Novel adenovirus vaccine therapy for the treatment of recurrent respiratory papillomatosis

Abstract

Multiantigenic human papillomavirus (HPV) molecular vaccine constructs for use and treatment of HPV-related disorders and conditions, e.g., HPV molecular vaccines targeting HPV6 and HPV11-associated recurrent respiratory papillomatosis (RRP).

Description

[Technical Field]

[0001] Background technology [Background technology]

[0002] Recurrent respiratory papillomatosis (RRP) is a rare, difficult-to-treat, and sometimes fatal neoplasm of the upper and lower respiratory tract. 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 Acad Sci 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 or adult-onset based on the age of onset. Juvenile-onset RRP has an incidence of 4 / 100,000 and tends to have an aggressive clinical course. Adult-onset RRP has an incidence of 2-3 / 100,000 and tends to have a more slowly progressive clinical course.

[0004] The pathological conditions and mortality of RRPs stem from the effects of the papilloma population on the vocal cords, trachea, and lungs. This can cause voice changes, wheezing, airway obstruction, loss of lung volume, and / or post-obstructive pneumonia. Silver RD, Rimell FL, Adams GL, Derkay CS, Hester R: Diagnosis and management of pulmonary metastasis from recurrent respiratory papillomatosis. Otolaryngol Head Neck Surg 2003, 129(6):622-629. Repeated procedures are required for weight loss and disease monitoring, which exposes participants to anesthetic and surgical risks, as well as psychological distress. The economic cost of RRPs is estimated at $150 million per year in the United States. Derkay CS, Wiatrak B: Recurrent respiratory papillomatosis: a review. Laryngoscope 2008, 118(7):1236-1247. Although rare (1–3 percent of cases), RRP can be converted to invasive squamous cell carcinoma. Dedo HH, Yu KC: CO(2) laser treatment in 244 patients with respiratory papillomas. Laryngoscope 2001, 111(9):1639-1644. Subsequent mortality is based on the clinical stage of the malignant tumor at the time of diagnosis.

[0005] There is no treatment for RRP, no approved drug therapy currently exists, and complete regression of RRP has not yet been observed using immunotherapy. The only hope for treatment of RRP is repeated endoscopic depletion by ablation or excision of the papillomatous lesions. According to surgical principles, in order to minimize the pathological state from the procedure, the papillomatous disease must be removed, but the epithelium that appears normal must not be removed. It is thought that latent HPV virus particles persist in an inactive state in clinically normal mucosa, then become 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] Participants with juvenile-onset RRP require an average of 20 surgical procedures throughout their lifetime to control the disease. Ibid. Participants with adult-onset RRP generally require fewer interventions, although more than 50% still require five or more procedures 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 surgical procedures throughout their lifetime to maintain usable voice and an open airway. Adjuvant systemic therapies, including systemic interferon-alpha as well as local injections of antivirals and anti-angiogenic agents, are being tested in clinical trials. The research findings are contradictory, and there is no adjuvant approach that is widely indicated or accepted as standard treatment.

[0007] Topical immunotherapy has failed to eradicate RRP, clearly due to the chronic persistence of latent HPV in seemingly normal mucosa. This idea is supported by studies demonstrating the presence of HPV DNA in clinically healthy mucosa of participants with RRP. 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 efforts to study systemic immunotherapy for RRP have been limited. Adjuvant IFN-α after papilloma treatment showed a short-term increase in time to relapse, but did not demonstrate long-term benefits.

[0008] Programmed death ligand 1 (PD-L1) expression in tumor cells is strongly associated with poor prognosis in various human cancers. (Healy GB, Gelber RD, Trowbridge AL, Grundfast KM, Ruben RJ, Price KN: Treatment of recurrent respiratory papillomatosis with human leukocyte interferon. Results of a multicenter randomized clinical trial. N Engl J Med 1988, 319(7):401-407). Recently, several drugs targeting the programmed death 1 (PD-1) / PD-L1 pathway have received regulatory approval and have demonstrated excellent duration of response for multiple tumor types, including head and neck cancers. 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. N Engl J Med 2016, 375(19):1856-1867.In particular, atezolizumab, durvalumab, and avelumab are all anti-PD-L1 antibodies with proven efficacy and regulatory approval. However, these anti-PD-L1 antibodies have not shown safe and effective treatment options for adults with RRP.

[0009] Therefore, in this field, there remains a need for relatively non-invasive methods for safely and effectively treating adults with RRP, considering both the cost and psychological benefits to patients, including methods that focus on priming the T cell response to HPV antigens.

[0010] Therapeutic vaccines are one immunotherapy strategy that can enhance novel T-cell responses from individuals with RRPs. Therapeutic vaccination of participants with a long peptide vaccine encoding the HPV-16 antigen induced complete disease regression in nearly 50% of participants with precancerous vulvar intraepithelial neoplasia. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer 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. The magnitude of the HPV 16-specific T-cell response correlated with the magnitude of the clinical response in these participants. 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. Permanent remission without recurrence of papillomatic lesions in children and adults with RRP was reported from Mexico City after frequent intrafocal injections (a total of 4 injections, once every 2 weeks, directly into the papilloma) of modified vaccinia ankara virus encoding bovine papillomavirus E2.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. This study demonstrated clinical activity, but further improvements are needed, and translational research defining the mechanism of papilloma clearance is lacking. [Overview of the Initiative]

[0011] Provided herein are non-naturally occurring polynucleotides (and polypeptides expressed thereby) encoding non-naturally occurring polypeptides, including immune response-inducing human papillomavirus (HPV) polypeptides. Also provided herein are non-naturally occurring, variably configured structures of HPV immune response-inducing polypeptides, linked by various polypeptide linker sequences, thereby containing fusion proteins useful as vaccine antigens.

[0012] The present invention includes, but is not limited to, compositions (e.g., substances comprising polynucleotides, polypeptides, vectors, vaccines, or cells), methods for preparing and delivering compositions, and the use of naturally occurring polynucleotides encoding naturally occurring polypeptides containing antigens for vaccines against human papillomavirus (HPV), particularly HPV6 and HPV11, as well as therapeutic approaches for treating pathological conditions caused by these specific HPV virulence factors.

[0013] In particular, the present invention includes polynucleotides encoding multiantigenic polypeptides derived from HPV6, HPV11, and HPV16, as well as other polypeptide sequences, and polypeptides encoded thereby, for use as vaccine components and for treating disorders associated with HPV infection.

[0014] For example, embodiments of the present invention relate to the use of the compositions described herein as therapeutic vaccines against HPV6 and / or HPV11 (HPV6 / 11)-induced or related diseases, for example, but not limited to, recurrent respiratory papillomatosis (RRP), anogenital warts, and other HPV6 / 11-related diseases, such as lower genital neoplasms (e.g., intraepithelial neoplasms of the cervix, vagina, and vulva), cervical cancer, vulvar cancer, anal cancer, penile cancer, and head and neck cancer. In one embodiment, the compositions described herein are used as therapeutic vaccines against RRP.

[0015] The present invention includes a unique and innovative HPV polypeptide vaccine design approach for HPV6 / 11-induced diseases. The present invention includes a mix of design strategies, such as the use of whole protein sequences, the use of peptide "fragments," hybrid polypeptide constructs, and the introduction of amino acid substitutions, insertions, deletions, and rearrangements of polypeptides and peptide HPV gene products (proteins).

[0016] As described above, the specific modifications of the antigens described herein were made, in particular, by introducing point mutations, substitution mutations, and / or rearrangements of the viral polypeptide sequence to prevent oncogenic gene expression and / or to disable essential viral functions (e.g., viral replication).

[0017] In certain embodiments, HPV initial proteins E2 and E4 are identified as novel antigens against HPV6 / 11 and incorporated into vaccine designs described herein.

[0018] In a particular embodiment, the present invention involves the novel incorporation of four HPV-derived antigenic components into a single HPV vaccine design.

[0019] In a particular embodiment, the present invention involves the novel incorporation of four HPV-derived antigenic components into a single HPV vaccine design.

[0020] In certain embodiments, the present invention includes the novel incorporation and combination of both high-cancer-risk and low-cancer-risk HPV genotype epitopes into a single HPV antigen construct.

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

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

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

[0024] In a particular embodiment, the vector includes a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 119 or a functional variant thereof (for example, 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 certain embodiments, the expression cassette includes a promoter, and the expression of the transgene is under the control of the promoter. In some such embodiments, the promoter is a cytomegalovirus promoter or a synthetic promoter.

[0026] In certain embodiments, the promoter is a synthetic promoter. In some such embodiments, the synthetic promoter includes a blocking sequence, an enhancer, and / or a response element. In certain embodiments, the enhancer includes 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 certain embodiments, the response element includes 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 certain embodiments, the expression cassette includes a 5'UTR. In some such embodiments, the 5'UTR includes 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 certain embodiments, the expression cassette includes a stop sequence. In some such embodiments, the stop 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 a particular embodiment, the vector is an adenovirus vector, and the expression cassette is located at the E1 region deletion junction.

[0030] In a particular embodiment, the expression cassette is cloned from right to left with respect to the adenovirus viral genome.

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

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

[0033] In part, the present invention also relates to pharmaceutical compositions comprising vectors and pharmaceutically acceptable carriers as described herein.

[0034] In certain embodiments, the composition further comprises additional therapeutic agents. In some such embodiments, the additional therapeutic agents treat conditions caused by HPV pathogens.

[0035] The present invention also relates in part to a kit comprising the pharmaceutical compositions described herein. In certain embodiments, the kit further includes labels.

[0036] In part, the present invention also relates to a method for treating a disease or disorder, including RRP, in a subject that requires it, the method comprising the step of administering a vector described herein to the subject.

[0037] In a particular embodiment of the method, the vector is approximately 0.1 × 10 11 ~Approx. 1×10 12 It is administered in amounts equivalent to individual virus particles.

[0038] In certain embodiments, the method includes the step of administering a composition containing a vector. In some such embodiments, the composition is administered subcutaneously, intramuscularly, intravenously, intracranially, intraarticularly, intradermally, transdermally, intratumorally, or intrafocally. 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 an aerosol spray or mist.

[0039] In certain embodiments, the method includes the step of administering a composition comprising multiple doses of vectors. In some embodiments, each dose is approximately 0.1 × 10⁻⁶ 11 ~Approx. 5×10 11 The formulation may contain viral particle units. In some embodiments, each dose is administered at least 11 days apart. In certain embodiments, a second dose is administered 2 weeks after the first dose, a third dose 6 weeks after the second dose, and a fourth dose 12 weeks after the third dose. In some embodiments, at least one dose is administered at least 1 month apart from a previous dose.

[0040] In some embodiments, the dose reduction procedure is performed before the first dose is administered. In some embodiments, one or more dose reduction procedures 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 before the first dose is administered. In one embodiment, three dose reduction procedures are performed before the first dose is administered. In another embodiment, one or more dose reduction procedures are performed 12 months before the first dose is administered. In a further embodiment, three dose reduction procedures are performed 12 months before the first dose is administered.

[0041] In some embodiments, one or more dose reduction procedures are performed after the first dose is administered and before the final dose is administered. In some embodiments, one or more dose reduction procedures are performed after the final dose has been administered. In some embodiments, one or more dose reduction procedures are performed between the administration of at least one dose of the composition.

[0042] In one embodiment, the patient may undergo at least one dose reduction procedure at least six months, one year, or two years after administration of the dose of the composition described herein, or at a timeframe determined by the patient's physician, in which case the patient may then receive one or more additional doses of the composition after such a dose reduction procedure. In some embodiments, the clinical efficacy of the treatment method is measured after the final dose has been administered.

[0043] In some embodiments, administration of the vectors described herein results in a reduction of the need for surgical intervention (e.g., debulking surgery).

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

[0045] In some embodiments, administration of the vectors described herein results in an improvement in the Derkay score.

[0046] In certain embodiments, the method further comprises the administration of one or more additional therapeutic agents. In some such embodiments, the additional therapeutic agents may be chemotherapeutic agents, anti-inflammatory agents, analgesics, and / or biological response modifiers.

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

[0048] In some embodiments, the fusion protein includes 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 includes 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 includes an HPV6 E7 protein containing the amino acid sequence of SEQ ID NO: 5 and an HPV6 E7 protein containing the amino acid sequence of SEQ ID NO: 9.

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

[0050] In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 70. In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 72. In some embodiments, the fusion protein includes 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 HPV16 E7 agonist enhancer.

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

[0053] Vectors comprising any of those described herein are also provided 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 deficient 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, an inverted terminal repeat (ITR), a poly(a) site, and a spacer. In some embodiments, the adenovirus vector is a gorilla adenovirus vector. In some embodiments, the adenovirus vector is a GC46 gorilla adenovirus vector.

[0054] Methods for inducing an anti-HPV immune response in a target requiring such response are also provided herein. In some embodiments, the method includes the step of administering a therapeutically effective amount of one of the vectors described herein to the target. In some embodiments, the therapeutically effective amount is approximately 1 × 10⁻⁶ 11 and approximately 5 × 10 11 Includes particle units (PU).

[0055] Methods for treating HPV-related diseases or disorders in subjects requiring such treatment are also provided herein. In some embodiments, the method includes the step of administering a therapeutically effective dose of one of the vectors described herein to the subject. In some embodiments, the HPV-related disease or disorder is HPV6-related disease or disorder or HPV11-related disease or disorder. In some embodiments, the HPV-related disease or disorder is HPV-related cancer. In some embodiments, the HPV-related disease or disorder is recurrent respiratory papillomatosis (RRP), anogenital warts, lower genital neoplasms, cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer. In some embodiments, the HPV-related disease or disorder is RRP. In some embodiments, the therapeutically effective dose is about 1 × 10⁻⁶ 11 and approximately 5 × 10 11 The method includes particle units (PU). In some embodiments, the method further includes a step of administering an additional therapy. In some embodiments, the additional therapy includes an administration of at least one of the following: an angiogenesis inhibitor, e.g., bevacizumab (AVASTIN®), and an immune checkpoint inhibitor, e.g., a PD-1 inhibitor (e.g., pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and semiprimab (LIBTAYO®)) and / or a PD-L1 inhibitor (e.g., atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®)). In some embodiments, the method further includes a dose reduction procedure.

[0056] Fusion proteins encoded by any of the polynucleotides described herein are also provided herein.

[0057] Compositions comprising any of the polynucleotides described herein are also provided herein. In some embodiments, any of the compositions described herein are for use in the treatment of a disease or disorder in a subject requiring such treatment.

[0058] The use of any of the polynucleotides described herein in the manufacture of a pharmaceutical product for use in the treatment of a disease or disorder in a subject requiring such use is also provided herein.

[0059] A kit containing any of the polynucleotides described herein is also provided herein.

[0060] Vaccines comprising any of the polynucleotides described herein are also provided herein. In some embodiments, the vaccines are intended for use in the treatment of diseases or disorders in subjects requiring them.

[0061] In part, the present invention also relates to the use of the vectors described herein in the manufacture of pharmaceuticals for use in the treatment of diseases or disorders such as RRP in subjects where such use is required.

