1-10 amino acid substitutions relative to SEQ ID NO:5 or 9 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid substitution). In some embodiments, the HPV6E7 protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:5 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5. In some embodiments, the HPV6E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5. In some embodiments, the HPV6E7 protein comprises an amino acid sequence having 1-10 amino acid substitutions relative to SEQ ID NO:5 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid substitution). In some embodiments, the HPV6E7 protein comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:9 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:9. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:9. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having 1-10 amino acid substitutions relative to SEQ ID NO:9 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid substitution). In some embodiments, the HPV6 E7 protein comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the HPV6 E7 protein comprises the amino acid sequence of SEQ ID NO:5 or 9.In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:109. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:109. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:109. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence having 1-10 amino acid substitutions relative to SEQ ID NO:109 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid substitution). In some embodiments, the HPV6 E7 protein comprises the amino acid sequence of SEQ ID NO:109. In some embodiments, the HPV6 E7 protein comprises an amino acid sequence selected from SEQ ID NO:5, 9, 109, and 189-196.

[0428] Exemplary HPV11 proteins include, but are not limited to, one or more HPV11 E6 proteins and one or more HPV11 E7 proteins. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with respect to SEQ ID NO:42. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO:42. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO:42. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:42. In some embodiments, the HPV11 E6 protein comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:108. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:108. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:108. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:108. In some embodiments, the HPV11 E6 protein comprises the amino acid sequence of SEQ ID NO:108. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:112. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:112. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:112. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:112. In some embodiments, the HPV11 E6 protein comprises the amino acid sequence of SEQ ID NO:112.In some embodiments, the HPV11 E6 protein comprises an amino acid sequence selected from SEQ ID NO:42, 108, 112, 180-188, and 213-220. In some embodiments, the HPV11 E6 protein comprises an amino acid sequence selected from SEQ ID NO:42, 108, and 112.

[0429] In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:45 or 106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:45 or 106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:45 or 106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:45 or 106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:45. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:45. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:45. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:45. In some embodiments, the HPV11 E7 protein comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:106. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:106. In some embodiments, the HPV11 E7 protein comprises the amino acid sequence of SEQ ID NO:106.In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:114. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:114. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:114. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:114. In some embodiments, the HPV11 E7 protein comprises the amino acid sequence of SEQ ID NO:114. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence selected from SEQ ID NO:45, 106, 114, 164-171, and 229-236. In some embodiments, the HPV11 E7 protein comprises an amino acid sequence selected from SEQ ID NO:45, 106, and 114.

[0430] A common sequence between the HPV6 E2 and HPV11 E2 protein sequences or fragments thereof can be identified and referred to as the HPVE2 protein. In some embodiments, the HPV E2 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:105. In some embodiments, the HPVE2 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:105. In some embodiments, the HPV E2 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:105. In some embodiments, the HPV E2 protein comprises an amino acid sequence having 1-36 amino acid substitutions (e.g., 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 amino acid substitutions) relative to SEQ ID NO:105. In some embodiments, the HPV E2 protein comprises an amino acid sequence selected from SEQ ID NO:105 and 154-163.

[0431] A common sequence between the HPV6 E4 and HPV11 E4 protein sequences or fragments thereof can be identified and referred to as the HPVE4 protein, which may be included in any polynucleotide or fusion protein described herein. In some embodiments, the HPV E4 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:107. In some embodiments, the HPV E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:107. In some embodiments, the HPV E4 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:107. In some embodiments, the HPV E4 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:107. In some embodiments, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO:107. In some embodiments, the HPV E4 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:111. In some embodiments, the HPV E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:111. In some embodiments, the HPV E4 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:111. In some embodiments, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO:111. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence selected from SEQ ID NO:107, 111, 172-179, and 205-212. In some embodiments, the HPV6E4 protein comprises an amino acid sequence selected from SEQ ID NO:107 and 111.

[0432] A common sequence between the HPV6 E6 and HPV11 E6 protein sequences or fragments thereof can be identified and referred to as the HPVE6 protein, which may be included in any polynucleotide or fusion protein disclosed herein. In some embodiments, the HPV E6 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:115. In some embodiments, the HPV E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:115. In some embodiments, the HPV E6 protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:115. In some embodiments, the HPV E6 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:115. In some embodiments, the HPV E6 protein comprises the amino acid sequence of SEQ ID NO:115. In some embodiments, the HPV E6 protein comprises an amino acid sequence selected from SEQ ID NO:115 and 237-246.