[0062] The features of this disclosure are set forth in particular in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained from the following detailed description illustrating exemplary embodiments in which the principles of this disclosure are utilized, and from reference to the appended drawings. [Brief explanation of the drawing]

[0063] [Figure 1-1]Figure 1A illustrates an engineered GC46 vector (AdV-HPV6 / 11) with deletions in the E1 and E4 regions and a CMV HPV6 / 11 expression cassette in the E1 region. Figure 1B illustrates the HPV-6 / 11 proteins (HPV-E2, HPV-E4, HPV-E6, and HPV-E7) expressed from the engineered GC46 vector (AdV-HPV6 / 11) with deletions in the E1 and E4 regions and a CMV HPV6 / 11 expression cassette in the E1 region. Figure 1C illustrates the proteins / antigens expressed from AdV-HPV6 / 11, a fusion of selective regions of HPV proteins (HPV-E2, HPV-E4, HPV-E6, and HPV-E7) expressed in HPV-6 and HPV-11 infected cells. Figure 1D is a schematic diagram of a vector skeleton containing an antigen open reading frame, flanked with a CMV promoter, an SV40 polyadenylation signal, a 3' untranslated region, and the following genes, which are sequenced from 5' to 3': HPV6 E6, HPV11 E7, HPV6 E7, HPV11 E6, HPV6 E4, HPV6 E6, HPV6 E7, HPV11 E6, HPV6 E4, HPV11 E7, and HPV6 E2. [Figure 1-2] Same as above. [Figure 2] This figure shows the fluorescence activity, as measured by flow cytometry, 24 hours after transduction of cultured autologous dendritic cells from participants with recurrent respiratory papillomatosis (RRP) with a basic gorilla adenovirus construct encoding GFP (5 × 10³ MOI). [Figure 3] This figure illustrates IFNg ELISA of T lymphocytes from three RRP participants who underwent three rounds of stimulation of dendritic cells transduced with AdV-HPV6 / 11 or a control. The x-axis shows the adenovirus construct tested (construct differences include encoded gene linkers and antigens). [Figure 4]Figure 4A illustrates the experimental design for evaluating the HPV antigen-specific immune response of peripheral T lymphocytes from wild-type C57BL / 6 mice inoculated with AdV-HPV6 / 11. Figure 4B illustrates a representative ELISpot well micrograph demonstrating the response to the HPV6 and 11 15-mer duplicate peptide pool and synthetic minimal peptide after in vivo inoculation with AdV-HPV6 / 11 instead of empty GC46. Figure 4C illustrates the quantification of IFNγ spots in vaccinated male (n=3) and female (n=3) mice. Figure 4D illustrates the quantification of IFNγ spots in an independently validated in vivo vaccination experiment in female wild-type C57BL / 6 mice (n=5). Figure 4E shows the quantification (left) and representative flow cytometry dot plot (right) of CD8+ T lymphocyte IFNγ production after evaluation of HPV antigen-specific T lymphocyte response from 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 mice inoculated with AdV-HPV6 / 11. [Figure 5] Figure 5 illustrates ELISpot, which evaluates peripheral T lymphocytes from mice (N-4) treated with AdV-HPV6 / 11 (top row) or an empty vector (bottom row) for their response to a 15-mer duplicated peptide or minimal covalent epitope IYSYAYKHLK (SEQ ID NO: 120) from HPV 6 or 11 E6. PMA / Iono is the positive control. Figure 5 discloses SEQ ID NO: 120. [Figure 6]Figure 6A is a schematic diagram illustrating retroviral transduction to produce MOC1 cells expressing HPV6 E6. Figure 6B is a flow cytometry dot plot demonstrating E6(NGFR) positivity in parental MOC1 or MOC1-E6 cells, quantified on the right. Figure 6C is a summary of tumor growth curves for mice (n=7 mice / group) carrying parental MOC1 or MOC1-E6 tumors treated with AdV-HPV6 / 11, empty C46, ​​or control (PBS). Red dots indicate treatment. Tumor volume at day 40 is quantified individually on the right. Figure 6D is a summary of growth curves for mouse-carrying MOC1-E6 tumors treated with AdV-HPV6 / 11 in the presence or absence of CD8 or CD4 depletion antibodies. Red dots indicate treatment, and blue dots indicate depletion. The tumor volume on day 40 is quantified individually on the right side. [Figure 7] Figure 7A depicts a representative dot plot of freshly digested parental pMOC1 or MOC1-E6 tumors taken from mice treated with AdV-HPV6 / 11 or empty GC46, evaluated for T lymphocyte accumulation by flow cytometry. Normalized quantifications are shown on the right. Figure 7B depicts representative H&E and immunofluorescence micrographs demonstrating the localization of T lymphocytes within MOC1-E6 tumors treated with AdV-HPV6 / 11 or empty GC46. Figure 7C depicts the quantification of total T lymphocytes per high-magnification field (HPF). mpIF multiplex immunofluorescence. Figure 7D depicts the quantification of tumor boundary localization of CD8+ and CD4+ T lymphocytes relative to tumor parenchymal localization. [Figure 8]Figure 8A is a schematic diagram of tumor-infiltrating lymphocytes (TILs) cultured from tumors of MOC1-E6 tumor-bearing mice (n=5 / group) treated with AdV-HPV6 / 11 or empty GC46 vector. Figure 8B is a diagram of representative impedance analysis of cultured TILs from MOC1-E6 tumors, treated with AdV-HPV6 / 11, empty GC46, or control (PBS), and then co-cultured with either parental MOC1 or MOC1-E6 target tumor cells (n=5 tumors per condition). TILs are added to target cells at experimental time point 0. The death percentage (expressed as a percentage decrease in the target cell index) is quantified 16 hours after TIL addition to the target and is shown on the right. Figure 8C is a diagram illustrating representative ELISpot well micrographs and IFNγ spot quantifications demonstrating the response of HPV6 and 11 to the 15-mer duplicate peptide pool and synthetic minimal peptides in TIL cultures from MOC1-E6 tumors treated with AdV-HPV6 / 11 or empty GC46. [Figure 9]Figure 9A is a bar graph illustrating the total number of clinically necessary interventions for patients treated in DL1 (patients 1-3) and DL2 (patients 4-15) during the 12 months before (left) and after (right) the study. Respondents are shown in blue, and non-respondents in gold. Patients who did not require intervention during the 12 months after the study treatment were considered complete responders, and patients who required less than 50% of the interventions during the 12 months after the study treatment compared to the 12 months before the study treatment were considered partial responders. Respondents are divided into complete responders and partial responders. Figure 9B is a waterfall plot illustrating the change in clinically necessary procedures in the year following the study treatment compared to the previous year. Patients are ranked from left to right in descending order of the decrease in intervention frequency. The solid horizontal line indicates the 50% reduction threshold that distinguishes responders from non-respondents. Figure 9C illustrates representative clinical endoscopic images demonstrating the appearance of the larynx in four of six patients with complete response (CR) who showed no visible lesions after AdV-HPV6 / 11 treatment. The Derkay score (upper left) and the time after completion of the study treatment (upper right) are included. The post-treatment images are from the most recent endoscopic examination at the data cutoff point. [Figure 10]Figure 10A is a dot plot illustrating the logarithmically transformed change in HPV-specific T cell response from peripheral blood 6 weeks after completion of the study-target treatment, compared to previous levels. Each dot represents the logarithmically transformed change in IFNγ concentration after peptide stimulation by individual pools of HPV peptides encoded in AdV-HPV6 / 11. Pools in which no IFNγ response was detected in pre- or post-treatment samples are not shown. Patient N=14; sufficient pre-treatment PBMCs were not available from patient 5. Respondents are shown in blue, and non-respondents in gold. Figure 10B is a dot plot illustrating the changes in HPV-specific peripheral blood response. Significance was determined by the Mann-Whitney two-tailed test. Figure 10C is a bar graph illustrating the fraction of the post-treatment TCRβ repertoire, represented by the top 10 CDR3 frequencies determined to be HPV-specific in the HPV 6 / 11 peptide-stimulated FEST assay. The Simpson clonal index is shown above each bar graph. The top horizontal bar indicates the response to the treatment. R: Respondent; NR: Non-respondent. Figure 10D is a dot plot showing the log-transformed change in the frequencies (determined by FEST assay) of the top 10 HPV-specific CDR3 sequences from peripheral blood 6 weeks after completion of AdV-HPV6 / 11 treatment compared to pre-treatment without peptide stimulation. Figure 10E is a dot plot showing a summary of the changes in the top 10 HPV-specific CDR3 sequences. Significance was determined by an unpaired two-sided t-test. Figure 10F is a bar graph summarizing the fractions (determined by FEST assay) of the top 10 peripheral blood HPV-specific CDR3 sequences detected in post-treatment peripheral blood, either not detected (appeared), detected at a lower frequency (enlarged), or detected at a higher frequency (contracted), compared to pre-treatment peripheral blood. The top horizontal bar indicates the effectiveness of the treatment. Figure 10G is a dot plot illustrating the log-2 converted change in HPV-specific papilloma-infiltrating lymphocytes (PILs) after completion of the study-target treatment compared to before. Each dot represents the log-converted change in the number of IFNγ spots after co-culture of antigen-presenting cells loaded with individual pools of HPV peptides encoded by AdV-HPV6 / 11 and PILs.Pools in which no IFNγ response was detected in pre- or post-treatment samples are not shown. Patient N=9; post-treatment biopsy material was unavailable for patients 7, 10, 11, and 13, and pre-treatment PIL culture establishment failed for patients 2 and 3. Figure 10H is a dot plot summarizing the changes in HPV-specific PIL response. Significance was determined by the Mann-Whitney two-tailed test. Figure 10I is a clinical endoscopic image showing the appearance of the pharynx of patient 5 before treatment and at 6 weeks. Red arrows indicate the pre-treatment and 6-week biopsy sites that produced PIL for the experiments shown in Figures 11J and 11K. Figure 10J is a representative IFNγELISpot well (with IFNγ spot numbers inserted) showing IFNγ spots after stimulation with pool 2 peptides and negative (DMSO alone) and positive (PMA / ionomycin) controls before treatment and at 6 weeks. Figure 10K is a bar graph showing the IFNγ concentration after co-culturing antigen-presenting cells loaded with individual peptides contained in Pool 2 with PIL samples for 6 weeks. [Figure 11]Figure 11A shows representative micrographs of T cell immunofluorescence in baseline papilloma biopsy material collected from responders (top row) and non-responders (bottom row). Figure 11B shows a dot plot showing the density of CD8 T cells in papillomas and stroma or in responders and non-responders. Significance was determined by the Mann-Whitney two-tailed test. Figure 11C shows a box plot showing the number of papilloma cells normalized to HPV gene transcripts in responders and non-responders, determined from single-cell RNA-seq. Significance was determined by two-way ANOVA. Figure 11D shows a box plot showing the cell-normalized reactome IFNγ signaling score in different cell types (x axis) in responders and non-responders, determined from single-cell RNA-seq. Significance was determined by two-way ANOVA. Figure 11E is a heatmap showing the number of bio-normalized chemokine transcripts in different cell types (y-axis). The bar graph on the right shows mean expression. The top horizontal bar shows the response to treatment. The significance of the difference between responders and non-responders was determined by two-way ANOVA. Figure 11F is a violin diagram showing the number of CXCR3 transcripts for CD8 and CD4 papilloma T cells determined from single-cell RNA-seq. Significance was determined by the Mann-Whitney two-tailed test. Figure 11G is a violin diagram showing the percentage of (total) cells positive for CXCL9 or CXCL10. Significance was determined by the Mann-Whitney two-tailed test. Representative micrographs of RNAscope immunofluorescence are shown. Figure 11H is a dot plot illustrating the expression of selected T cell-related genes across T lymphocyte clusters, identified by single-cell RNA-seq and sorted by the ratio of change in the number of cells detected in responders and non-responders (responders / non-responders; bar graph below). T cells that were more common in non-responders are in the left column, and cells that were more common in responders are in the right column. The color of the circles corresponds to the scaled average expression, and the size of the circles indicates the percentage of cells with non-zero gene expression for the corresponding gene. The bar graph at the top represents the total number of cells. [Figure 12]Figure 12A is a dot plot illustrating the expression of selected myeloid cells-related genes across myeloid clusters, identified by single-cell RNA-seq and sorted by the ratio of change in cell number detected in responders and non-responders (responders / non-responders; bar graph below). Myeloid cells frequently observed in non-responders are in the left column, and cells frequently observed in responders are in the right column. The color of the circles corresponds to scaled mean expression, and the size of the circles indicates the percentage of cells with non-zero gene expression for the corresponding gene. The bar graph at the top represents the total number of cells. Figure 12B is a heatmap illustrating the number of bio-normalized chemokine transcripts in different cell types (y-axis). The bar graph on the right shows mean expression. The horizontal bar at the top indicates the response to treatment. The significance of the difference between responders and non-responders was determined by two-way ANOVA. Figure 12C is a heatmap illustrating the number of bio-normalized VEGF transcripts in different cell types (y-axis). The bar graph on the right shows mean expression. The top horizontal bar shows the response to treatment. The significance of the difference between responders and non-responders was determined by two-way ANOVA. Figure 12D is a violin diagram illustrating the percentage of (total) cells positive for CXCL8. Significance was determined by a two-sided Mann-Whitney test. Representative micrographs of RNAscope immunofluorescence are shown. Figure 12E is a diagram illustrating representative micrographs of bone marrow cell immunofluorescence in baseline papilloma biopsy material for responders (top row) and non-responders (bottom row). Figure 12F is a dot plot illustrating the density of neutrophil-derived cells (PMNs) and macrophages (Mθ) in papillomas and stroma or in responders and non-responders. Significance was determined by the Mann-Whitney two-tailed test. [Figure 13] This figure illustrates micrographs of H%E-stained slides from each of the 15 patients enrolled in this study. The patient number is inserted in the upper left corner of each image. [Figure 14]This figure illustrates a dot plot showing the Derkay score (y-axis) obtained from clinical endoscopic images available 12 months prior to, during, and 12 months after the study (x-axis) for each patient. The lines are color-coded according to response. [Figure 15] Figures 15A-15C illustrate representative pre- and post-procedure clinical endoscopic images of the larynx and trachea, where applicable, for (A) patients with complete response (CR) but with visible residual lesions after the procedure, (B) patients with partial response, and (C) patients with no response. The Derkay score is inserted in the upper left, and the time of imaging is inserted in the upper right of each image. Post-procedure images for CR patients are from the most recent endoscopic examination at the data cutoff point. Images for PR and non-response patients are from the first clinically deemed necessary procedure after completion of the study-eligible procedure. [Figure 16] Figure 16A shows a representative immunofluorescence micrograph of T cell staining. Figure 16B shows a representative immunofluorescence micrograph of T cell phenotype. Figures 16C-16G show the density of (C) Ki67+CD8 T cells, (D) all CD4 T cells, (E) Ki67+CD4 T cells, and (F) regulatory T cells (Treg) in the papilloma and stroma in responders and non-responders. The PD-L1 H score of papilloma cells is shown in (G). Significance was determined by the Mann-Whitney two-tailed test. [Figure 17]Figure 17A is a scatter plot showing UMAP embedding in all sequenced cells from all 13 patients, annotated by cell type. Figure 17B is a bar graph showing mean chemokine expression in monocytic or neutrophil-like cells based on single-cell RNA-seq. Figure 17C is a representative immunofluorescence micrograph of chemokine RNAscope staining. Figure 17D is a scatter plot showing UMAP embedding in CD8 T cells, with individual clusters identified by color. Figure 17E is a scatter plot showing UMAP embedding in CD4 T cells, with individual clusters identified by color. Figure 17F is a dot plot showing the expression of selected T cell-related genes across T lymphocyte clusters, identified by single-cell RNA-seq and sorted by the ratio of change in the number of cells detected in responders and non-responders (responders / non-responders; bar graph below). The left column shows T cells that were frequently observed in non-responders, and the right column shows cells that were frequently observed in responders. The color of the circles corresponds to the scaled average expression, and the size of the circles indicates the percentage of cells with non-zero gene expression for the corresponding gene. The bar graph at the top represents the total number of cells. [Figure 18] Figures 18A and 18B depict dot plots showing terms that were more prevalent in responders and non-responders by gene set enrichment analysis of (A) all papillomamonocyte-derived cells or (N) all papillomaneutrophil-derived cells. p-values ​​were calculated based on the hypergeometric distribution and adjusted using the Benjamin-Hochberg method. [Figure 19] Figure 19A is a bar graph showing the mean chemokine expression in monocyte or neutrophil cells based on single-cell RNA-seq. Figure 19B is a representative immunofluorescence micrograph of bone marrow cell marker staining. [Figure 20]This figure illustrates bar graphs showing the results of neutralizing antibody assays of serum samples collected from Phase 1 clinical trial participants at baseline (before treatment) and at 43 days (6 weeks), 12 weeks, and 24 weeks after treatment with AdV-HPV6 / 11, further categorized by the participants' overall clinical response (complete response (CR), partial response (PR), or no response (NR)). [Figure 21] This figure shows a dot plot of the titers of neutralizing HIV 6 / 11 antibodies induced in Phase 1 clinical study participants before (baseline) and after treatment with AdV-HPV6 / 11 DL1 (1 × 10¹¹ particle units) and DL2 (5 × 10¹¹ particle units). [Figure 22] This is a diagram illustrating the Phase II study protocol. [Figure 23] This figure shows a graph comparing the number of surgeries performed in the 12 months prior to AdV-HPV6 / 11 treatment with the number of surgeries performed in the 12 months following treatment. [Figure 24] This graph shows bar charts representing the number of months since the completion of surgery and the number of pre-treatment surgeries performed in the 12 months prior to AdV-HPV6 / 11 treatment. Each bar represents an individual patient who has not undergone surgery since treatment. [Modes for carrying out the invention]

[0064] It should be understood that this disclosure is not limited to the specific embodiments described herein and is subject to modification. While various features of this disclosure may be described in the context of a single embodiment, features may also be provided separately or in any preferred combination. Those skilled in the art will recognize that variations and modifications of this disclosure exist that fall within their scope.

[0065] All terms are intended to be understood in the same way as they are understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are generally understood by those skilled in the art in which this disclosure relates.

[0066] Section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described. definition

[0067] The following definitions are supplementary to the definitions in the Art and apply to this application; they should not be attributed to any related or unrelated cases, such as any generally owned patent or application. Therefore, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0068] In this application, the use of the singular form includes the plural form unless otherwise specifically indicated. It should be noted that, as used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise.

[0069] In this application, the use of “or” means “and / or” unless otherwise specified. The terms “and / or” and “any combination thereof,” as well as their grammatical equivalents, can be used interchangeably as used herein. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjugate or disjunctively unless the context specifically refers to a disjunctive use.

[0070] The use of the term "including," as well as other forms such as "include," "includes," and "included," is not restrictive; that is, "including" does not mean "limited to."

[0071] References to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in conjunction with embodiments are included in at least some embodiments of this disclosure, but not necessarily in all embodiments of this disclosure.

[0072] As used herein and in the claims, the terms “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude additional possible components, elements, or steps of the method. Any embodiment discussed herein is intended to be performed with respect to any method or composition of the Disclosure, and vice versa. Furthermore, compositions of the Disclosure may be used to achieve the methods of the Disclosure.

[0073] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on the method of measuring or determining the value, i.e., the limits of the measuring system. For example, “about” may mean within 1 or a standard deviation greater than 1, according to practice in the art. Alternatively, “about” may mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the quantity “about 10” includes 10 and any quantities from 9 to 11. In yet another example, the term “about” in relation to a reference number may also include a range of values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or plus or minus 1% from that value. Alternatively, particularly in relation to biological systems or processes, the term “about” may mean within one order of magnitude of the value, preferably within five times, more preferably within two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” means within an acceptable margin of error for the particular value to be assumed.

[0074] With respect to the numerical range limitations specified herein, each intervening number between them is explicitly intended to be of the same precision. For example, in the range of 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 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 intended.