[0433] A common sequence between the HPV6 E7 and HPV11 E7 protein sequences or fragments thereof can be identified and referred to as the HPVE7 protein, which may be included in any polynucleotide or fusion protein disclosed herein. In some embodiments, the HPV E7 protein comprises an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with respect to SEQ ID NO:113. Exemplary variants include the amino acid sequences of SEQ ID NO:221-228. In some embodiments, the HPV E7 protein comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO:113. In some embodiments, the HPV E7 protein comprises an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO:113. In some embodiments, the HPV E7 protein comprises an amino acid sequence having 1-5 amino acid substitutions (e.g., 1-4, 1-3, 1-2, or 1 amino acid substitution) relative to SEQ ID NO:113. In some embodiments, the HPV E7 protein comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the HPV E7 protein comprises an amino acid sequence selected from SEQ ID NO: 113 and 221-228.

[0434] In some embodiments, the fusion protein comprises copies of one or more HPV6 and / or HPV11 proteins. For example, the fusion protein comprises copies of one or more HPV6 E4, HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 proteins. In some embodiments, the fusion protein comprises an HPV6 E4 protein containing an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:3 and an HPV6 E4 protein containing an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:7. In some embodiments, the fusion protein comprises an HPV6 E4 protein containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3 and an HPV6 E4 protein containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7. In some embodiments, the fusion protein comprises an HPV6 E4 protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO:3 and an HPV6 E4 protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO:7. In some embodiments, the fusion protein comprises an HPV6 E4 protein containing the amino acid sequence of SEQ ID NO:3 and an HPV6 E4 protein containing the amino acid sequence of SEQ ID NO:7. In some embodiments, the fusion protein comprises an HPV6 E6 protein containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO:11 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) and an HPV6 E6 protein containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO:40 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the fusion protein comprises an HPV6 E6 protein having an amino acid sequence having at least 90% sequence identity with SEQ ID NO:11 and an HPV6 E6 protein having an amino acid sequence having at least 90% sequence identity with SEQ ID NO:40. In some embodiments, the fusion protein comprises an HPV6 E6 protein having an amino acid sequence having at least 95% sequence identity with SEQ ID NO:11 and an HPV6 E6 protein having an amino acid sequence having at least 95% sequence identity with SEQ ID NO:40.In some embodiments, the fusion protein comprises an HPV6 E6 protein containing the amino acid sequence of SEQ ID NO:11 and an HPV6 E6 protein containing the amino acid sequence of SEQ ID NO:40. In some embodiments, the fusion protein comprises an HPV6 E7 protein containing an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:5 and an HPV6 E7 protein containing an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity) with SEQ ID NO:9. In some embodiments, the fusion protein comprises an HPV6 E7 protein containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5 and an HPV6 E7 protein containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:9. In some embodiments, the fusion protein comprises an HPV6 E7 protein having an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5 and an HPV6 E7 protein having an amino acid sequence having at least 95% sequence identity with SEQ ID NO:9. In some embodiments, the fusion protein comprises an HPV6 E7 protein having the amino acid sequence of SEQ ID NO:5 and an HPV6 E7 protein having the amino acid sequence of SEQ ID NO:9.

[0435] In one exemplary embodiment, the polynucleotide-encoded polypeptide construct or fusion protein of the present invention comprises the amino acid sequence of SEQ ID NO:66, 68, 70, 72, or 74 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the following: SEQ ID NO:66, 68, 70, 72, or 74, or a conserved substitution variant of SEQ ID NO:66, 68, 70, 72, or 74). In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:68 (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:66 (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:70 (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:72 (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:74 (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity).

[0436] In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 85% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 95% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 96% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 97% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 98% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises an amino acid sequence having at least 99% identity with SEQ ID NO:68. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:68.

[0437] In one exemplary embodiment, the polypeptide construct of the present invention comprises the sequence of SEQ ID NO:68 or a functional variant thereof (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity: SEQ ID NO:68, or a conserved substitution variant of SEQ ID NO:68, or a non-conserved substitution variant of SEQ ID NO:68).

[0438] In some embodiments, the polypeptide constructs of the present invention have functional variants of SEQ ID NO:68 that, compared to SEQ ID NO:68, have similar or enhanced binding affinity to HPV6 / 11-related proteins and / or elicit similar or enhanced immunogenic responses. Such variants can be readily identified using sequence alignment software such as ClustalW.