[0075] The term “isolated” and its grammatical equivalent, as used herein, refers to the removal of nucleic acids, proteins, polypeptides, cells, or other materials from their natural environment. The term “purified” and its grammatical equivalent, as used herein, refers 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, not “absolute purity.” However, it should be understood that nucleic acids and proteins may be formulated with diluents or adjuvants and still be isolated for practical purposes. For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used for introduction into cells. The term “substantially purified” and its grammatical equivalent, as used herein, refers to nucleic acid sequences, polypeptides, proteins, or other compounds that essentially do not contain, i.e., no more than about 50%, no more than about 70%, or no more than about 90% of the polynucleotides, proteins, polypeptides, and other molecules to which the nucleic acid, polypeptide, protein, or other compound relates in nature.

[0076] "Nucleic acid," "nucleic acid molecule," "polynucleotide," "polynucleotide construct," "oligonucleotide," and their grammatical equivalents, as used herein, refer to polymeric forms of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of molecules. Therefore, this term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. This also includes polynucleotides modified, for example, by methylation and / or capping, as well as polynucleotides in their unmodified forms. This term also means that it includes molecules, including synthetic and semi-synthetic nucleotides and polynucleotides that do not exist in nature, as well as nucleotide analogs. When discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein in accordance with the usual convention of giving the sequence only in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). "Recombinant polynucleotide" is a polynucleotide that has undergone molecular biological manipulation. The polynucleotide sequences and vectors disclosed or intended herein can be introduced into cells, for example, by transfection, transformation, or transduction.

[0077] When applied to a polynucleotide or nucleic acid sequence, the term "fragment" refers to a nucleotide sequence that is shorter than a reference nucleic acid and, across its common portion, contains the same nucleotide sequence as the reference nucleic acid. Such a nucleic acid fragment according to the present invention may, where appropriate, be contained within a longer polynucleotide of which it is a component. Such fragments include, or alternatively consist of, oligonucleotides having a length range of 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 longer consecutive nucleotides of nucleic acid according to the present invention.

[0078] As used herein, “isolated polynucleotide” or “isolated nucleic acid fragment” refers to a polymer of RNA or DNA that is single-stranded or double-stranded, optionally containing synthetic, non-natural, or modified nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may constitute one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0079] The term “gene” and its grammatical equivalent refer to polynucleotides containing nucleotides that code for functional molecules, including functional molecules produced solely by transcription (e.g., bioactive RNA species) or functional molecules produced by both transcription and translation (e.g., polypeptides). The term “gene” encompasses cDNA and genomic DNA nucleic acids. “Genes” also refer to nucleic acid fragments that express specific RNA, proteins, or polypeptides, including regulatory sequences preceding the coding sequence (5' non-coding sequence) and subsequent regulatory sequences (3' non-coding sequence). “Native gene” refers to a gene found naturally together with its own regulatory sequences. “Chimera gene” refers to any gene that is not a native gene and contains regulatory sequences and / or coding sequences that are not found together in nature. Thus, a chimeric gene may contain regulatory sequences and coding sequences from different origins, or regulatory sequences from the same origin but arranged in a different manner than those found in nature. A chimeric gene may contain coding sequences and / or regulatory sequences from different origins. “Endogenous gene” refers to a native gene in its natural location within the genome of an organism. An "external" gene or "other" gene refers to a gene that is not normally found in the host organism but has been introduced into the host organism through gene transfer. External genes can include native genes inserted into non-native organisms or chimeric genes. A "transgene" is a gene that has been introduced into the genome through a transformation procedure.

[0080] 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 base-pair with a complementary single-stranded target nucleic acid to form a double-stranded molecule.

[0081] "Heterogeneous DNA" refers to DNA that is not naturally located in a cell or in a chromosomal region of a cell. Heterogeneous DNA may include exogenous genes. "Exogenous genes" means genes that are foreign to the subject, i.e., genes introduced into the subject by a transformation process, an unmutated version of an endogenous mutated gene, or a mutated version of an endogenous unmutated gene. Exogenous genes may be either native or synthetic genes introduced into the subject in the form of DNA or RNA that can function by a DNA intermediate, for example by reverse transcriptase. Such genes may be introduced into target cells, directly into the subject, or indirectly by the transfer of transformed cells into the subject.

[0082] A "primer" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double-stranded nucleic acid region that can function as an initiation site for DNA synthesis under favorable conditions. Such primers can be used in polymerase chain reactions or for DNA sequencing.

[0083] A DNA "coding sequence" or "coding region" refers to a double-stranded DNA sequence that codes for a polypeptide and, under the control of a suitable regulatory sequence, can be transcribed and translated into a polypeptide in cells, ex vivo, in vitro, or in vivo. A "suitable regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), internally, or downstream (3' non-coding sequence) of the coding sequence that influences transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of a coding sequence are determined by a 5' (amino) terminal start codon and a 3' (carboxy) terminal translation termination codon. Codextic sequences may include, but are not limited to, prokaryotic sequences, mRNA-derived cDNA, genomic DNA sequences, and synthetic DNA sequences. When a coding sequence is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence are typically located at 3' of the coding sequence.

[0084] An "open reading frame," abbreviated as ORF, refers to a nucleic acid sequence of a certain length, whether DNA, cDNA, or RNA, that contains a translation start signal or start codon, e.g., ATG or AUG, and a stop codon, and is potentially translated into a polypeptide sequence.

[0085] The term "downstream" refers to a nucleotide sequence located 3' relative to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally refer to sequences that follow the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0086] The term "upstream" refers to a nucleotide sequence located 5' relative to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally refer to sequences located 5' to the coding sequence or the transcription start site. For example, most promoters are located upstream of the transcription start site.

[0087] The term "response element" refers to one or more cis-acting DNA elements that confer responsiveness to a promoter mediated by interaction with the DNA-binding domain of a transcription factor. This DNA element may be palindromic (complete or incomplete) in its sequence, or it may consist of a sequence motif or half-regions separated by a variable number of nucleotides. The half-regions may be similar or identical and may be arranged as serial or reverse repeat sequences, as a single half-region, or as a serial multimer of adjacent half-regions. Depending on the nature of the cell or organism into which the response element is incorporated, the response element may include minimal promoters isolated from different organisms. The DNA-binding domain of the transcription factor binds to the DNA sequence of the response element, in the presence or absence of a ligand, to initiate or repress the transcription of downstream genes regulated by this response element. Examples of DNA sequences for the response elements of natural ecdysone receptors 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. Cell Biol. 14:4465 1994).

[0088] The term “operably ligated,” as used herein, refers to the physical and / or functional ligation of a DNA segment to another DNA segment in such a manner that the segments function in their intended manner. A DNA sequence encoding a gene product is operably ligated to a regulatory sequence if it is ligated to a regulatory sequence, such as a promoter, enhancer, and / or silencer, in a manner that allows for the modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably ligated to a promoter if it is ligated to a promoter downstream of the transcription start site of the promoter in the correct reading frame relative to the transcription start site, allowing transcription elongation to proceed by the DNA sequence. An enhancer or silencer is operably ligated to a DNA sequence encoding a gene product if it is ligated to the DNA sequence in a manner that increases or decreases the transcription of the DNA sequence, respectively. Enhancers and silencers may be located upstream, downstream, or embedded within the coding region of the DNA sequence. The DNA for the signal sequence is operably ligated to the DNA encoding the polypeptide, if the signal sequence is expressed as a preprotein that participates in polypeptide secretion. Ligation of the DNA sequence to the regulatory sequence is typically achieved by ligation at a suitable restriction site or via an adapter or linker inserted into the sequence using a restriction endonuclease known to those skilled in the art.

[0089] As used herein, the term “codon degenerate variant” refers to a modified nucleic acid sequence that codes for the same amino acid sequence as the original sequence, but with different specific nucleotides containing codons. The genetic code is degenerate, meaning that multiple codons can code for the same amino acid. For example, the amino acid leucine can be coded by six different codons: CTG, CTT, CTC, CTA, TTG, and TTA. A codon degenerate table, also known as a genetic code table or codon table, is a chart that provides information about the relationships between codons (sequences of three nucleotides) and the corresponding amino acids they code for. The table lists 64 possible codons and indicates which amino acid each codon represents. Table 1 is an example of a codon degenerate table.

[0090] [Table 1]

[0091] The following definitions are supplementary to the definitions in the Art and apply to this application; they should not be attributed to any related or unrelated cases, such as any generally owned patent or application. Therefore, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0092] Furthermore, publicly available software resources are readily available for the “reverse translation” of polypeptide sequences, also known as computer-generated “reverse transcription,” which is the conversion of a polypeptide sequence into the nucleotide sequence that encodes it. 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).

[0093] As used herein, codon degenerate variants may be utilized to optimize gene expression or enhance protein production. By modifying codons within a nucleic acid sequence, it is possible to utilize codons that are more frequently used or preferred by the translational mechanisms of the host organism. This may result in increased efficiency of protein expression or improved compatibility with specific host organisms.

[0094] As used herein, the term "expression" refers to the transcription and stable accumulation of sense RNA (mRNA) or antisense RNA derived from nucleic acids or polynucleotides. Expression may also refer to the translation of mRNA into proteins or polypeptides.

[0095] 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 by specific restriction sites or by homologous recombination. The DNA segment contains a polynucleotide encoding the polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in a proper reading frame for transcription and translation. A “transformation cassette” refers to a specific vector containing a polynucleotide encoding the polypeptide of interest, and having elements in addition to the polynucleotide that promotes transformation of a particular host cell. The cassettes, expression cassettes, gene expression cassettes, and transformation cassettes of the present invention may also include elements that enable enhanced expression of the polynucleotide encoding the polypeptide of interest in a host cell. These elements may include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, and polyadenylation sequences. The expression cassettes described herein are approximately 500-10,000 bp, 1,000-5,000 bp, 1,500-4,500 bp, 1,800-4,400 bp, 2,000-4,500 bp, 2,100-4,400 bp, 2,200-4,300 bp, 2,300-4,200 bp, 2,400-4,100 bp, and 2,500-4,000 bp. The expression cassette may contain a total length of 0 bp, approximately 2,600 to 3,900 bp, approximately 2,700 to 3,800 bp, approximately 2,800 to 3,800 bp, approximately 2,900 to 3,700 bp, approximately 3,000 to 3,600 bp, approximately 3,100 to 3,500 bp, approximately 3,150 to 3,450 bp, approximately 3,200 to 3,400 bp, approximately 3,250 to 3,350 bp, or approximately 3,300 bp. Alternatively, the expression cassette may contain any number of base pairs within these ranges.For example, the expression cassettes are approximately 500 bp, 750 bp, 1,000 bp, 1,250 bp, 1,500 bp, 1,750 bp, 2,000 bp, 2,250 bp, 2,500 bp, 2,550 bp, 2,600 bp, 2,650 bp, 2,700 bp, 2,750 bp, 2,800 bp, 2,850 bp, 2,900 bp, 2,950 bp, 3,000 bp, 3,050 bp, 3,100 bp, 3,150 bp, 3,200 bp, 3,250 bp, and 3,300 bp. The expression may contain approximately 3,350 bp, approximately 3,400 bp, approximately 3,450 bp, approximately 3,500 bp, approximately 3,550 bp, approximately 3,600 bp, approximately 3,650 bp, approximately 3,700 bp, approximately 3,750 bp, approximately 3,800 bp, approximately 3,850 bp, approximately 3,900 bp, approximately 3,950 bp, approximately 4,000 bp, approximately 4,050 bp, approximately 4,100 bp, approximately 4,150 bp, approximately 4,200 bp, approximately 4,250 bp, approximately 4,300 bp, approximately 4,350 bp, approximately 4,400 bp, approximately 4,450 bp, or approximately 4,500 bp. In one embodiment, the expression cassette contains approximately 2,800 bp. In another embodiment, the expression contains 2,825 bp.

[0096] As used herein, the term “vector” refers to any vehicle for cloning and / or transferring nucleic acids into host cells. A vector may be a replicon to which another DNA segment can be attached, resulting in the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replicating under its own control. The term “vector” includes both viral and nonviral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors known in the art can be used to manipulate nucleic acids, to incorporate response elements and promoters into genes, etc. Possible vectors include, for example, bacteriophages, e.g., lambda derivatives, or plasmids, e.g., pBR322 or pUC plasmid derivatives, or Bluescript vectors, e.g., plasmids or modified viruses. Another example of a vector useful in the present invention is the ULTRAVECTOR® Production System (Intrexon Corp., Blacksburg, VA), described in International Publication No. 2007 / 038276. For example, insertion of DN fragments corresponding to response elements and promoters into a suitable vector can be achieved by ligating a suitable DNA fragment into a selected vector having complementary adherent ends. Alternatively, the ends of the DNA molecule may be enzymatically modified, or any site may be generated by ligating a nucleotide sequence (linker) to the DNA ends. Such vectors may be engineered to contain a selection marker gene that provides selection of cells into which the marker has been incorporated into the cellular genome. Such markers enable the identification and / or selection of host cells that incorporate and express the protein encoded by the marker.

[0097] As used herein, the term “plasmid” refers to an extrachromosomal element, often containing genes, that is typically in the form of a circular double-stranded DNA molecule, rather than being part of the cell’s central metabolism. Such elements may be single-stranded or double-stranded DNA or RNA, of any origin, autologous replication sequences, genomic integration sequences, phages or nucleotide sequences, linear, circular, or higher-order coils, where some nucleotide sequences are conjugated to or recombined into a unique construct that can introduce into the cell a promoter fragment and DNA sequence for a selected gene product, along with a suitable 3’ untranslated sequence.

[0098] As used herein, the terms “cloning vector” and “replicon” refer to a unit-length nucleic acid, preferably DNA, such as a plasmid, phage, or cosmid, which replicates sequentially and contains an origin of replication, to which another nucleic acid segment may be attached in such a way that it results in replication of the attached segment. A cloning vector may be capable of replicating in one cell type and expressing in another cell type (a “shuttle vector”). A cloning vector may contain one or more sequences that can be used as a vector for insertion of a sequence of interest and / or for selection of cells containing one or more multiple cloning sites.

[0099] As used herein, the term "viral vector" refers to a virus, viral particle, or derivative thereof that can transfer nucleic acid into a cell or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements mainly derived from viruses. Viral vectors, particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells and living animal subjects. Viral vectors that can be used include, but are not limited to, retroviruses, adeno-associated viruses, poxviruses, baculoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, adenoviruses, geminiviruses, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytfectins), DNA-protein complexes, and biopolymers. In addition to nucleic acids, vectors may also contain one or more regulatory regions, and / or selectable markers useful for selection, measurement, and monitoring of the results of nucleic acid transfer (such as which tissues to transfer into, duration of expression, etc.).

[0100] As used herein, the terms "adenovirus" and "adenoviral vector" refer to adenoviruses that retain the ability to participate in the adenovirus life cycle and / or are physically inactivated, for example, by disruption (such as sonication), denaturation (such as using heat or solvents), or cross-linking (such as via formalin cross-linking). The "adenovirus life cycle" includes (1) virus binding and entry into cells, (2) transcription of the adenovirus genome and translation of adenovirus proteins, (3) replication of the adenovirus genome, and (4) assembly of viral particles (e.g., Fields Virology, 5 thSee, Knipe et al. (eds.), Lippincott Williams & Wilkins, Philadelphia, PA (2006). Adenoviruses may also be deficient in replication by the deletion of one or more portions of the naturally occurring viral genome (i.e., they do not retain the ability to participate in the adenovirus life cycle), as used and described herein. "Adenovirus" and "adenovirus vector" may include adenoviruses whose adenovirus genome has been engineered to accommodate nucleic acid sequences that are non-native with respect to the adenovirus genome. Typically, adenovirus vectors are constructed, for example, by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus genome of an adenovirus to accommodate the insertion of a non-native nucleic acid sequence into the adenovirus for gene transfer.

[0101] As used herein, the terms “MOI” or “infection multiplicity” refer to the average number of viral particles (e.g., recombinant virus or control virus) that infect a single cell in a given experiment.

[0102] As used herein, the term “transfection” refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell is “transfected” with exogenous or heterologous RNA or DNA if such RNA or DNA is introduced into the cell. A cell is “transformed” with exogenous or heterologous RNA or DNA if the transfected RNA or DNA causes a phenotypic alteration. The RNA or DNA that transforms a cell may be incorporated (covalently linked) into the chromosomal DNA that makes up the cell’s genome.

[0103] As used herein, the term “transformation” refers to the introduction of a nucleic acid fragment into the genome of a host organism that results in genetically stable inheritance. A host organism containing a transformed nucleic acid fragment is referred to as a “transgenic,” “recombinant,” or “transformed” organism.

[0104] As used herein, the term “electroporation” refers to the use of transmembrane electric field pulses that transiently increase the permeability of the cell membrane, enabling the introduction of exogenous biological materials into cells, such as DNA, RNA, peptides, polypeptides, proteins, enzymes, or ribonucleoproteins (RNPs). The electric field pulses create transient pores in the cell membrane, facilitating the uptake of biological materials. Electroporation can be performed using specialized buffers and devices that control pH, conductivity, osmolality, and other parameters to optimize the process and enhance transfection efficiency while minimizing cell damage. Electroporation can be used to introduce exogenous materials (e.g., biomolecules, plasmids, oligonucleotides, expression cassettes, siRNAs, drugs, and ions) into various cell types, including primary human hematopoiesis, immune cells, pluripotent progenitor cells, fibroblasts, and endothelial cells, for applications in gene therapy, cell therapy, and biotechnology research.

[0105] As used herein, the terms “inducing,” “inducing,” and their grammatical equivalents refer to an increase in nucleic acid sequence transcription, promoter activity, and / or expression, brought about by a transcription regulator, compared to some baseline level of transcription.