[0439] In some embodiments, the polypeptide construct of the present invention comprises any of SEQ ID NO:105-115 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any of the following: any of SEQ ID NO:105-115, or a conserved substitution variant of any of SEQ ID NO:105-115, or a non-conserved substitution variant of any of SEQ ID NO:105-115). In one exemplary embodiment, the polypeptide construct of the present invention comprises each of SEQ ID NO:105-115.

[0440] In some embodiments, the polynucleotide construct variant may comprise one or more conserved substitution variants from any of the antigenic regions of SEQ ID NO:105-115. For example, any hydrophilic amino acid may be substituted with any other hydrophilic amino acid, any aliphatic amino acid may be substituted with any other aliphatic amino acid, any basic amino acid may be substituted with any other basic amino acid, any aromatic amino acid may be substituted with any other aromatic amino acid, and any combination thereof. Exemplary polynucleotide construct variants containing conserved substitution mutations include, but are not limited to, SEQ ID NO:143 and SEQ ID NO:144.

[0441] In some embodiments, polynucleotide construct variants may include one or more amino acid additions or deletions. For example, one or more amino acids may be added to or removed from any antigenic segment of the polynucleotide construct described herein. Exemplary polynucleotide construct variants that include amino acid additions and / or deletions include, but are not limited to, SEQ ID NO:144-148.

[0442] In some embodiments, the polypeptide construct variant has the same HPV6 / 11 antigen region as SEQ ID NO:68, but the antigen region of SEQ ID NO:68 is shuffled and rearranged in a different order than that of SEQ ID NO:68. In some embodiments, the variant includes the antigen regions of HPV E2 (SEQ ID NO:105), HPV11 E7 (SEQ ID NO:106), HPV E4 (SEQ ID NO:107), HPV11 E6 (SEQ ID NO:108), HPV6 E7 (SEQ ID NO:109), HPV6 E6 (SEQ ID NO:110), HPV E4 (SEQ ID NO:111), HPV11 E6 (SEQ ID NO:112), HPV E7 (SEQ ID NO:113), HPV11 E7 (SEQ ID NO:114), and HPV E6 (SEQ ID NO:115) of SEQ ID NO:68, but the antigen regions are scrambled and rearranged compared to the antigen region order of SEQ ID NO:68. Exemplary peptide construct variant sequences include, but are not limited to, SEQ ID NO:134 (antigen region sequence: HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, HPV E7, HPV11 E7, HPV E6, HPV E2, and HPV11 E7), SEQ ID NO:135 (antigen region sequence: HPV E6, HPV11 E7, HPV E2, HPV11 E7, HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, and HPV E7), and SEQ ID NO:136 (antigen region sequence: HPV E7, HPV11 E7, HPV E6, HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, HPV E2, and HPV11 E7).

[0443] The polypeptide construct contains about 2 to about 20 antigenic regions (e.g., about 5 to about 15, about 10 to about 12). For example, the polypeptide construct contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more antigenic regions. In some embodiments, the polypeptide construct variant has fewer antigenic regions compared to SEQ ID NO:68. For example, the polypeptide construct variant has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fewer antigenic regions than SEQ ID NO:68. A polypeptide construct variant containing fewer antigenic regions than SEQ ID NO:68 may omit any of the antigenic regions of SEQ ID NO:68. Exemplary peptide construct variants containing fewer antigenic regions than SEQ ID NO:68 include, but are not limited to, SEQ ID NO:137 (antigen region sequence: HPV E2, HPV11 E7, HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, HPV E7, and HPV E6), SEQ ID NO:138 (antigen region sequence: HPV E4, HPV11 E6, HPV6 E7, HPV E4, HPV11 E6, HPV E7, HPV11 E7, HPV E6, HPV E2, and HPV11 E7), and SEQ ID NO:139 (antigen region sequence: HPV E7, HPV11 E7, HPV E6, HPV11 E6, HPV6 E7, HPV6 E6, HPV11 E6, HPV E2, and HPV11 E7). In some embodiments, the peptide construct variant has more antigenic regions compared to SEQ ID NO:68. For example, the peptide construct variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more antigenic regions than SEQ ID NO:68. A peptide construct variant containing more antigenic regions than SEQ ID NO:68 may add one or more antigenic regions (e.g., N-terminal, C-terminal, between other antigenic regions, inserted into and interrupted by other antigenic regions, and combinations thereof) to any portion of the antigenic region portion of the peptide construct.Exemplary peptide construct variants containing more antigenic regions than SEQ ID NO:68 include, but are not limited to, SEQ ID NO:140 (antigen region sequence: HPV E2, HPV11 E7, HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, HPV E7, HPV11 E7, HPV E6, and HPV11 E6), SEQ ID NO:141 (antigen region sequence: HPV E6E7, HPV E7, HPV11 E7, HPV E6, HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, HPV E2, HPV11 E7), and SEQ ID NO:142 (antigen region sequence: HPV E2, HPV11 E7, HPV E4, HPV11 E6, HPV6E7, HPV E4, HPV6 E6, HPV...). E4, HPV11 E6, HPV E7, HPV11 E7, HPV E7, HPV E6).

[0444] In some embodiments, the antigen region or fusion protein may also include any of the linker peptides described herein (e.g., rigid linker peptides, flexible linker peptides, or combinations thereof).

[0445] In some embodiments, the antigen region or fusion protein may also include any of the agonist peptides described herein (also referred to as enhancer agonist peptides or agonist enhancers). Agonist peptides are modified versions of immunogenic epitopes that enhance the immune response. For example, agonist peptides can improve T cell recognition while maintaining compatibility with native peptide-MHC interactions on tumor cells. See, for example, Tsang et al., Vaccine 35(19):2605-2611(2017). Exemplary agonist peptides include, but are not limited to, HPV6 agonist peptides (e.g., HPV6 E2 agonist peptide, HPV6 E4 agonist peptide, HPV6 E6 agonist peptide, and HPV11 agonist peptide), HPV11 agonist peptides (e.g., HPV11 E6 agonist peptide and HPV11 E7 agonist peptide), and HPV16 agonist peptide. In some embodiments, if the agonist peptide is included, the agonist peptide is an HPV16 E6 agonist peptide, for example, an agonist peptide comprising the amino acid sequence of SEQ ID NO:48 or SEQ ID NO:50. In some embodiments, if the agonist peptide is included, the agonist peptide is an HPV16 E7 agonist peptide, for example, an agonist peptide comprising the amino acid sequence of SEQ ID NO:52 or SEQ ID NO:54.

[0446] IV. Vaccines

[0447] This disclosure provides a vaccine comprising a polynucleotide encoding the fusion protein described herein. In some embodiments, the vaccine comprises a polynucleotide encoding a fusion protein comprising an HPV6 protein selected from HPV6 E2, HPV6 E4, HPV6 E6, and HPV6 E7 proteins; and an HPV11 protein selected from HPV11 E6 and HPV11 E7 proteins. The vaccine of this disclosure can be used for the prevention and / or treatment of HPV infection and HPV-related diseases.

[0448] In some embodiments, the vaccine comprises a polynucleotide encoding a fusion protein, said fusion protein comprising HPV6 E2, HPV6 E4, HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7 proteins. In some embodiments, the HPV6 E2 protein comprises the amino acid sequence of SEQ ID NO:1, the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO:3 or 7, the HPV6 E6 protein comprises the amino acid sequence of SEQ ID NO:11 or 40, the HPV6 E7 protein comprises the amino acid sequence of SEQ ID NO:5 or 9, the HPV11 E6 protein comprises the amino acid sequence of SEQ ID NO:42, and the HPV11 E7 protein comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:68, or comprises the amino acid sequence of SEQ ID NO:68 or a conserved substitution variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 66, 70, 72, or 74. In some embodiments, the fusion protein further comprises a rigid linker polypeptide and / or an HPV16 E6 or E7 agonist enhancer.

[0449] The vaccine's polynucleotides are operatively linked to elements that promote the expression of the fusion protein, such as a promoter, 5' untranslated region (UTR), transcription start site (TSS), 3' UTR, tetracycline response element, and / or Kozak segment. In some embodiments, the promoter is operatively linked to a promoter enhancer segment. The vaccine of this disclosure can be administered via any suitable route, including, for example, intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intranasal, oral, or transdermal administration. The vaccine can be administered as a single dose or as multiple doses over time. The vaccine can be administered alone or in combination with other therapeutic agents, such as chemotherapeutic agents, immunomodulators, or other vaccines.

[0450] In some embodiments, the vaccine comprises a vector containing a polynucleotide encoding the fusion protein. The vector can be any suitable vector, such as a plasmid, viral vector, bacterial vector, or yeast vector. In some embodiments, the vector is a plasmid vector, such as a DNA plasmid vector. In some embodiments, the vector is a viral vector, such as an adenovirus vector, adeno-associated virus vector, retroviral vector, lentiviral vector, vaccinia virus vector, or herpesvirus vector. In some embodiments, the vector is an adenovirus vector, such as a chimpanzee adenovirus vector.