[0106] As used herein, the terms “promoter” and “promoter sequence” are interchangeable and refer to DNA sequences capable of controlling the expression of coding sequences or functional RNA. Generally, coding sequences are located 3' relative to promoter sequences. Promoters may be entirely derived from native genes, or they may consist of different elements derived from different promoters found in nature, or they may further include synthetic DNA segments. Those skilled in the art will understand that different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that express genes in most cell types are generally referred to as “constitutive promoters.” Promoters that express genes in specific cell types are generally referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that express genes at specific stages of development or cell differentiation are generally referred to as “development-specific promoters” or “cell differentiation-specific promoters.” Promoter induces and triggers gene expression after exposure or treatment of cells with virulence factors, biomolecules, chemicals, ligands, light, etc., which induce the promoter; these are generally referred to as "inducible promoters" or "regulatory promoters." It is further recognized that, in most cases, the precise boundaries of regulatory sequences are not fully defined, and DNA fragments of different lengths may have the same promoter activity.

[0107] The promoter sequence typically extends upstream (5' direction) at its 3' end, bordered by the transcription start site, to contain the minimum number of bases or elements necessary to initiate transcription at a level detectable above the background. Within the promoter sequence, the transcription start site (conveniently defined, e.g., by mapping to a nuclease SI) and the protein-binding domain (consensus sequence) that leads to RNA polymerase binding are found.

[0108] The origin of the promoter may be natural or synthetic, and the origin of the promoter should not limit the scope of the invention as described herein. In other words, the promoter may be cloned directly from cells, or it may have been previously cloned from a different origin, or it may be synthetic.

[0109] As used herein, the term “transcriptional regulator” refers to a biological element that acts to prevent or inhibit the transcription of promoter-driven DNA sequences under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to allow or stimulate the transcription of promoter-driven DNA sequences under certain environmental conditions (e.g., an inducer or enhancer).

[0110] As used herein, the term “enhancer” refers to a DNA sequence that increases transcription, for example, of a operably linked nucleic acid sequence. Enhancers can be located several kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, the pattern of DNA methylation, or changes in DNA structure. Numerous enhancers from various different origins are well known in the art and are available as cloned polynucleotides or within them (e.g., from contract laboratories, e.g., ATCC, and other commercial or personal origins). Some polynucleotides containing promoters (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, 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 (mu) 5' enhancers, light chain (kappa) 5' enhancers, kappa and mu intron enhancers, and 3' enhancers (see, in general, Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353–363; and U.S. Patent No. 5,885,827)).

[0111] 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 certain embodiments, the TSP is constitutive, i.e., continuously active. A constitutive TSP may be constitutive-ubiquitous (i.e., generally functioning in any tissue or cell without the need for additional factors or regulators) or constitutive-tissue or cell-specific (i.e., generally functioning in a specific tissue or cell type without the need for additional factors or regulators). In certain embodiments of the present invention, the TSP is activated under conditions associated with a disease, disorder, or condition. In certain 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, disorder, or condition” include, but are not limited to, disease-specific promoters, promoters that respond to specific physiological, developmental, differentiation, or pathological conditions, promoters that respond to specific biomolecules, and promoters that are specific to specific tissues or cell types associated with a disease, disorder, or condition, such as tumor tissue or malignant cells. TSPs may include sequences of naturally occurring promoters, modified sequences derived from naturally occurring promoters, or synthetic sequences (e.g., insertion of a response element into a minimal promoter sequence to alter the promoter's responsiveness).

[0112] A therapeutic switch promoter useful in the present invention may include any promoter useful for treating, restoring, or preventing a specific disease, disorder, or condition. Examples include, but are not limited to, promoters of genes that show increased expression only during a specific disease, disorder, or condition, and promoters of genes that show increased expression under specific cellular conditions (e.g., proliferation, apoptosis, pH changes, oxidative states, oxygen levels). In some embodiments in which the gene switch includes two or more transcription factor sequences, the specificity of the therapeutic method can be increased by combining the disease or condition-specific promoter with a tissue or cell-type-specific promoter that restricts the expression of the therapeutic product to the tissue in which it is expressed. Thus, tissue or cell-type-specific promoters are encompassed within the definition of a therapeutic switch promoter.

[0113] 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 modulates gene expression in response to a ligand that binds 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. Additional examples of chimeric ecdysone receptor systems are described in U.S. Patent Publication No. 7,091,038, U.S. Patent Publication Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, as well as International Publication Nos. 01 / 70816, 02 / 066612, 02 / 066613, 02 / 066614, 02 / 066615, 02 / 29075, and 2005 / 108617. In one embodiment, the system is the RheoSwitch® therapeutic system (RTS), which contains two fusion proteins: the DEF domain of a mutagenic ecdysone receptor (EcR) fused to the Gal4 DNA-binding domain, and the EF domain of a chimeric RXR fused to the VP16 transcriptional activation domain, both expressed under a constitutive promoter.

[0114] When RNA polymerase transcribes the coding sequence into mRNA, it is "under the control" of transcriptional and translational regulatory sequences in the cell, which are then trans-spliced ​​(if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

[0115] "Transcriptional and translational regulatory sequences" refer to DNA regulatory sequences, such as promoters, enhancers, and terminators, that provide expression of coding sequences in host cells. In eukaryotic cells, polyadenylation signals are regulatory sequences. Enhancers that may be used in embodiments of the present invention include, but are not limited to, SV40 enhancers, cytomegalovirus (CMV) enhancers, elongation factor 1 (EF 1) enhancers, yeast enhancers, and viral gene enhancers.

[0116] The terms “3' non-coding sequence” and “3' untranslated region (UTR)” refer to DNA sequences located downstream (3') of the coding sequence and may include polyadenylation [poly(A)] recognition sequences and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by influencing the addition of polyadenylate tracts to the 3' end of mRNA precursors.

[0117] 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 a sequence that is naturally responsible for the expression of a particular nucleic acid (homologous region), or it may include a sequence of a different origin that is responsible for the expression of a different protein or even a synthetic protein (heterologous region). In particular, the sequence may be a sequence of a prokaryotic, eukaryotic, or viral gene, or an induced sequence, that stimulates or represses the transcription of a gene in a specific or nonspecific manner, and in an inducible or non-inducible manner. A regulatory region may include an origin of replication, an RNA splice site, a promoter, an enhancer, a transcription termination sequence, and a signal sequence that directs a polypeptide to the secretory pathway of a target cell.

[0118] As used herein, the term “modulate” means to induce, reduce, or inhibit nucleic acid or gene expression, resulting in the induction, reduction, or inhibition of protein or polypeptide production, respectively.

[0119] As used herein, the term “inducible promoter” refers to a promoter whose activity is induced by the presence or absence of a transcription factor, such as a biological or abiotic factor. Inducible promoters are useful because the expression of a gene operably linked to them can be turned on or off at a particular stage of development of an organism or a particular tissue. Non-limiting examples of inducible promoters include alcohol-regulating promoters, tetracycline-regulating promoters, steroid-regulating promoters, metal-regulating promoters, pathogenesis-regulating promoters, temperature-regulating promoters, and light-regulating promoters. An inducible promoter may be part of a gene switch or a gene switch. An inducible promoter may be a gene switch ligand-inducible promoter. In some cases, an inducible promoter may be an ecdysone receptor-based gene switch of two small molecule ligand-inducible polypeptides.In some cases, gene switches are, but are not limited to, International Publication Nos. 2001 / 070816; International Publication Nos. 2002 / 029075; International Publication Nos. 2002 / 066613; International Publication Nos. 2002 / 066614; International Publication Nos. 2002 / 066612; International Publication Nos. 2002 / 066615; International Publication Nos. 2003 / 027266; ​​International Publication Nos. 2003 / 027289; International Publication Nos. 2005 / 108 Publication No. 617; International Publication No. 2009 / 045370; International Publication No. 2009 / 048560; International Publication No. 2010 / 042189; International Publication No. 2010 / 042189; International Publication No. 2011 / 119773; and International Publication No. 2012 / 122025; and U.S. Publication No. 7,091,038; U.S. Publication No. 7,776,587; U.S. Publication No. 7,807,417; U.S. Publication No. 8,202,718 Specification; Specification No. 8,105,825; Specification No. 8,168,426; Specification No. 7,531,326; Specification No. 8,236,556; Specification No. 8,598,409; Specification No. 8,715 ,959 specification; 7,601,508 specification; 7,829,676 specification; 7,919,269 specification; 8,030,067 specification; 7,563,879 specification; You can choose from ecdysone-based receptor components described in any of the systems described in Specification No. 8,021,878; Specification No. 8,497,093; Specification No. 7,935,510; Specification No. 8,076,454; Specification No. 9,402,919; Specification No. 9,493,540; Specification No. 9,249,207; and Specification No. 9,492,482.

[0120] As used herein, two or more individually operable gene regulatory systems are referred to as “orthogonal” if a) each modulation of a given system by its respective ligand at a selected concentration results in a measurable change in the magnitude of gene expression in that system, and b) regardless of whether the actual modulations are synchronous or sequential, the change is statistically significantly different from the change in expression of all other simultaneously operable systems in the cell, tissue, or organism. Preferably, the modulation of each individual operable gene regulatory system results in a change in gene expression that is at least twice as large, e.g., at least five times, ten times, 100 times, or 500 times larger than all other operable systems in the cell, tissue, or organism. Ideally, each modulation of a given system by its respective ligand at a selected concentration results in a measurable change in the magnitude of gene expression in that system, and no measurable change in expression of all other operable systems in the cell, tissue, or organism. In such cases, multiple inducible gene regulatory systems are referred to as “fully orthogonal.” Useful orthogonal ligand and orthogonal receptor-based gene expression systems are described in U.S. Patent Application Publication No. 2002 / 0110861.

[0121] As used herein, the term “gene switch” refers to a combination of a promoter-related response element and a ligand-dependent transcription factor-based system that modulates the expression of the gene into which the response element and promoter are incorporated in the presence of one or more ligands. The term “polynucleotide encoding a gene switch” refers to a combination of a promoter-related response element and a ligand-dependent transcription factor-based system that modulates the expression of the gene into which the response element and promoter are incorporated in the presence of one or more ligands. Closely regulated inducible gene expression systems or gene switches, such as EcR-based systems, are useful in a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, and functional genomics and regulation of traits in transgenic plants and animals. Such inducible gene expression systems may include ligand-induced heterologous gene expression systems.

[0122] As used herein, the term "CAP" or "cap" refers to a modified nucleotide, generally 7-methylguanosine (7meG-ppp-G) ligated at the 5' end of eukaryotic mRNA, typically from 3' to 5', which plays a necessary element in the normal translation initiation pathway during protein expression from that mRNA.

[0123] As used herein, the term “Sleeping Beauty (SB) transposon system” refers to a synthetic DNA transposon system for introducing DNA sequences into vertebrate chromosomes. Some exemplary embodiments of the system are described, for example, in U.S. Patent Nos. 6,489,458, 8,227,432, 9,228,180 and International Publication No. 2016 / 145146. The Sleeping Beauty transposon system comprises a Sleeping Beauty (SB) transposase and an SB transposon. In embodiments, the Sleeping Beauty transposon system may include an SB11 transposon system, an SB100X transposon system, or an SB110 transposon system.

[0124] As used herein, the terms “transposon” or “transposition element” (TE) refer to vector DNA sequences that can alter their position within the genome, sometimes by producing or reversing mutations, thereby changing the size of the cell’s genome. Transposition often results in duplication of TEs. Class I TEs are copied in two steps: first, they are transcribed from DNA to RNA, and the resulting RNA is then reverse-transcribed back into DNA. This copied DNA is then inserted into the genome at the new location. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the TE itself. The characteristics of retrotransposons are similar to those of retroviruses, e.g., HIV. The cut-and-paste transposition mechanism of Class II TEs does not involve an RNA intermediate. Transposition is catalyzed by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA, while others bind to specific DNA sequence targets. Transposases perform staggered cuts at the target site, resulting in a single-stranded 5' or 3' DNA overhang (sticky end). This step excises the DNA transposon, which is then ligated to a new target site. This process involves the activity of DNA polymerase to fill the gap and DNA ligase to close the sugar-phosphate backbone. This results in duplication of the target site. The insertion site of a DNA transposon can be identified by a short series of repeat sequences that can be created by staggered cuts and DNA polymerase filling in the target DNA, followed by a series of reverse repeat sequences that are crucial for subsequent transposase-mediated TE excision. Cut-and-paste TEs can overlap if their transposition occurs during the S phase of the cell cycle, when the donor site has already replicated but the target site has not yet. Transpositions can be classified as either autonomous or non-autonomous in both class I and class II TEs. Autonomous TEs can move on their own, while non-autonomous TEs require the presence of another TE to move.This is often because non-autonomous TEs lack transposases (for class II) or reverse transcriptases (for class I).

[0125] As used herein, the term “transposase” refers to an enzyme that binds to the end of a transposon and catalyzes the transposon’s movement to another part of the genome via a cut-and-paste mechanism or a replication-transposition mechanism.

[0126] As used herein, the terms “polypeptide,” “peptide,” “polypeptide construct,” and “peptide construct,” as well as their grammatical equivalents, refer to polymer compounds composed of covalently linked amino acid residues. “Mature protein” is a full-length protein, optionally including glycosylation or other modifications typical of a protein in a given cellular environment. Embodiments of the present invention, as disclosed herein, include the HPV antigen / antigenic polypeptides, peptides, and mature proteins described herein, and also include polynucleotides (DNA or RNA) encoding them. Polypeptides and proteins disclosed herein (including their functional fragments and functional variants) may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 Examples include 2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0127] As used herein, the term “polypeptide fragment” means a polypeptide whose amino acid sequence is shorter than that of a reference polypeptide and which contains the same amino acid sequence throughout the entire portion having these reference polypeptides. Such fragments may, where appropriate, be contained within a larger polypeptide of which they are part. Such fragments of polypeptides according to the present invention may have amino acid lengths of 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 longer.

[0128] As used herein, the terms “isolated polypeptide,” “isolated peptide,” or “isolated protein” refer to polypeptides or proteins that are substantially free from compounds normally associated with them in their natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” does not mean the exclusion of artificial or synthetic mixtures with other compounds, or the presence of impurities that do not impede biological activity, for example, resulting from incomplete purification, the addition of stabilizers, or formulation into pharmaceutically acceptable preparations.

[0129] As used herein, the terms “identical” or “sequence identity” in the context of two nucleic acid or amino acid sequences of polypeptides refer to residues in two sequences that are identical when aligned for maximum match across a defined comparison window. “Comparison window,” as used herein, refers to a segment of at least about 20, typically about 50 to about 200, and more commonly about 100 to about 150 consecutive positions over which the sequences can be compared to the same number of consecutive positions of a reference sequence after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art.The optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J Mal. Biol., 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Nat. Acad Sci USA., 85:2444 (1988); or by computer execution of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program from Intelligentics, Mountain View Calif, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA, GAP, BESTFIT, BLAST, FASTA, and TFASTA); the CLUSTAL program is described in Higgins and Sharp, Gene, 73:237-244. Alignment is also frequently performed by laboratory and manual alignment. (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); 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).

[0130] In one class of embodiments, the polypeptides herein are 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, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example. Similarly, nucleic acids can also be described by reference to an initiating nucleic acid, for example, a nucleic acid may be 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, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example. 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 will find matching residues in the larger molecule in the order in which the two molecules are optimally aligned.

[0131] As used herein, the term “identity percentage” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing their sequences, as is known in the art. In the art, “identity” also means the degree of sequence relevance between polypeptide or polynucleotide sequences, determined by matching strings of such sequences. "Identity" and "similarity" are not limited to those described above, or can be readily calculated by known methods, including, for example, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Methods for determining identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity percentage calculations may be performed using sequence analysis software, such as the MegAlign (or more recently, MegAlign Pro) program from the LASERGENE Bioinformatics Computer Suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of sequences may be performed using Clustal's alignment method (Higgins et al., CABIOS. 5:151 1989) with default parameters (gap penalty=10, gap length penalty=10). Default parameters for pairwise alignment using Clustal's method may be selected: KTUPLE 1, gap penalty=3, window=5, and DIAGONALS SAVED=5.

[0132] As used herein, the term “substantially similar” and its grammatical equivalents, applied to nucleic acids or amino acid sequences, mean that the nucleic acid or amino acid sequence has sequence identity of at least 90% or higher relative to a reference sequence, e.g., at least 95%, at least 98%, at least 99%, 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%, relative to a reference sequence, using standard parameters and comparison programs described above, e.g., BLAST. The term “substantially identical” and its grammatical equivalent, applied to nucleic acids or amino acid sequences, means that a nucleic acid or amino acid sequence contains a sequence that, using standard parameters and a comparison program described above, e.g., BLAST, has at least 99% sequence identity with respect to a reference sequence, e.g., 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%. For example, the BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and comparisons of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.The percentage is calculated by determining the number of positions that give the number of matched positions, where the same nucleic acid base or amino acid residue is present in both sequences, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In embodiments, substantial identity exists over a region of sequence that is at least about 50 residues long, over a region of at least about 100 residues, and in embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding region.

[0133] Where used herein, the term “functional fragment” or its grammatical equivalent is used herein to mean a portion, fragment, or segment of a biomolecule that retains essential functional characteristics or activity of the original biomolecule. The term “functional variant” or its grammatical equivalent is used herein to mean a modified form of a biomolecule that retains essential functional characteristics or activity of the original molecule, while exhibiting some degree of variation. This includes biomolecules that have been modified, for example, by genetic engineering or mutagenesis techniques, to introduce specific changes while preserving the overall functionality of the biomolecule. Functional variants may have one or more amino acid substitutions, insertions, or deletions compared to the original molecule, while still maintaining the desired biological activity or function. Techniques for obtaining these variants, including genetic (repression, deletion, mutation, etc.), chemical, and enzymatic techniques, are known to those skilled in the art. In one embodiment, a biological variant comprises at least about 14 monomers (e.g., nucleotides or amino acids).