[0451] In some embodiments, the vaccine comprises a polypeptide encoded by the polynucleotide. The polypeptide can be prepared by any suitable method, such as expression in host cells, expression in a cell-free system, or chemical synthesis. The polypeptide can be purified by any suitable method, such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, or high-performance liquid chromatography (HPLC). The polypeptide can be formulated with a pharmaceutically acceptable carrier for administration as a vaccine.

[0452] In some embodiments, the vaccine comprises a composition containing the polynucleotide, carrier, or polypeptide. The composition may also contain additional components, such as adjuvants, stabilizers, preservatives, or other therapeutic agents. Suitable adjuvants include, for example, aluminum salts, oil-in-water emulsions, Toll-like receptor (TLR) agonists, and saponins. The choice of adjuvant may depend on factors such as the desired immune response, route of administration, and target population.

[0453] In some embodiments, the vaccine comprises cells containing the polynucleotide, carrier, polypeptide, or composition. The cells can be any suitable cell type, such as bacterial cells, yeast cells, insect cells, or mammalian cells. In some embodiments, the cells are immune cells, such as dendritic cells, macrophages, monocytes, B cells, T cells, or natural killer (NK) cells. In some embodiments, the cells are dendritic cells, such as human dendritic cells. In some embodiments, the cells are T cells, such as CD4+ T cells or CD8+ T cells. The T cells can be naive T cells, effector T cells, or memory T cells. The T cells can be regulatory T cells (Treg) or γδ T cells. The cells can be autologous or allogeneic relative to the recipient. The cells can be modified to express the fusion protein by any suitable method, such as transfection, transduction, or electroporation. The cells can be administered as a vaccine via any suitable route, such as intravenous, intradermal, or subcutaneous administration. Using immune cells such as dendritic cells or T cells as a vaccine can enhance the immune response against HPV antigens and improve vaccine efficacy.

[0454] V. Treatment Methods

[0455] This invention relates in part to a method of treating a disease or condition in a subject, comprising administering to said subject the polynucleotides, peptides, carriers, compositions, vaccines, or cells of the invention. In some embodiments, the method relates to administering to a subject suffering from anogenital warts, lower genital tract tumors (e.g., cervical, vaginal, and vulvar intraepithelial neoplasia), cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer the polynucleotides, peptides, carriers, compositions, vaccines, or cells of the invention. In some embodiments, the method relates to administering to a subject suffering from a malignant tumor caused by HPV 6 / 11 the polynucleotides, peptides, carriers, compositions, vaccines, or cells of the invention.

[0456] The present invention also relates in part to a method for eliciting a T-cell response in HPV-infected (e.g., HPV 6 / 11+) cells in a desired subject (e.g., a subject with RRP), the method comprising administering the vector of the present invention to the subject. In some embodiments, the method relates to administering the polynucleotide, polypeptide, vector, composition, vaccine, or cells of the present invention to a subject suffering from a malignancy caused by HPV 6 / 11.

[0457] This invention also relates to inducing an anti-HPV immune response in a desired subject (e.g., a subject suffering from RRP or other HPV-related diseases or conditions, including those related to HPV6 or HPV11). Inducing this immune response may involve increasing the recruitment, number, or proliferation of various immune cells, including but not limited to dendritic cells, Langerhans cells, natural killer cells, natural killer T cells, and keratinocytes, compared to an HPV immune response without the administration of the polynucleotide, carrier, fusion protein, or composition described herein. In some embodiments, inducing an anti-HPV immune response includes administering to the subject a therapeutically effective amount of any of the polynucleotides, carriers, fusion proteins, or compositions thereof described herein. In some embodiments, inducing an anti-HPV immune response includes administering to the subject a therapeutically effective amount of any of the carriers described herein (e.g., a carrier comprising SEQ ID NO: 68). In some embodiments, the therapeutically effective amount of the carrier comprises about 1 x 103 11 5x10 11 Particle unit (PU).

[0458] In some implementations, the disease or condition to be treated is RRP and the route of administration is subcutaneous.

[0459] In some embodiments, the method of the present invention prevents disease progression with a lower PU dose than that of prior methods known in the art. For example, in some embodiments, the method uses a PU dose of 5e 9 The PU dose is delivered via a carrier, composition, or vaccine to achieve protection against the disease. In other embodiments, the method uses 5e 10The carrier, composition, or vaccine administered at a dose of PU achieves protection against disease. In some embodiments, the method of the present invention prevents disease progression by administering the therapeutic composition fewer times than prior methods known in the art. For example, in some embodiments, the method achieves protection against disease with only a single administration of the carrier, composition, or vaccine.

[0460] In some implementations, the object of treatment is a mammal, such as a primate. In some implementations, the object of treatment is a human.

[0461] The methods may involve administering the polynucleotide, polypeptide, carrier, composition, vaccine, or cell in a therapeutically effective amount to treat the disease or condition. The methods may involve administering the polynucleotide, polypeptide, carrier, composition, vaccine, or cell in a therapeutically effective amount to enhance the activity of T-cell responses against specific HPV proteins or antigens (e.g., HPV6 / 11-specific proteins or antigens). The methods may involve administering the polynucleotide, polypeptide, carrier, composition, vaccine, or cell in a therapeutically effective amount to treat RRP. The methods may involve administering the polynucleotide, polypeptide, carrier, composition, vaccine, or cell in a therapeutically effective amount to reduce a subject's Delkay score.

[0462] The effective dose may vary depending on the subject's condition, age, sex, medical history, and / or weight. The dose may also vary depending on the condition being treated, the encoded anti-inflammatory agent, the carrier used for administration, the type of cell and / or vaccine, and the route of administration.

[0463] In some embodiments, the carrier, composition, or vaccine is administered in dose form. In some embodiments, the amount administered in a single dose may contain about 0.1 x 10⁻⁶. 9 To approximately 10x10 12 Particle unit, 0.1x10 9 From approximately 1.0x10 12 Particle unit, approximately 0.1 x 10⁻⁶ 9 To approximately 10x10 11 Particle unit, approximately 0.1 x 10⁻⁶ 9 From approximately 1.0x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 9 To approximately 0.5x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 9 To approximately 0.1x10 11 Particle unit, approximately 1.0 x 10⁻⁶ 10 To approximately 10x10 11 Particle unit, approximately 1.0 x 10⁻⁶ 10 To approximately 0.1x10 11 Particle unit, approximately 0.1 x 10⁻⁶ 11To approximately 10x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 11 Approximately 9x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 11 Approximately 8x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 11 Approximately 7x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 11 Approximately 6x10 11 Particle unit, approximately 0.5 x 10⁻⁶ 12 To approximately 10x10 12 Particle unit, approximately 0.5 x 10⁻⁶ 12 From approximately 1.0x10 12 Particle unit, approximately 1.0 x 10⁻⁶ 11 To approximately 0.1x10 12 Particle unit, approximately 0.1 x 10⁻⁶ 12 To approximately 10x10 12 Particle unit, approximately 1x10 10 Particle unit, approximately 5x10 10 Particle unit, approximately 5x10 11 Particle unit, approximately 6 x 10 11 Particle unit, approximately 7 x 10 11 Particle unit, approximately 8 x 10 11 Particle unit, approximately 9x10 11 Particle unit, approximately 10 x 10 11 Particle unit, approximately 1x10 12 Particle unit, approximately 2 x 10 12 Particle unit, approximately 3 x 10 12 Particle unit, approximately 4 x 10 12 Particle unit, approximately 5x10 12 Particle unit, approximately 6 x 10 12 Particle unit, approximately 7 x 10 12 Particle unit, approximately 8 x 10 12 Particle unit, approximately 9x10 12 Particle unit or approximately 10x10 12 Particle unit.

[0464] In some embodiments, the dosage may comprise approximately 1.0 x 10⁻⁶. 5 From approximately 1.0x10 10 Plaque-forming units (PFU), for example, about 0.5 x 10⁻⁶. 5 To approximately 0.5x10 10 PFU, approximately 0.1 x 10 5 x 0.1x10 10 PFU, approximately 1x106 To approximately 1x10 9 PFU, approximately 0.5 x 10 6 To approximately 0.5x10 9 PFU, approximately 0.1 x 10 6 To approximately 0.1x10 9 PFU, approximately 1x10 7 To approximately 1x10 8 PFU, approximately 0.5 x 10 7 To approximately 0.5x10 8 PFU, approximately 0.1 x 10 7 To approximately 0.1x10 8 PFU, approximately 1.0 x 10 6 From approximately 1.0x10 9 PFU, approximately 0.5 x 10 6 To approximately 0.5x10 9 PFU, approximately 1.0 x 10 7 To approximately 1x10 8 PFU, approximately 1.0 x 10 6 From approximately 1.0x10 8 PFU, approximately 0.5 x 10 6 To approximately 0.5x10 8 PFU or approximately 0.1x10 6 To approximately 0.1x10 8 PFU.