[0134] As used herein, the term “homology” in all its grammatical and spelling variations refers to the percentage of identity between two polynucleotides or two polypeptide segments. The degree of sequence agreement between one segment and another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of sequence information between two polypeptide molecules by aligning the sequence information using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form a stable double helix between homologous regions, followed by digestion by a single-strand specific nuclease and sizing of the digested fragments.

[0135] When used in the context of amino acid sequences, the term "substitution" refers to a variation in an amino acid sequence in which one amino acid is replaced by another. The nomenclature used to represent amino acid substitutions follows a standard format. For example, in "L50G," "L" represents the original amino acid leucine (abbreviated as "L"), "50" indicates the position of the amino acid in the amino acid sequence relative to its N-terminus (in this case, the amino acid is the 50th amino acid from the N-terminus of the sequence), and "G" indicates the substituted amino acid, in this example, glycine (abbreviated as "G"). Therefore, "L50G" represents a substitution in which leucine at position 50 (relative to its N-terminus) of the amino acid sequence is replaced by glycine.

[0136] As used herein, the terms “conservative amino acid substitution” or “conservative mutation” refer to the substitution of one amino acid with another amino acid that shares common properties. A functional method for defining the common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (see Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, a group of amino acids can be defined as those within a group in which the amino acids preferentially exchange with each other, and therefore their effects on the overall protein structure are most similar to each other (Schulz, GE and Schirmer, RH, above). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, e.g., lysine and arginine, and vice versa, in which a positive charge can be maintained; glutamic acid and aspartic acid, and vice versa, in which a negative charge can be maintained; serine and threonine, in which free -OH can be maintained; and glutamine and asparagine, in which free -NH2 can be maintained. Examples of conservative amino acid substitutions are shown in the chart below.

[0137] [Table 2]

[0138] An amino acid sequence that differs from a reference amino acid sequence solely through conservative amino acid substitutions is referred to herein as a “conservatively substituted variant” of the reference sequence. Given the established knowledge and well-known techniques in protein science, determining the functional impact of a “conservatively substituted variant” compared to a reference amino acid sequence is well within the skill of those skilled in the art.

[0139] In some embodiments, the functional variant may be a conservatively substituted variant of the reference sequence. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 100 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 90 or fewer amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 80 or fewer amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 70 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 60 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 50 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference protein by 40 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 30 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 20 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 10 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted 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 conservative amino acid substitution.In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by at least 100 and 150 conservative amino acid substitutions.

[0140] An amino acid sequence that differs from a reference amino acid sequence by at least one non-conservative amino acid substitution is referred to herein as a “non-conservatively substituted variant” of the reference sequence. As used herein, the term “non-conservative amino acid substitution” refers to amino acid substitutions between different groups, such as the substitution of tryptophan by lysine or serine by phenylalanine. In this case, it is preferable that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the homologous parent protein. The substitutability of amino acids is discussed in more detail, for example, LY Yampolsky and A. Stoltzfus, “The Exchangeability of Amino acids in Proteins,” Genetics 2005 Aug.; 170(4):1459-1472. Given the established knowledge and well-known techniques in protein science, determining the functional effect of non-conserved amino acid substitutions in a functional variant compared to a reference amino acid sequence is well within the skill of those skilled in the art.

[0141] In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least one non-conserved amino acid substitution. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 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. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 10 to 20 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 21 to 30 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 31 to 40 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 41 to 50 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 51 to 60 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 61 to 70 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 71 to 80 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 81 to 90 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 91 to 100 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least 100 non-conservative amino acid substitutions.

[0142] As used herein, the term “antibody” refers to either a monoclonal antibody or a polyclonal antibody. “Monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of a B cell and bound to the same epitope. In contrast, “polyclonal antibody” refers to a group of antibodies produced by different B cells and bound to different epitopes of the same antigen. A total antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, while each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have similar general structures, and each region contains four framework regions whose sequences are relatively conserved. The framework region is connected by three complementarity-determining regions (CDRs). These three CDRs, known as CDR1, CDR2, and CDR3, form the antibody's "hypervariable region," which is responsible for antigen binding.

[0143] As used herein, the terms “functional antibody fragment” and “functional fragment of an antibody” or their grammatical equivalents are used interchangeably to mean a portion, fragment, or segment of an antibody that retains essential functional characteristics or activity of the original antibody. In one embodiment, this activity is the ability to specifically bind to an antigen (see Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005) for general reference). A functional antibody fragment may include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or a combination thereof. Non-limiting examples of functional antibody fragments include: (i) an antigen-binding fragment (Fab), which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide crosslinking in the stem region; (iii) a variable fragment ("Fv") consisting of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment joined by a synthetic linker that allows the two domains to synthesize a single polypeptide chain (e.g., 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: 778). Examples include dimers of polypeptide chains, (see 1998), and (v) dimers of polypeptide chains, where each polypeptide chain contains a VH connected to the VL by a peptide linker that is too short to allow pairing between the 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 No. 8,603,950.

[0144] As used herein, the term “antibody-like molecule” can be, for example, a protein that is a member of the Ig superfamily capable of selectively binding to a partner. MHC molecules and T cell receptors are such molecules. In one embodiment, the antibody-like molecule is a TCR. In one embodiment, the TCR is modified to increase its MHC binding affinity.

[0145] As used herein, the terms “antigen-recognizing moiety” or “antigen-recognizing domain” refer to a molecule or part of a molecule that specifically binds to an antigen. In one embodiment, the antigen-recognizing moiety is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0146] As used herein, the term “immune cells” includes dendritic cells, macrophages, neutrophils, mast cells, eosinophils, basophils, natural killer cells, and lymphocytes (e.g., B and T cells).

[0147] As used herein, the terms “T cell” or “T lymphocyte” refer to a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on their cell surface. The “TCR” is a protein molecule found on the surface of T cells, and it is a type of leukocyte involved in adaptive immune responses. The variable domain of the TCR contains a highly pleomorphic loop called the complementarity-determining region (CDR), which is responsible for binding to peptide-presenting MHC. There are two main forms of TCR: αβ TCR and γδ TCR. Both forms consist of two protein chains, known as alpha (α) and beta (β) chains for αβ TCR and gamma (γ) and delta (δ) chains for γδ TCR. These chains together form a heterodimer structure. The majority of T cells in the human immune system express αβ TCR. The α and β chains of αβ TCRs are encoded by separate gene segments, which undergo recombination during T cell development to produce diverse TCR specificities. The α and β chains contain variable (V), diverse (D), and conjugating (J) gene segments, respectively, similar to the antibody gene rearrangement process. The combination of the V, D, and J gene segments contributes to the unique antigen-binding specificity of αβ TCRs. αβ TCRs recognize antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. In contrast to αβ TCRs, γδ TCRs are less dominant in the immune system but still play an important role. The γ and δ chains of γδ TCRs are also encoded by separate gene segments and undergo recombination during T cell development. The γδ TCR gene rearrangement process differs from that of αβ TCRs. γδ T cells often exhibit tissue-specific distributions and are found in epithelial tissues, such as the skin and intestines. γδ TCRs can recognize various antigens, including certain peptide and non-peptide molecules, independently of MHC presentation. Both αβ TCRs and γδ TCRs participate in and respond to immune surveillance, but they have different functions and specificities.While αβ TCRs are primarily involved in the recognition of peptides presented by major histocompatibility complex (MHC) molecules, γδ TCRs may possess a wider range of antigen recognition capabilities.

[0148] TCRs and constructs encoding TCRs that recognize MHC-antigen complexes can be created and introduced into T cells (known as TCR T cells), and the following TCR-peptide-MHC interactions can be utilized to induce an immune response. Greenbaum et al., Cancer Immunol Res 1 November 2021; 9 (11): 1252-1261. There is interest in the use of TCRs with higher affinity than the normal range for peptide-MHC antigens (type I), referred to as high-affinity TCRs, to generate soluble TCRs that can be used directly against target cells, either 1) to drive the activity of CD4 helper T cells (which lack the CD8 coreceptor), or 2) by attaching to "effector" molecules (e.g., antibody Fc region, toxic drug, or antibody scFv for forming bispecific proteins, e.g., anti-CD3 antibody) (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 Opin Pharmacol, 5:438-43 (2005); Richman and Kranz, Biomol Eng, 24:361-73 (2007)). This approach may also overcome the problem faced by some cancer patients due to their T cells not expressing TCRs with sufficient specificity and binding affinity to basic tumor antigens. For example, more than 300 MHC-restricted T cells that define tumor antigens have been identified (Cheever et al., Clin Cancer Res. 2009;15(17):5323-5337). These tumor antigens include mutated peptides, differentiation antigens, and overexpressed antigens, all of which function as targets for therapy.Since most cancer antigens described to date are derived from intracellular proteins that can only be targeted on the cell surface in the context of MHC molecules, TCRs are ideal candidates for therapy because they have evolved to recognize this class of antigens. Similarly, TCRs can detect peptides derived from viral proteins that are processed naturally in infected cells and displayed on the cell surface by MHC molecules. However, patients with these diseases may not have optimized TCRs that bind to and destroy infected cells. Finally, in a highly specific manner, TCRs can be used as receptor antagonists for autoimmune targets or as delivery agents to immunosuppress local immune cell responses, thereby avoiding general immunosuppression.

[0149] As used herein, the term “helper T cells” (TH or Th cells) refers to cells that assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of 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 peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI, TH2, TH3, TH9, TH17, TH22, or TFH (follicular helper T cells), which secrete different cytokines that promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes.

[0150] As used herein, the terms “cytotoxic T cells” (TC cells, or CTLs) or “cytotoxic T lymphocytes” refer to cells that destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells are also known as 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 anerogenic state that prevents autoimmune diseases by IL-10, adenosine, and other molecules secreted by regulatory T cells.

[0151] As used herein, the term “memory T cells” refers to a subset of antigen-specific T cells that persist for a long period after an infection has resolved. They rapidly expand into a large number of effector T cells upon re-exposure to their congener antigens, thus providing an immune system with memory of past infections. Memory T cells include 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 either CD4+ or CD8+. Memory T cells typically express the cell surface proteins CD45RO, CD45RA, and / or CCR7.

[0152] As used herein, the term “regulatory T cells” (Treg cells), previously known as suppressor T cells, refers to T cells that play a role in maintaining immune tolerance. Their primary roles are to shut down T cell-mediated immunity toward termination of the immune response and to suppress autoreactive T cells that have evaded the process of negative selection in the thymus.

[0153] As used herein, the term “natural killer T cell” (NKT cell – not to be confused with natural killer cells of the innate immune system) refers to cells that bridge the adaptive immune system to the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CDs. When activated, these cells can perform functions attributed to both helper T cells (TH) and cytotoxic T cells (TC) (i.e., cytokine production and release of cytotoxic / death-causing molecules). They can also recognize and eliminate certain tumor cells and cells infected with herpesviruses.

[0154] As used herein, the term “proliferative disorders” refers to a unified concept in which excessive cell proliferation and / or turnover of the intracellular matrix significantly contribute to the pathogenesis of the disease, including cancer.

[0155] Where used herein, “patient” or “subject” refers to a mammalian subject that has been diagnosed with, has, or is suspected of having, a disease or disorder, such as cancer. In some embodiments, the term “patient” refers to a mammalian subject that has a higher-than-average likelihood of developing a proliferative disorder, such as cancer. Exemplary patients may be humans, apes, dogs, pigs, cattle, cattle, horses, goats, sheep, rodents, and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female. “Patient in need of it” or “subject in need of it” is used herein to refer to a patient who has been diagnosed with, or is suspected of having, a disease or disorder, but is not limited to, for example, human papillomavirus (HPV) infection.

[0156] "Administering" is used herein to refer to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration of a composition, e.g., by injection, may be carried out by intravenous, subcutaneous, intradermal, intraperitoneal, or intramuscular injection. One or more such routes may be used. Parenteral administration may be, for example, by bolus injection or by progressive perfusion over time. Alternatively, or in combination, administration may be by an oral route. In addition, administration may also be by surgical deposition or placement of a medical device. Pharmaceutical compositions may include the compositions of the present invention described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline or phosphate-buffered saline; carbohydrates, such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0157] As used herein, the term “therapeutic product” refers to a therapeutic polypeptide or therapeutic polynucleotide that confers a beneficial function to a host cell on which such product is expressed. Therapeutic polypeptides may include, but are not limited to, small peptides of approximately three amino acids in length, single-stranded or multi-stranded proteins, and fusion proteins. Therapeutic polynucleotides may include, but are not limited to, antisense oligonucleotides, small interfering RNAs, ribozymes, and RNA external guide sequences. Therapeutic products may include naturally occurring sequences, synthetic sequences, or combinations of natural and synthetic sequences.

[0158] As used herein, the terms “treatment,” “to treat,” or their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms or pathological signs that may contribute to the disease. For this purpose, the method of the present invention comprises the step of administering a therapeutically effective amount of a composition of the present invention expressing the nucleic acid sequence of the present invention, or a vector comprising the nucleic acid sequence of the present invention.

[0159] As used herein, “treatment interval” refers to a treatment cycle, for example, a course of administration of a therapeutic agent that may be repeated on a regular schedule. In some embodiments, the dosage regimen may have one or more periods during the treatment interval in which no therapeutic agent is administered.

[0160] As used herein, “dosage regimen” or “medication regimen” includes a treatment regimen based on a determined set of doses. The terms “dose” and “medicate” as used herein refer to the administration of a substance to achieve a therapeutic objective (e.g., treatment of a tumor).

[0161] The terms “administered in combination,” “simultaneously administered,” “to administer simultaneously,” or “to provide simultaneously,” as used herein, mean that two (or more) different treatments are delivered to a subject during the course of the subject’s suffering due to a disease or disorder, for example, that two or more treatments are delivered after the subject has been diagnosed with a disease or disorder and before the disease or disorder is cured or eliminated, or before the treatments are discontinued for any other reason. In some embodiments, the delivery of one treatment is still taking place when the delivery of a second treatment is initiated, resulting in an overlap in the duration of administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment is initiated. In some embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation is observed with the first treatment. In some embodiments, the delivery is such that the reduction of symptoms or other parameters relating to the impairment is greater than that observed with a treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or more than additive. The delivery may be such that the effect of the delivered first treatment is still detectable when the second treatment is delivered.

[0162] In some embodiments of the present invention, the first and second treatments may be administered simultaneously (e.g., at the same time) or sequentially, using the same or separate compositions. Sequential administration refers to the administration of one treatment prior to the administration of an additional (e.g., 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 hours or longer before; 4, 5, 6, 7, 8, 9 days or longer before; or 1, 2, 3, 4, 5, 6, 7, 8 weeks or longer before). The order of administration of the first and secondary treatments may also be reversed.

[0163] The terms “therapeutic effective dose,” “therapeutic dose,” “immunological effective dose,” “antiotumor effective dose,” and “tumor inhibitory effective dose,” or their grammatical equivalents, refer to an effective dose, which is the amount and duration of medication required to achieve the desired therapeutic outcome. The therapeutic effective dose may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compositions described herein to induce a desired response in one or more subjects.

[0164] Alternatively, the pharmacological and / or physiological effects of administering one or more compositions described herein to a patient or subject may be “preventive,” meaning the effect completely or partially prevents the disease or its symptoms. “Preventive effective dose” refers to the effective dose and duration required to achieve the desired preventive outcome (e.g., prevention of disease or prevention of signs of a target condition).

[0165] The term "Derkay score" refers to a scoring system used to assess the severity of recurrent respiratory papillomatosis (RRP) in children. The Derkay score assigns points based on factors such as age of onset, frequency of surgery, location of the papilloma, and tracheostomy dependence. This helps clinicians determine the extent 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. I. Vectors

[0166] Gene therapy, which involves introducing a transgene expressing an exogenous protein into a target (e.g., via vaccination), has demonstrated its usefulness in treating diseases and disorders in those who need it. For such introduction, vectors, such as viral vectors, containing the transgene encoding such a protein are typically used.

[0167] In part, this invention relates to vectors containing expression cassettes and expression cassettes containing transgenes that encode HPV antigen design.

[0168] In certain embodiments, the vector is a plasmid.

[0169] Another suitable vector is an integrated expression vector. Such a vector may be randomly integrated into the DNA of a host cell or may contain a recombination site that enables specific recombination between the expression vector and the chromosomes of the host cell. Such an integrated expression vector can utilize endogenous expression regulatory sequences on the chromosomes of the host cell to produce the expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNATM5 / FRT), or components of the cre-lox system, which can be found in, for example, the pExchange-6 Core vector from Stratagene (La Jolla, Calif.). Examples of vectors that integrate randomly into host cell chromosomes include, for example, pcDNA3.1 from Invitrogen (Carlsbad, Calif.) (when introduced in the absence of the T antigen), and pCI or pFN10A(ACT)FLEXITM from Promega (Madison, Wis.). A. Methods for introducing nucleic acids into cells

[0170] Methods for introducing and expressing genes in cells are well known. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, 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 a desired polynucleotide into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362. 1.Physical method

[0171] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Methods for producing 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 embodiments, the method for introducing polynucleotides into host cells is calcium phosphate transfection or polyethyleneimine (PEI) transfection.