[0465] In some implementations, the viral vector can be quantified by quantitative PCT analysis (Q-PCR) or analytical HPLC.

[0466] For treating HPV-related pathology, the dose of the carrier can, for example, be approximately 1 x 10⁻⁶. 9 To approximately 1x10 13 Particle unit, approximately 5x10 9 Approximately 5x10 12 Particle unit, approximately 1x10 10 To approximately 1x10 12 Particle unit, approximately 1x10 11 Approximately 9x10 11 Particle unit approximately 1x10 11 Approximately 9x10 11 Particle unit approximately 1x10 11 Approximately 9x10 11 Particle unit, approximately 1x10 10 To approximately 1x10 12 Particle unit, approximately 1x10 11 Approximately 9x10 11 Particle unit, approximately 2 x 1011 Approximately 8x10 11 Particle unit, approximately 3 x 10 11 Approximately 7x10 11 Particle unit, approximately 4 x 10 11 Approximately 6x10 11 Particle unit or approximately 5x10 11 Particle unit.

[0467] For treating RRP, the dose of the carrier can be, for example, about 0.1 x 10⁻⁶. 9 To approximately 10x10 12 Virus particles, approximately 0.5 x 10⁻⁶ 9 Approximately 9x10 12 Virus particles, approximately 0.5 x 10⁻⁶ 9 Approximately 8x10 12 Virus particles, approximately 0.5 x 10⁻⁶ 9 Approximately 7x10 12 Virus particles, approximately 0.5 x 10⁻⁶ 9 Approximately 6x10 12 Virus particles, approximately 0.5 x 10⁻⁶ 9 Virus particles approximately 5x10 10 Virus particles, approximately 0.1 x 10⁻⁶ 10 Virus particles approximately 10x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 10 Approximately 9x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 10 Approximately 8x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 10 Approximately 7x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 10 Approximately 6x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 10 Virus particles approximately 5x10 11 Virus particles, approximately 0.1 x 10⁻⁶ 11 To approximately 10x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 11 To approximately 9.0x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 11 To approximately 8.0x10 11 Virus particles, approximately 0.5 x 10⁻⁶ 11 To approximately 7.0x10 11 Virus particles are appro...

Claims

1. A polynucleotide encoding a fusion protein, said fusion protein comprising: (a) an HPV6 protein selected from HPV6 E2, HPV6 E4, HPV6 E6 and HPV6 E7 proteins; and (b) an HPV11 protein selected from HPV11 E6 and HPV11 E7 proteins.

2. The polynucleotide of claim 1, comprising: HPV6 E2 protein; HPV6 E4 protein; HPV6 E6 protein; HPV6 E7 protein; HPV11 E6 protein; and HPV11 E7 protein.

3. The polynucleotide of claim 1, wherein the HPV6 E2 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

1.

4. The polynucleotide of claim 1, wherein the HPV6 E2 protein comprises the amino acid sequence of SEQ ID NO:

1.

5. The polynucleotide of claim 1, wherein the HPV6 E4 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 or 7.

6. The polynucleotide of claim 1, wherein the HPV6 E4 protein comprises the amino acid sequence of SEQ ID NO: 3 or 7.

7. The polynucleotide of claim 1, wherein the HPV6 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 or 40.

8. The polynucleotide of claim 1, wherein the HPV6 E6 protein comprises the amino acid sequence of SEQ ID NO: 11 or 40.

9. The polynucleotide of claim 1, wherein the HPV6 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5 or 9.

10. The polynucleotide of claim 1, wherein the HPV6 E7 protein comprises the amino acid sequence of SEQ ID NO: 5 or 9.

11. The polynucleotide of claim 1, wherein the HPV11 E6 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

42.

12. The polynucleotide of claim 1, wherein the HPV11 E6 protein comprises the amino acid sequence of SEQ ID NO:

42.

13. The polynucleotide of claim 1, wherein the HPV11 E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

45.

14. The polynucleotide of claim 1, wherein the HPV11 E7 protein comprises the amino acid sequence of SEQ ID NO:

45.

15. The polynucleotide of claim 1, wherein the fusion protein comprises an HPV6 E4 protein comprising the amino acid sequence of SEQ ID NO: 3 and an HPV6 E4 protein comprising the amino acid sequence of SEQ ID NO:

7.

16. The polynucleotide of claim 1, wherein the fusion protein comprises the HPV6 E6 protein comprising the amino acid sequence of SEQ ID NO: 11 and the HPV6 E6 protein comprising the amino acid sequence of SEQ ID NO:

40.

17. The polynucleotide of claim 1, wherein the fusion protein comprises an HPV6 E7 protein comprising the amino acid sequence of SEQ ID NO: 5 and an HPV6 E7 protein comprising the amino acid sequence of SEQ ID NO:

9.

18. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

68.

19. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

68.

20. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

68.

21. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 97% identity with SEQ ID NO:

68.

22. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 98% identity with SEQ ID NO:

68.

23. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 99% identity with SEQ ID NO:

68.

24. The polynucleotide of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:68 or a variant thereof with conserved substitutions.

25. The polynucleotide of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

68.

26. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

66.

27. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

70.

28. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

72.

29. The polynucleotide of claim 1, wherein the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

74.

30. The polynucleotide of claim 1, wherein the fusion protein further comprises a rigid linker polypeptide.

31. The polynucleotide of claim 1, wherein the fusion protein further comprises an HPV16 E6 agonist enhancer.

32. The polynucleotide of claim 1, wherein the fusion protein further comprises an HPV16 E7 agonist enhancer.

33. The polynucleotide of claim 1, operatively linked to at least one of: a promoter; a 5′ untranslated region (UTR); a transcription start site (TSS); a 3′ UTR; a tetracycline-responsive element; and a Kozak region.

34. The polynucleotide of claim 33, wherein the polynucleotide is operatively linked to a promoter enhancer region.

35. A vector comprising the polynucleotide of any one of claims 1–34.

36. The vector of claim 35, wherein the vector is a plasmid, a viral vector, or a non-viral vector.

37. The vector of claim 36, wherein the viral vector is an adenovirus vector.

38. The vector of claim 37, wherein the adenovirus vector lacks one or more elements selected from the E1–E4 region and the L1–L5 region.

39. The vector of claim 37, wherein the adenovirus vector comprises one or more elements selected from E2B, L1, L2, L3, E2A, L4, E3, L5, inverted terminal repeat (ITR), poly(a) site, and spacer sequence.

40. The vector of claim 37, wherein the adenovirus vector is a chimpanzee adenovirus vector.

41. The vector of claim 37, wherein the adenovirus vector is a GC46 gorilla adenovirus vector.

42. A method for inducing an anti-HPV immune response in a subject in need, comprising administering to the subject a therapeutically effective amount of the carrier of claim 30.

43. The method of claim 42, wherein the therapeutically effective amount comprises about 1 × 10⁻⁶. 11 Approximately 5×10 11 Particle unit (PU).

44. A method for treating HPV-related diseases or conditions in a subject in need, comprising administering a therapeutically effective amount of the carrier of claim 35 to the subject.

45. The method of claim 44, wherein the HPV-related disease or condition is an HPV6-related disease or condition or an HPV11-related disease or condition.

46. ​​The method of claim 44, wherein the HPV-related disease or condition is HPV-related cancer.

47. The method of claim 44, wherein the HPV-related disease or condition is recurrent respiratory papillomavirus (RRP), genital perianal warts, lower genital intraepithelial neoplasia, cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer.

48. The method of claim 44, wherein the HPV-related disease or condition is RRP.

49. The method of claim 44, wherein the therapeutically effective amount comprises about 1 × 10⁻⁶. 11 Approximately 5×10 11 Particle unit (PU).

50. The method of claim 44, wherein the method further comprises administering an additional therapy.

51. The method of claim 44, wherein the additional therapy comprises administering at least one of the following: an angiogenesis inhibitor, such as bevacizumab (AVASTIN®); and an immune checkpoint inhibitor, such as a PD-1 inhibitor (e.g., pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and cimipril (LIBTAYO®)) and / or a PD-L1 inhibitor (e.g., atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®)).

52. The method of claim 44 further includes performing a debulking surgery.

53. A fusion protein encoded by any one of claims 1–34.

54. A composition comprising any one of the polynucleotides of claims 1–34.

55. The composition of claim 49, for treating a disease or ailment in a person in need.

56. Use of the polynucleotide as described in any one of claims 1–34 in the preparation of a medicament for treating a disease or condition in a person in need.

57. A kit comprising the polynucleotide of any one of claims 1–34.

58. A vaccine comprising any one of the polynucleotides claimed in claims 1–34.

59. The vaccine of claim 58, for treating a disease or condition in a person in need.

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