[0172] In some embodiments, the method for introducing polynucleotides into host cells is electroporation. Electroporation is a technique that uses electrical pulses to temporarily increase the permeability of the cell membrane, allowing the uptake of nucleic acid molecules into the cell. This process enhances the delivery and expression of biological materials (e.g., peptides or nucleic acids) in the target cells, potentially improving the immune response to HPV. In some embodiments, the biological material is the HPV antigen. In other embodiments, the biological material is the nucleic acid encoding the HPV antigen.

[0173] The electroporation buffer may contain water, sugar, sugar alcohol, chloride salt, and buffering agent. The pH, conductivity, and osmolality of the buffer are carefully controlled. The buffer may be used with UltraPorator® electroporation apparatus and cartridges. UltraPorator® electroporation apparatuses are designed for the rapid production of gene and cell therapies and can be used as a scale-up and commercialization solution for decentralized cell production. See, for example, PCT / US20 / 59984 (filed November 11, 2020) and U.S. Patent Application No. 17 / 095,028 (filed November 11, 2020).

[0174] In some embodiments, a suspension is formed by combining human-derived cells with exogenous biological material in a buffer, and then an electric current is applied to the suspension to facilitate the introduction of the biological material into the cells. The voltage pulse has an electric field strength of 1–10 kV / cm, a duration of 5–250 μs, and at least 2 A / cm 2 The current density may be such that the method can introduce biological materials, such as nucleic acids, peptides, polypeptides, proteins, enzymes, or RNPs, into primary human blood cells, pluripotent progenitor cells, fibroblasts, and endothelial cells. In some embodiments, the method can introduce biologically active materials into primary human blood cells, pluripotent progenitor cells of human blood, and primary human fibroblasts and endothelial cells. In some embodiments, the cells are human blood cells, such as immune cells. In certain 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 a combination of some of them. In some embodiments, the lymphocytes are T cells. In certain embodiments, the cells are obtained from a patient.

[0175] In some embodiments, the transfection yield and the retrieval yield of transfected cells using the electroporation buffer may be 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 about 1.1 times higher than the transfection yield with the control buffer, and is, 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 than the transfection yield of the control buffer.

[0176] In some of the methods described herein, the HPV antigen is administered to a subject. In some embodiments, these methods include the step of introducing the nucleic acid molecule of the present invention to a subject, followed by electroporation. In specific embodiments, the nucleic acid molecule (e.g., a plasmid encoding the antigen of interest or a therapeutic protein) is injected into the target tissue of the subject, e.g., skin or muscle, using a conventional needle or needle-free injection device. Immediately after injection, a handheld electroporation device is applied to the injection site, e.g., by bringing it into contact with the skin or tissue. The device delivers a short, controlled electrical pulse to the tissue, creating transient pores in the cell membrane, making them 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 produced antigen stimulates an immune response, which may provide protection against the targeted pathogen. In the case of therapeutic proteins, they may exert their intended effects within the cell or tissue.

[0177] In some embodiments, the method may include a step of administering a single nucleic acid molecule, e.g., multiple copies of a single plasmid, or two or more different nucleic acid molecules, e.g., multiple copies of 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 distinct nucleic acid sequences. This approach enhances the immune response by enabling the delivery of multiple HPV antigens or the simultaneous delivery of additional immunostimulators.

[0178] Gene constructs containing nucleic acids encoding the HPV antigen can be administered using a variety of methods, including electroporation devices, conventional syringes, standard needles, side-port needles (as described in U.S. Patent Application Publication No. 2023 / 0017972), needle-free injection devices, or "microprojectile bombardment gene guns." Each of these methods has its advantages and can be selected based on factors such as target tissue, desired level of gene expression, and specific application.

[0179] Several minimally invasive electroporation devices and methods have been described in the literature. These include devices and methods disclosed in U.S. Patent Publication No. 20080234655; U.S. Patent No. 6,520,950; U.S. Patent No. 7,171,264; U.S. Patent No. 6,208,893; U.S. Patent No. 6,009,347; U.S. Patent No. 6,120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; U.S. Patent No. 6,763,264; and U.S. Patent No. 20240123052. These devices and methods are designed to efficiently deliver nucleic acid molecules to cells while minimizing tissue damage and discomfort to the target. By using these minimally invasive electroporation techniques, nucleic acids encoding HPV antigens can be efficiently introduced into target cells, potentially leading to HPV antigen production and stimulation of an immune response. 2.Chemical method

[0180] Chemical methods for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). B. Virus-based delivery systems

[0181] Viral-based delivery systems for delivering nucleic acids, such as viral vectors, are also provided herein. Representative viral vectors include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, and herpes virus-based vectors. Viral vectors may be used as delivery vehicles for therapeutic molecules, such as nucleic acids encoding anti-inflammatory agents, while also avoiding immune surveillance by host cells. Retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus are all suitable for viral vector applications. Robbins et al., Pharmacology & Therapeutics, 80:35-47 (1998). In particular, recombinant adenovirus vectors provide high levels of transgene expression, where the vector remains as episomal DNA without integration 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 may be rendered replication-deficient by deletion of essential viral genes and their replacement with expression cassettes containing foreign therapeutic genes.

[0182] Viral vectors that are not infectious in humans or that have been engineered to remove or inactivate their infectious properties are desired for use in gene therapy because they are efficient in delivering transgenes and can deliver high nucleic acid payloads to dendritic cells.

[0183] However, the effectiveness of treatments using viral vectors is limited by their immunogenicity. For example, human adenoviral vectors are commonly used in gene therapy, but since the majority of the US population has been exposed to the wild-type form of such viruses, many in the population have existing immunity against them. As a result, such vectors and the transgenes carried therein are rapidly removed from the bloodstream. Furthermore, the immunogenicity of such vectors limits their effectiveness in the case of repeated dosing. 1. Retroviral vector

[0184] In certain 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 long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia virus, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. 2. Adeno-associated virus vector

[0185] In certain embodiments, the viral vector is an adeno-associated virus vector. Such vectors are derived from adeno-associated viruses. An advantage of the use of such vectors is that they have low immunogenicity in humans. Another advantage of using such vectors is that they are small and compact and can thus be used efficiently in delivering genes to cells. However, their size also limits the payload capacity compared to the use of adenoviral vectors. They are also more difficult to produce compared to adenoviral vectors. In addition, they have a narrow tissue tropism. 3. Herpesvirus-based vectors

[0186] In certain embodiments, the viral vector is a herpesvirus-based vector. Such vectors are derived from herpes simplex virus (HSV). Such vectors are known to be able to infect a wide variety of cell types and to have long persistence in the host. However, they are more difficult to manipulate compared to adenovirus vectors. 4. Adenovirus-based vectors

[0187] In certain embodiments, the viral vector is an adenovirus vector. Such vectors are derived from adenoviruses, such as human adenovirus (e.g., human Ad5 adenovirus), triadenovirus, or gorilla adenovirus. Adenoviruses are generally associated with benign conditions in humans, and the genomes of adenoviruses isolated from various species, including humans, have been extensively studied. Adenovirus vectors are advantageous because they can infect a wide variety of cell types. They are also relatively easy to handle and can carry high payloads.

[0188] Adenovirus vectors can be produced with high titers and efficiently transfer DNA into replicating and non-replicating cells. Adenovirus vector genomes can be constructed using any species, strain, subtype, mixture of species, strains, or subtypes, or chimeric adenoviruses as the source of vector DNA. Adenovirus stocks that can be used as the source of adenoviruses can be amplified from adenovirus serotypes 1 to 51, currently available from the United States Cell Culture Lineage Preservation Center (ATCC, Manassas, Va.), or from any other serotype of adenovirus available from any other source. For example, an adenovirus could 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.

[0189] An adenovirus vector can be any adenovirus vector that can grow in a cell, which lies in some prominent (but not necessarily substantial) part of the adenovirus genome derived from or based on. An adenovirus vector can be based on the genome of any suitable wild-type adenovirus. In certain embodiments, the adenovirus vector is derived from the genome of a wild-type adenovirus of group C, particularly serotype 2 or 5. Adenovirus vectors are well known in the art, for example, 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 Publication Nos. 95 / 34671, 97 / 21826, and 00 / 00628, as well as in Thomas Shenk, “Adenoviridae and their Replication,” and MS This is described in Horwitz, “Adenoviruses,” Chapters 67 and 68, Virology, BN Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996).

[0190] Adenoviruses are medium-sized (90-100 nm) non-enveloped icosahedral viruses containing approximately 36 kb of double-stranded DNA. The adenovirus capsid mediates crucial interactions in the early stages of viral infection of cells and is required to package the adenovirus genome at the end of the adenovirus life cycle. The capsid contains 252 capsomeres, which contain 240 hexons, 12 penton base proteins, and 12 fibers. Ginsberg et al., Virology, 28: 782-783 (1966). Each hexon contains three identical proteins, namely polypeptide II. Roberts et al., Science, 232: 1148-1151 (1986). Each penton base contains five identical proteins, and each fiber contains three identical proteins. Proteins IIIa, VI, and IX are thought to be present in the adenovirus coat and 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 is dependent on the adenovirus polymerase protein. Therefore, the major components of the adenovirus particle are expressed from the genome only if the polymerase protein gene is present and expressed.

[0191] Several characteristics of adenoviruses make them ideal as vehicles for introducing genetic material into cells for therapeutic applications. For example, adenoviruses have high titers (e.g., about 10). 13Adenoviruses can be produced in particle units (PUs) and can deliver genetic material to non-replicating and replicating cells. In addition, adenovirus genomes can be engineered to carry large amounts of exogenous DNA (up to approximately 8 kb), and adenovirus capsids can further enhance the delivery of longer sequences. (Curiel et al., Hum. Gene Ther., 3: 147-154 (1992)). Furthermore, adenoviruses generally do not integrate into host cell chromosomes but are instead maintained as linear episomes, thereby minimizing the possibility that recombinant adenoviruses may interfere with normal cellular function.

[0192] Adenoviruses may be modified, for example, by methods known in the art, to be used as adenovirus vectors, for example, gene delivery vehicles. Adenoviruses and adenovirus vectors may be replicable, conditionally replicable, or lacking in replication.

[0193] A replicable adenovirus or adenovirus vector can replicate in a typical host cell, i.e., a cell typically susceptible to adenovirus infection. A replicable adenovirus or adenovirus vector may have one or more mutations (e.g., one or more deletions, insertions, and / or substitutions) in its adenovirus genome compared to wild-type adenovirus, without inhibiting viral replication in the host cell. For example, an adenovirus or adenovirus vector may have a partial or complete deletion of the early adenovirus region, known as the E3 region, which is not essential for the replication of the adenovirus or adenovirus genome.

[0194] A conditionally replicating adenovirus or adenovirus vector is an adenovirus or adenovirus vector that is engineered to replicate under certain conditions. For example, a replication-essential gene function, such as a gene function encoded by an adenovirus early region, can be operably linked to an inducible, repressive, or tissue-specific transcriptional regulatory sequence, such as a promoter. In such embodiments, replication requires the presence or absence of a specific factor that interacts with the transcriptional regulatory sequence. Conditionally replicating adenovirus vectors are further described in U.S. Patent No. 5,998,205.

[0195] A replication-deficient adenovirus or adenovirus vector is an adenovirus or adenovirus vector that requires the completion of one or more gene functions or regions of the adenovirus genome necessary for replication, for example, as a result of a deletion in one or more replication-essential gene functions or regions, so that the adenovirus or adenovirus vector does not replicate in typical host cells, particularly human cells, to which it infects.

[0196] Deletion of gene function or genomic region, as used herein, is defined as a disruption (e.g., deletion) of sufficient genetic material in the adenovirus genome, such as when the nucleic acid sequence is disrupted (e.g., deleted) in whole or in part, eliminating or impairing the function of the gene (e.g., reducing the function of the gene product to about half, one-fifth, one-tenth, one-twentieth, one-thirtieth, or one-fiftieth or less). Deletion of an entire gene region is often not necessary for disrupting the function of replication-essential genes. However, removal of a large portion of one or more gene regions may be desirable for the purpose of providing sufficient space in the adenovirus genome for one or more transgenes. While deletion of gene material is preferred, mutation of gene material by addition or substitution is also appropriate for disrupting gene function. Essential replication gene functions are gene functions required for adenovirus replication (e.g., proliferation) and are encoded by, for example, 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).

[0197] Whether an adenovirus or adenovirus vector is replicable or replication-deficient, an adenovirus or adenovirus vector typically holds at least a portion of the adenovirus genome. An adenovirus or adenovirus vector may contain any portion of the adenovirus genome, including protein-coding regions and / or non-protein-coding regions. An adenovirus or adenovirus vector may contain, for example, at least one nucleic acid sequence encoding an adenovirus protein. An adenovirus or adenovirus vector may contain a nucleic acid sequence encoding any suitable adenovirus protein, for example, a protein encoded by any one of the early region genes (i.e., E1A, E1B, E2A, E2B, E3, and / or E4 regions), or a protein encoded by any one of the late region genes encoding viral structural proteins (i.e., L1, L2, L3, L4, and L5 regions).

[0198] It should be recognized that deletions in different regions of adenovirus vectors can alter the mammalian immune response. In particular, deletions in different regions can reduce the inflammatory response induced by adenovirus vectors. Furthermore, the coat protein of adenovirus vectors can be modified to reduce the ability or inability of the adenovirus vector to be recognized by neutralizing antibodies against the wild-type coat protein, as described in International Publication No. 98 / 40509.

[0199] In certain embodiments, the adenovirus or adenovirus vector comprises one or more nucleic acid sequences encoding a pIX protein, a DNA polymerase protein, a penton protein, a hexon protein, and / or a fiber protein. The adenovirus or adenovirus vector may include a full-length nucleic acid sequence encoding the full-length amino acid sequence of an adenovirus protein. Alternatively, the adenovirus or adenovirus vector may include a portion of a full-length nucleic acid sequence encoding a portion of the full-length amino acid sequence of an adenovirus protein. The “portion” 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 amino acids (for example, 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), but includes fewer than 1,200 amino acids (for example, 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). Preferably, a portion of the amino acid sequence is defined by approximately 3 to approximately 500 amino acids (e.g., approximately 10, 100, 200, 300, 400, or 500 amino acids), approximately 3 to approximately 300 amino acids (e.g., approximately 20, 50, 75, 95, 150, 175, or 200 amino acids), or approximately 3 to approximately 100 amino acids (e.g., approximately 15, 25, 35, 40, 45, 60, 65, 70, 80, 85, 90, 95, or 99 amino acids), or any two of the aforementioned values. More preferably, a “portion” of the amino acid sequence includes approximately 500 amino acids or less (e.g., approximately 3 to approximately 400 amino acids, approximately 10 to approximately 250 amino acids, or approximately 50 to approximately 100 amino acids, or any two of the aforementioned values).

[0200] The adenovirus pIX protein is present in the adenovirus capsid and has been shown to enhance hexon nocamer interactions, making it essential for full-length genome packaging. For example, Boulanger et al., J Gen. Virol., 44: 783-800 (1979); Horwitz MS, “Adenoviridae and their replication” in Virology, 2 nd See, ed., BN Fields et al. (eds.), 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 the adenovirus structure, pIX has also been shown to exhibit transcriptional properties, such as stimulation of adenovirus major late promoter (MLP) activity. See, for example, Lutz et al., J. Virol., 71(7): 5102-5109 (1997). The nucleic acid sequences encoding all or part of the adenovirus pIX protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0201] The adenovirus DNA polymerase protein is essential for viral DNA replication both in vitro and in vivo. The polymerase is co-purified in complex with a terminal protein (TP) precursor (pTP), which is covalently attached to the 5' end of adenovirus DNA. (Field et al., J. Biol. Chem., 259: 9487-9495 (1984)). Both adenovirus DNA polymerase and pTP are encoded by the E2 region. The polymerase protein is required for the expression of all structural proteins except pIX. Without the gene sequence for the polymerase protein, the polymerase protein cannot be produced. As a result, the viral genome will not replicate, the major late promoter will not be activated, and the capsid protein will not be expressed. The nucleic acid sequences encoding all or part of the adenovirus DNA polymerase protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0202] The adenovirus hexon protein is the largest and most abundant protein in the adenovirus capsid. The hexon protein is essential for the construction of the viral capsid, the determination of the icosahedral symmetry of the capsid (which then defines limitations on capsid volume and DNA packaging size), and the integrity of the capsid. In addition, the hexon is a primary 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). While the main structural features of the hexon protein are shared by adenoviruses across serotypes, the hexon protein differs in size and immunological properties between serotypes. Jornvall et al., J. Biol. Chem., 256(12): 6181-6186 (1981). A comparison of 15 adenovirus hexon proteins revealed that the main antigenic and serotype-specific regions of the hexon appear to be located in loops 1 and 2 (i.e., LI or l1 and LII or l2, respectively), where there are seven distinct hypervariable regions (HVR1-HVR7) of varying lengths and sequences among adenovirus serotypes. Crawford-Miksza et al., J. Virol., 70(3): 1836-1844 (1996). Nucleic acid sequences encoding all or part of the adenovirus hexon proteins are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0203] The adenovirus fiber protein is a homotrimer of adenovirus polypeptide IV that has three domains: a tail, a shaft, and a knob. Devaux et al., J. Molec. Biol., 215: 567-88 (1990), Yeh et al., Virus Res., 33: 179-98 (1991). The fiber protein mediates viral binding to receptors on the cell surface mainly via the knob and shaft domains. Henry et al., J. Virol., 68(8): 5239-46 (1994). The amino acid sequence for trimerization is located in the knob and appears to be required for the amino terminus of the fiber (tail) to associate properly with the penton base. Novelli et al., Virology, 185: 365-76 (1991). In addition to cell receptor recognition and penton base binding, the fiber contributes to serotype identity. Fiber proteins from different adenovirus serotypes are quite different. 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, the fiber protein has multiple functions important in the adenovirus life cycle. Nucleic acid sequences encoding all or part of the adenovirus fiber protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0204] The adenovirus penton base protein is located at the vertices of the icosahedral capsid and contains five identical monomers. The penton base protein provides the structure for crosslinking hexon proteins on multiple faces of the icosahedral capsid and provides an essential interface for the incorporation of fiber proteins into the capsid. Each monomer of the penton base contains an RGD tripeptide motif. Neumann et al., Gene, 69: 153-157 (1988). The RGD tripeptide mediates binding to αv integrin, and adenoviruses with point mutations in the RGD sequence of the penton base limit their ability to infect cells. Bai et al., J. Virol., 67: 5198-5205 (1993). Therefore, the penton base protein is essential for the structure of the capsid and for the maximum efficiency of viral-cellular interaction. The nucleic acid sequences encoding all or part of the adenovirus penton base protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0205] An adenovirus or adenovirus vector may contain one, two, three, four, or all five of the aforementioned sequences, either alone or in any combination. In this regard, an adenovirus or adenovirus vector may contain any combination of any two of the aforementioned sequences, any combination of any three of the aforementioned sequences, any combination of any four of the aforementioned sequences, or all five of the aforementioned sequences.

[0206] In certain embodiments, the adenovirus or adenovirus vector is replication-deficient, and consequently, the replication-deficient adenovirus or adenovirus vector requires the complementation of the replication-essential gene function of at least one region of the adenovirus genome for replication (e.g., to form adenovirus vector particles).

[0207] A replication-deficient adenovirus or adenovirus vector can be modified in any preferred manner to induce a deficiency in the function of one or more replication-essential genes in one or more regions of the adenovirus genome for replication. Complementation of the deficiency in the function of one or more replication-essential genes in one or more regions of the adenovirus genome refers to the use of exogenous means to provide the deficiency in the replication-essential gene function. Such compensation can be brought about in any preferred manner, for example, by using complementary cells and / or exogenous DNA (e.g., helper adenovirus) encoding the disrupted replication-essential gene function.

[0208] In some embodiments, the adenovirus or adenovirus vector may lack the function of one or more replication-essential genes in the early region of the adenovirus genome (i.e., E1–E4 region), the late region of the adenovirus genome (i.e., L1–L5 region), both the early and late regions of the adenovirus genome, or all adenovirus genes (i.e., high-volume 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). Adenovirus vectors can also essentially have the entire adenovirus genome removed, in which case at least one of the viral inverted terminal repeat sequences (ITRs) and one or more promoters, or the viral ITRs and packaging signals, remain intact (i.e., the adenovirus amplicon). The larger the region of the adenovirus genome removed, the larger the piece of exogenous nucleic acid sequence that can be inserted into the genome. For example, given that the adenovirus genome is 36kb, the capacity of the adenovirus exogenous insert, by leaving the viral ITRs and one or more promoters intact, is approximately 35kb. Alternatively, an augmented-deficient adenovirus vector containing only the ITRs and packaging signals can efficiently allow for the insertion of approximately 37-38kb of exogenous nucleic acid sequences. Naturally, the inclusion of spacer elements in any or all of the deficient adenovirus regions reduces the capacity of the adenovirus vector for larger inserts.

[0209] In certain embodiments, an adenovirus vector is an "enhanced deletion," meaning that the adenovirus vector lacks the function of one or more genes required for viral replication in each of two or more regions of the adenovirus genome. For example, the aforementioned E1-deficient or E1 / E3-deficient adenovirus vector may further lack the function of at least one replication-essential gene in the E4 region (represented as an E1 / E4-deficient adenovirus vector). An adenovirus vector with an entire E4 region deleted may induce a lower host immune response.

[0210] Examples of replication-deficient 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 in International Publication Nos. 1994 / 028152, 1995 / 002697, 1995 / 016772, 1995 / 034671, 1996 / 022378, 1997 / 012986, 1997 / 021826, and 2003 / 022311.

[0211] The early region of the adenovirus genome includes the E1, E2, E3, and E4 regions. The E1 region includes the E1A and E1B subregions, and one or more deletions of replication-essential gene function in the E1 region may include one or more deletions of replication-essential gene function in either or both of the E1A and E1B subregions, thereby requiring the completion of the E1A and / or E1B subregions of the adenovirus genome for the adenovirus or adenovirus vector to grow (e.g., to form adenovirus vector particles). The E2 region includes the E2A and E2B subregions, and one or more deletions of replication-essential gene function in the E2 region may include one or more deletions of replication-essential gene function in either or both of the E2A and E2B subregions, thereby requiring the completion of the E2A and / or E2B subregions of the adenovirus genome for the adenovirus or adenovirus vector to grow (e.g., to form adenovirus vector particles).

[0212] The E3 region does not contain any replication-essential gene function, and consequently, deletion of the E3 region does not require the replacement of any gene function in the E3 region for the adenovirus or adenovirus vector to replicate (e.g., to form adenovirus vector particles), either partially or entirely. In the context of this disclosure, the E3 region is defined as a region beginning with an open reading frame encoding a protein with high homology to the 12.5K protein derived from the E3 region of human adenovirus 5 (NCBI reference sequence AP_000218) and ending with an open reading frame encoding a protein with high homology to the 14.7K protein derived from the E3 region of human adenovirus 5 (NCBI reference sequence AP_000224.1). The E3 region may be deleted entirely or partially, or retained entirely or partially. The size of the deletion may be adapted so that the genome retains an adenovirus or adenovirus vector that closely matches the optimal genome packaging size. Larger deletions are adapted to the insertion of larger heterologous nucleic acid sequences in adenoviruses or adenovirus genomes. In some embodiments of this disclosure, the L4 polyadenylation signal sequence located in the E3 region is retained.

[0213] The E4 region contains multiple open reading frames (ORFs). An adenovirus or adenovirus vector having deletions of all open reading frames in the E4 region except ORF6, and in some cases ORF3, does not require any gene function in the E4 region to replicate (e.g., to form adenovirus vector particles). Conversely, adenoviruses or adenovirus vectors having disruption or deletion of ORF6 in the E4 region, and in some cases ORF3 (e.g., having a deficiency in replication-essential gene function based on ORF6 and / or ORF3 in the E4 region), require the completion of the E4 region (specifically, ORF6 and / or ORF3 in the E4 region) with or without disruption or deletion of any other open reading frame in the E4 region, or the native E4 promoter, polyadenylation sequence, and / or right-side inverted terminal repeat (ITR), for the adenovirus or adenovirus vector to replicate (e.g., to form adenovirus vector particles).

[0214] The late regions of the adenovirus genome include the L1, L2, L3, L4, and L5 regions. Adenoviruses or adenovirus vectors may also have mutations in the major late promoter (MLP), as discussed in International Publication No. 2000 / 000628, which can optionally result in replication defects in the adenovirus or adenovirus vector.

[0215] The one or more regions of the adenovirus genome containing the deletion of one or more replication-essential gene functions are preferably one or more early regions of the adenovirus genome, namely the E1, E2, and / or E4 regions. Therefore, in certain embodiments, the adenovirus vector lacks all or part of such regions.

[0216] A replication-deficient adenovirus or adenovirus vector may have one or more mutations (e.g., one or more deletions, insertions, and / or substitutions) in the adenovirus genome compared to wild-type adenovirus, even if these mutations do not inhibit viral replication in host cells. Therefore, in addition to the deletion of one or more replication-essential gene functions, an adenovirus or adenovirus vector may also have deletions of other elements that are not essential for replication. For example, an adenovirus or adenovirus vector may have a partial or complete deletion of the early adenovirus region, known as the E3 region, which is not essential for the proliferation of the adenovirus or adenovirus genome.

[0217] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most the E1 or E4 region of the adenovirus genome for replication (e.g., to form adenovirus vector particles). In some such embodiments, the adenovirus vector may lack all or part of the E1 and / or E4 regions. Thus, the replication-deficient adenovirus or adenovirus vector requires the complementation of at least one replication-essential gene function in the E1A subregion and / or E1B region of the adenovirus genome (represented as an E1-deficient adenovirus vector) or the E4 region of the adenovirus genome (represented as an E4-deficient adenovirus vector) for replication (e.g., to form adenovirus vector particles). Adenoviruses or adenovirus vectors can lack the function of at least one replication-essential gene in the E1 region of the adenovirus genome (preferably all replication-essential gene functions) and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E1 / E3-deficient adenovirus vector). Adenoviruses or adenovirus vectors can lack the function of at least one replication-essential gene in the E4 region of the adenovirus genome (preferably all replication-essential gene functions) and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E3 / E4-deficient adenovirus vector).

[0218] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires complementation of at most the E2 region, preferably the E2A subregion, of the adenovirus genome for replication (e.g., to form 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 for replication (e.g., to form adenovirus vector particles) (represented as an E2A-deficient adenovirus vector). The adenovirus or adenovirus vector may be replication-deficient (preferably all replication-essential gene functions) of at least one replication-essential gene function in the E2A region of the adenovirus genome, and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E2A / E3-deficient adenovirus vector).

[0219] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most the E1 and E4 regions of the adenovirus genome to replicate (e.g., to form adenovirus vector particles). In some such embodiments, the adenovirus vector may lack all or part of the E1 and / or E4 regions. Thus, the replication-deficient adenovirus or adenovirus vector requires the complementation of at least one replication-essential gene function in both the E1 and E4 regions of the adenovirus genome to replicate (e.g., to form adenovirus vector particles) (represented as an E1 / E4-deficient adenovirus vector). The adenovirus or adenovirus vector may lack at least one replication-essential gene function in the E1 region of the adenovirus genome (preferably all replication-essential gene functions), at least one replication-essential gene function in the E4 region of the adenovirus genome, and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E1 / E3 / E4-deficient adenovirus vector). The adenovirus or adenovirus vector preferably requires, at most, completion of the E1 region of the adenovirus genome for replication, and does not require completion of any other deletions in the adenovirus genome for replication. More preferably, the adenovirus or adenovirus vector requires, at most, completion of the E1 and E4 regions of the adenovirus genome for replication, and does not require completion of any other deletions in the adenovirus genome for replication.

[0220] Adenoviruses or adenovirus vectors may contain spacer sequences to provide viral growth in complementary cell lines, similar to what is achieved by adenoviruses or adenovirus vectors lacking a single essential replication gene function (e.g., E1-deficient adenovirus vectors), if multiple essential replication gene functions of the adenovirus genome are deficient (e.g., E1 / E4-deficient adenovirus vectors). The spacer sequences may contain any one or more nucleotide sequences of a desired length, for example, at least about 15 base pairs (e.g., about 15 to 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, most preferably about 2,000 to about 3,000 nucleotides, or sequences within a range defined by any two of the aforementioned values. Spacer sequences may be coding or noncoding with respect to the adenovirus genome, and may be native or non-native, but the replication-essential function of the deleted region is not restored. Spacers may also contain expression cassettes. More preferably, spacers include polyadenylated sequences and / or genes that are non-native with respect to the adenovirus or adenovirus vector. The use of spacers in adenovirus vectors is further described, for example, in U.S. Patent No. 5,851,806 and International Publication No. 1997 / 021826.

[0221] By removing all or part of the adenovirus genome, for example, the E1, E3, and E4 regions of the adenovirus genome, the resulting adenovirus or adenovirus vector can accept the insertion of an exogenous nucleic acid sequence while retaining the ability to be packaged into an adenovirus capsid. The exogenous nucleic acid sequence can be inserted at any location in the adenovirus genome, insofar as the insertion at that location enables the formation of an adenovirus or adenovirus vector particle. Preferably, the exogenous nucleic acid sequence is located in the E1, E3, or E4 region of the adenovirus genome.

[0222] The replication-deficient adenoviruses or adenovirus vectors of this disclosure can be produced in complementary cell lines that provide gene functions not present in the replication-deficient adenoviruses or adenovirus vectors but required for viral replication, in order to produce high-titer viral vector stocks. Such complementary 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 Publication No. 1997 / 000326, and U.S. Patent Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (e.g., described in International Publication No. 95 / 34671 and Broough et al., J. Virol., 71: 9206-9213 (1997)). Other suitable complementary cell lines for producing replication-deficient adenoviruses or adenovirus vectors of this disclosure include complementary cells prepared to grow adenovirus vectors whose expression encodes a transgene that inhibits viral growth in host cells (see, for example, U.S. Patent Application Publication No. 2008 / 0233650). Additional suitable complementary cells are described, for example, in U.S. Patent Nos. 6,677,156 and 6,682,929, and International Publication No. 2003 / 020879.

[0223] In some cases, the cell genome does not need to contain a nucleic acid sequence, and its gene product compensates for all the deficiencies in the replication-deficient adenovirus vector. One or more replication-essential gene functions that are missing in the replication-deficient adenovirus vector can be supplied by a helper virus, for example, an adenovirus vector that trans-supplies one or more essential gene functions required for replication of the replication-deficient adenovirus or adenovirus vector. Alternatively, the adenovirus or adenovirus vector of the present invention may contain non-native replication-essential genes that compensate for one or more replication-essential gene functions that are missing in the replication-deficient adenovirus or adenovirus vector of the present invention. For example, the E1 / E4-deficient adenovirus vector can be manipulated to contain a nucleic acid sequence encoding E4 ORF 6 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). a. Gorilla-based adenovirus vector

[0224] In some embodiments, the adenoviruses described herein are isolated from gorillas. Western gorillas include the subspecies of Western lowland gorillas (Gorilla gorilla gorilla) and Cross River gorillas (Gorilla gorilla diehli), and eastern gorillas include the subspecies of mountain gorillas (Gorilla beringei beringei) and eastern lowland gorillas (Gorilla beringei graueri). For example, Wilson and Reeder, eds., Mammalian Species of the World, 3 rdSee ed., Johns Hopkins University Press, Baltimore, Md. (2005). In some embodiments, the adenoviruses of this disclosure have been isolated from mountain gorillas (Gorilla beringei beringei). Previous studies have characterized numerous gorilla adenoviruses and their genome sequences (see, for example, International Publication Nos. 2013 / 052832, 2013 / 052811, 2013 / 052799, 2019 / 173465, and 2022 / 115470).

[0225] Gorilla adenoviruses share similarities with human adenoviruses in terms of vector design and safety, offering advantages such as efficient transgene delivery and non-replicability through targeted deletions. Importantly, pre-existing human immunity to gorilla adenoviruses is minimal compared to human adenoviruses. This lack of recognition by the human immune system minimizes potential pre-existing immunity hurdles in gene therapy and vaccine application.

[0226] In certain embodiments, the adenovirus vector is derived from gorilla adenovirus type 40 (GAd40), e.g., GC44, GC45, or GC46. In certain embodiments, the adenovirus vector exhibits the functional adaptations described above. These adaptations may encompass their constituent elements, e.g., sequences encoding functional variants of the E2B, E2A, E3, and L1-L5 regions, as well as inverted terminal repeat sequences. Functional adaptations of such vectors featuring codon degenerate variants of the sequences encoding the E2B, E2A, E3, and L1-L5 regions are also conceivable.

[0227] In a particularly preferred embodiment, the adenovirus vector is derived from GC46, a newly isolated, intrinsic gorilla adenovirus strain isolated from fecal samples of healthy African gorillas. This adenovirus is closely related to and phylogenetically clusters with human adenovirus type C based on comparisons of hexon, DNA polymerase, and exon 4 ORF6 protein sequences. Duncan et al., Virology, 444:119-123 (2013). The seroprevalence of gorilla adenovirus type GC46 is less than approximately 6% in the United States. In comparison, the seroprevalence of type Ad5 is approximately 57%, and most seropositive individuals have high titers (IC90 above 200). Johnson et al., Molecular Therapy, 22:196-205 (2014). Therefore, compared to conventional adenovirus therapies based on the Ad5 serotype, existing neutralizing activity against gorilla adenovirus type GC46 is rare and weak in the United States. In addition, comparative studies from human serum samples originating from sub-Saharan Africa confirmed the rare and weak existing neutralizing activity in human populations. These data suggest that the existing neutralizing activity against GC46 does not significantly interfere with the structure of molecular vaccines and therapeutics on this platform, making gorilla adenovirus GC46 sufficiently suitable as a viral vector scaffold.

[0228] In a preferred embodiment of the present invention, the gorilla adenovirus vaccine encodes a fusion of selected regions of HPV proteins (e.g., HPV-E2, HPV-E4, HPV-E6, and HPV-E7) expressed in HPV-6 and HPV-11 infected cells.

[0229] In a specific embodiment of the present invention, the gorilla adenovirus vaccine encodes 791 amino acids of the HPV protein, of which 731 amino acids (92.4%) are derived from HPV-6 and 60 amino acids (7.6%) are derived from HPV-11.

[0230] In certain embodiments, the adenovirus vector is a gorilla adenovirus vector manipulated to delete part or all of the E1 and / or E4 regions. A deletion in the E1 region, for example, makes the adenovirus vector a replication defect and may include bases 459–3411, resulting in the deletion of the E1A and E1B promoters and the open reading frame. A deletion in the E4 region, for example, may include bases 34144–36824, removing all E4 open reading frames (ORFs) and thus eliminating essential elements for gorilla adenovirus replication. (The gorilla adenovirus coordinates provided herein are based on a wild-type adenovirus genome of size 37,213 base pairs.)

[0231] Modified gorilla adenovirus vectors having deletions in and / or within the E1 and / or E4 regions may offer one or more advantages over unmodified vector scaffolds. For example, extended deletions in the adenovirus genome may provide enhanced payload capacity for the adenovirus vector. A second potential advantage is a reduced risk of replicative adenovirus (RCA) development during adenovirus vector production. A third advantage is that the exclusion of E1 and E4 expression products may work to further silence other regions of the viral genome.

[0232] Therefore, in one embodiment, the gorilla adenovirus vector described herein has a deletion in or a portion of the E1 region. In another embodiment, the gorilla adenovirus vector described herein has a deletion in or a portion of the E4 region. In yet another embodiment, the gorilla adenovirus vector described herein has a deletion in or a portion of both the E1 and E4 regions. In one embodiment, the deletion in the E1 and / or E4 region comprises 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 region may comprise 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 in length compared to the wild type. In some embodiments, deletions in the E1 and / or E4 regions include 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 specific embodiments, deletions in the E1 and / or E4 regions include a length of approximately 3,000 bp compared to the wild type.

[0233] In certain embodiments, deletion of the E4 region removes all predictive open reading frames (ORFs) within it. To avoid the possibility of low-level production of adenovirus vectors with E4 deletions, a spacer sequence may be inserted into the E4-deleted region, as shown in Figure 1, to halt any potential transcription initiated from the retained E4 promoter. In one embodiment, the gorilla adenovirus vector described herein includes a spacer sequence inserted in place of the deleted portion of the E4 region. In one embodiment, the spacer sequence includes a bovine growth hormone polyadenylation (BGH polyA) signal sequence inserted in place of the deleted E4 ORF, but any suitable spacer sequence may be used. In some embodiments, the spacer sequence is about 10 to about 500 base pairs (bp) in length. For example, the spacer array is approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, The spacer sequence may be approximately 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or approximately 500 bp in length. Alternatively, the spacer sequence may be approximately 50 bp to 100 bp, 100 bp to 150 bp, 150 bp to 200 bp, 200 bp to 250 bp, 250 bp to 300 bp, 300 bp to 350 bp, 350 bp to 400 bp, 400 bp to 450 bp, or approximately 450 bp to 500 bp compared to the wild type. The spacer may also be of any length within these ranges. For example, the spacer may be approximately 250 bp to 350 bp, approximately 260 bp to 340 bp, approximately 270 bp to 330 bp, approximately 280 bp to 320 bp, or approximately 290 bp to 310 bp in length. In one embodiment, the spacer sequence is approximately 300 base pairs long. In yet another embodiment, the spacer sequence is 278 bp.

[0234] In another embodiment, the spacer sequence is located at approximately 34,700 to 35,000 base pairs in the vector genome compared to the wild type. In yet another embodiment, the spacer sequence is located at 34,692 to 34,969 base pairs in the vector genome compared to the wild type. In yet another embodiment, the spacer sequence contains the nucleic acid sequence of Sequence ID No. 95.

[0235] In certain embodiments, the vector is missing both the E1 and E4 regions. In some such embodiments, the E1 or E4 region is replaced by an expression cassette containing a transgene or spacer. In some such embodiments, the E1 region is replaced by an expression cassette, and the E4 region is replaced by a spacer.

[0236] In one embodiment, the E1 / E4-deficient GC46 adenovirus vector can be produced in any complementary cell line providing E1 and E4 ORF6 functionality, e.g., engineered 293 cells. Such cells may be cultured, for example, in a serum-free suspension in a shaker flask and infected with a master virus bank at an infection multiplicity (MOI) of 100 PU / cell. The culture sample may be subjected to downstream processing and purified using three rounds of cesium chloride density gradient ultracentrifugation to obtain a highly purified material. This material may then be frozen, then thawed, sterile filtered, and packed into vials, which can be stored in a freezer at approximately -60 to approximately -90°C.

[0237] In some embodiments, the vector of the present invention may be constructed by isolating a GC46 gorilla adenovector from a non-human primate origin, cloning the isolated GC46 genome, deleting the E1 and E4 regions of GC46, and expressing a human papillomavirus (HPV) 6 / 11 antigen design, and inserting an expression cassette in the E1 region under the control of a cytomegalovirus (CMV) pre-early promoter. In one embodiment described below, the CMV-HPV 6 / 11 antigen design contains key immunogenic peptides derived from HPV 6 and 11 epitopes, namely E2(HPV6), E4(HPV6), E6(HPV6 / 11), and E7(HPV6 / 11), where the HPV6-derived peptide has high sequence similarity to HPV11.

[0238] In some embodiments, the vector of the present invention encodes one of the HPV antigen regions or variants thereof described herein. For example, the vector may contain a nucleic acid sequence having at least 80% identity with SEQ ID NO: 68. C. Non-virus-based delivery systems

[0239] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0240] The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that associate with both liposomes and oligonucleotides, captured within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or in "broken-down" structures. They can also simply be dispersed in solution and, if applicable, form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include a class of compounds containing naturally occurring lipid droplets in the cytoplasm, as well as long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0241] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -200°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol.

[0242] "Liposome" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, as well as the capture of water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, compositions that have structures different in solution from the usual vesicular structure are also included. For example, lipids can be assumed to have a micelle structure, or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0243] In some examples, polynucleotides encoding polypeptides can also be introduced into cells using non-viral-based delivery systems, such as the Sleeping Beauty (SB) transposon system. In embodiments, the modified effector cells and other genetic elements described herein are delivered to cells using the SB11 transposon system, the SB100X transposon system, the SB110 transposon system, the piggyBac transposon system (see, e.g., Wilson et al., “PiggyBac Transposon-mediated Gene Transfer in Human Cells,” Molecular Therapy 15:139-145 (2007)) and / or the piggyBac transposon system (see, e.g., 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)). Additional transposase and transposon systems are described in 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 Application Publication No. 2011 / 0117072; Mates et al., Nat Genet, 41(6): 753-61 (2009). doi: 10.1038 / ng.343. Epub 2009 May 3, Gene Ther., 18(9):849-56 (2011). doi: 10.1038 / gt.2011.40. Epub 2011 Mar. 31 and Ivies et al., Cell, It is available in 91(4):501-10, (1997).

[0244] Additional suitable nonviral systems may include embedded expression vectors, which may contain recombination sites that can be randomly integrated into the host cell's DNA or that allow for specific recombination between the expression vector and the host cell's chromosomes. Targeted integration of a transgene to a given locus is a desirable goal for many applications. First, a first recombination site for a site-specific recombinase is inserted into a genomic site, either randomly or at a predetermined location. Subsequently, the cell is transfected with a plasmid containing the gene or DNA of interest, a second recombination site, and an origin for the recombinase (expression plasmid, RNA, protein, or viral expression recombinase). Recombination between the first and second recombination sites results in the integration of plasmid DNA. Such embedded expression vectors can utilize endogenous expression regulatory sequences on the host cell's chromosomes to produce the expression of the desired protein.

[0245] In some embodiments, targeted integration is facilitated by the presence of a sequence on a donor polynucleotide homologous to the sequence flanking at the integration site. For example, targeted integration using donor polynucleotides as described herein can be achieved according to conventional transfection techniques, such as those used to produce gene knockout or knock-in by homologous recombination. In other embodiments, targeted integration is facilitated both by the presence of a sequence on a donor polynucleotide homologous to the sequence flanking at the integration site, and by contacting the cell with the donor polynucleotide in the presence of a site-specific recombinase. Site-specific recombinase, or simply recombinase, means a polypeptide that catalyzes conserved site-specific recombination between its compatible recombination sites. As used herein, site-specific recombinase includes not only native polypeptides but also derivatives, variants, and / or fragments that retain activity, as well as native polynucleotides, derivatives, variants, and / or fragments that encode the recombinase that retains activity.

[0246] Also provided herein are systems for incorporating heterologous genes into host cells, wherein the system comprises one or more gene expression cassettes. In some examples, the system comprises a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide construct. In other examples, the system may include a second gene expression cassette comprising a second polynucleotide encoding a second polypeptide construct. In yet another example, the system may include a third gene expression cassette. In one embodiment, one of the gene expression cassettes may include a gene switch 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) an ecdysone receptor binding domain. In another embodiment, the system further comprises a recombination attachment site and a serine recombinase, wherein the heterologous gene is incorporated into the host cell in the presence of the serine recombinase when the host cell comes into contact with at least the first gene expression cassette.

[0247] In some examples, the system further includes a ligand, and as a result, upon contact with the host cell, the heterologous gene is expressed in the host cell in the presence of the ligand. In one example, the system also includes a recombination attachment site. In some examples, one recombination attachment site is a phage genome recombination attachment site (attP) or a bacterial genome recombination attachment site (attB). In one example, the host cell is a eukaryotic cell. In another example, the host cell is a human cell. In yet another example, the host cell is a T cell or an NK cell. II. Expression Cassette A. Introduced gene

[0248] The vector of the present invention may include an expression cassette for expressing a transgene. The “transgene” comprises a non-native nucleic acid sequence operably linked to a suitable regulatory element (e.g., a promoter), as a result of the expression of the non-native nucleic acid sequence, which may produce a protein (e.g., a peptide or polypeptide). The regulatory element (e.g., a promoter) may be native or non-native to the adenovirus or adenovirus vector.

[0249] A “non-native” nucleic acid sequence is any nucleic acid sequence (e.g., a DNA, RNA, or cDNA sequence) in which there is no naturally occurring nucleic acid sequence in the adenovirus at its naturally occurring location. Therefore, a non-native nucleic acid sequence may be found naturally in the adenovirus but may be located at a non-native position within the adenovirus genome and / or may be operably linked to a non-native promoter. The terms “non-native nucleic acid sequence,” “heterogeneous nucleic acid sequence,” and “exogenous nucleic acid sequence” are synonymous and can be used interchangeably in the context of this disclosure. A non-native nucleic acid sequence is preferably DNA and preferably encodes a protein (i.e., one or more nucleic acid sequences encoding one or more proteins).

[0250] Non-native nucleic acid sequences can encode therapeutic proteins that can be used to treat mammals preventively or therapeutically against disease. Suitable examples of therapeutic proteins include anti-inflammatory agents, such as cytokines, toxins, tumor suppressor proteins, growth factors, hormones, receptors, mitogens, immunoglobulins, neuropeptides, neurotransmitters, and enzymes. Alternatively, non-native nucleic acid sequences can encode antigens of pathogens (e.g., bacteria or viruses), and adenoviruses or adenovirus vectors can be used as vaccines. B. Promoter

[0251] The gene regulatory components of a therapeutic expression cassette are selected to confer high levels of expression of the transgene. Therefore, another aspect of this disclosure is an expression cassette further comprising a promoter. A promoter is a region of polynucleotides that initiates transcription of a coding sequence. Promoters are located on the same strand of DNA and upstream (in the direction of the 5' region of the sense strand), near the transcription start site of the gene. Some promoters are constitutive, so they are active under all circumstances in the cell, while others are regulated to become active in response to specific stimuli, such as inductive promoters. Further promoters include, but are not limited to, tissue-specific promoters or activation promoters, and include T cell-specific promoters.

[0252] The term "promoter activity" and its grammatical equivalent, as used herein, refer to the degree of expression of a nucleotide sequence operably ligated to a promoter whose activity is being measured. Promoter activity can be measured directly, for example, by determining the amount of RNA transcript produced by Northern blot analysis, or indirectly, by determining the amount of product encoded by a ligated nucleic acid sequence, such as a reporter nucleic acid sequence ligated to the promoter. 1. Inducible promoter

[0253] In certain embodiments, the promoter is an inductive promoter. An inductive promoter is a promoter whose activity is induced by the presence or absence of transcription factors, such as biological or abiotic factors. Inductive promoters are useful because the expression of a gene operably linked to an inductive promoter can be turned on or off at a particular stage of development of an organism or a particular tissue. Examples of inductive promoters include alcohol-regulating promoters, tetracycline-regulating promoters, steroid-regulating promoters, metal-regulating promoters, pathogenesis-regulating promoters, temperature-regulating promoters, and light-regulating promoters. In some embodiments, the inductive promoter is part of a gene switch. The inductive promoter may be a gene switch ligand-inductive promoter. In some cases, the inductive promoter may be an ecdysone receptor-based gene switch of two small molecule ligand-inductive polypeptides, such as the RHEOSWITCH® gene switch, for example, the system described in International Publication No. 2018 / 132494. Additional examples of gene switch systems include, but are not limited to, the systems described in U.S. Patent Nos. 6,258,603, 7,045,315, U.S. Patent Publication Nos. 2006 / 0014711, 2007 / 0161086, and International Publication No. 01 / 70816.

[0254] In some cases, gene switches are, but are not limited to, PCT / US2001 / 009050 (International Publication No. 2001 / 070816); U.S. Patent No. 7,091,038; U.S. Patent No. 7,776,587; U.S. Patent No. 7,807,417; U.S. Patent No. 8,202,718; PCT / US2001 / 030608 (International Publication No. 2002 / 029075); U.S. Patent No. 8,105,825; U.S. Patent No. 8,168,426; PCT / US2002 / 005235 (International Publication No. 2002 / 066613); U.S. Patent Publication No. Application No. 10 / 468,200 (US Patent Application Publication No. 20120167239); PCT / US2002 / 005706 (International Publication No. 2002 / 066614); US Patent No. 7,531,326; US Patent No. 8,236,556; US Patent No. 8,598,409; PCT / US2002 / 005090 (International Publication No. 2002 / 066612); US Patent No. 8,715,959 (US Patent Application Publication No. 20060100416); PCT / US2002 / 005234 (International Publication No. 2003 / 027 266); U.S. Patent No. 7,601,508; U.S. Patent No. 7,829,676; U.S. Patent No. 7,919,269; U.S. Patent No. 8,030,067; PCT / US2002 / 005708 (International Publication No. 2002 / 066615); U.S. Patent Application No. 10 / 468,192 (U.S. Patent Application Publication No. 20110212528); PCT / US2002 / 005026 (International Publication No. 2003 / 027289); U.S. Patent No. 7,563,879; U.S. Patent No. 8,021,878; U.S. Patent No. 8,497,093 Document; PCT / US2005 / 015089 (International Publication No. 2005 / 108617); U.S. Patent No. 7,935,510; U.S. Patent No. 8,076,454; PCT / US2008 / 011270 (International Publication No. 2009 / 045370); U.S. Patent Application No. 12 / 241,018 (U.S. Patent Application Publication No. 20090136465); PCT / US2008 / 011563 (International Publication No. 2009 / 048560); U.S. Patent Application No. 12 / 247,738 (U.S. Patent Application Publication No. 20090123441);The system can be selected from any of the ecdysone-based receptor components described in PCT / US2009 / 005510 (International Publication No. 2010 / 042189); U.S. Patent Application No. 13 / 123,129 (U.S. Patent Application Publication No. 20110268766); PCT / US2011 / 029682 (International Publication No. 2011 / 119773); U.S. Patent Application No. 13 / 636,473 (U.S. Patent Application Publication No. 20130195800); PCT / US2012 / 027515 (International Publication No. 2012 / 122025); and U.S. Patent No. 9,402,919.

[0255] Inducible promoters typically utilize ligands for dose-regulatory control of the expression of at least two of the aforementioned genes. In some cases, ligands include ecdysteroids, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazine, oxadiazoline, dibenzoylalkylcyanohydrazine, N-alkyl-N,N'-dialoylhydrazine, N-acyl-N-alkylcarbonylhydrazine, N-aloyl-N-alkyl-N'-aloylhydrazine, arnidoketones, 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylhalpadide, oxysterol, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, and 5-alpha-6-alpha-epoxycholesterol-3-sulfate. The following can be selected: ethyl phosphate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, RG-115819 (3,5-dimethylbenzoate N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazide), RG-115932 ((R)-3,5-dimethylbenzoate N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-dimethylbenzoate N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and any combination thereof. In one embodiment, the gene switch is such that the level of gene expression depends on the level of the ligand present. Examples of ligand-dependent transcription factor complexes that can be used in the gene switch of the present invention include, but are not limited to, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysones, and their analogs and mimetics), and rTTA activated by tetracyclines.In one aspect of the present invention, the gene switch is an EcR-based gene switch. 2. Non-inducible promoters

[0256] In certain embodiments, the promoter is a non-inducible promoter, including, for example, tissue-specific promoters, strongly constitutive promoters, or minimal promoters known in the Art. Suitable non-inducible promoters include, for example, the CMV promoter, the SV40 promoter, the CAG promoter, or others. In certain embodiments, the promoter is the CMV promoter. 3. Tissue-Specific Promoters

[0257] In certain embodiments, the promoter may be a tissue-specific promoter. Herein, “tissue-specific” refers to the regulated expression of a gene in a subset of tissues or cell types. In some cases, a tissue-specific promoter may be spatially regulated so that the promoter drives expression only in a particular tissue or cell type of an organism. In other cases, a tissue-specific promoter may be temporally regulated so that the promoter drives expression in different cell types or tissues over time, including during the development of an organism. In some cases, a tissue-specific promoter is regulated both spatially and temporally. In certain embodiments, a tissue-specific promoter is activated in a particular cell type either constitutively or intermittently at a specific time or stage of the cell type. For example, a tissue-specific promoter may be a promoter that is activated when a specific cell, such as a T cell or NK cell, is activated. T cells may be activated in various ways, for example, when presented with a peptide antigen by an MHC class II molecule. 4. Synthetic promoters and manipulated promoters

[0258] Synthetic promoters for use in expression cassettes described herein are also intended and may be manipulated to improve expression characteristics. Synthetic promoters may include various subcomponents, including, but are not limited to, blocking sequences, enhancers, and various response elements.

[0259] In certain embodiments, the promoter is an engineered promoter or a variant thereof. As described herein, the promoter may incorporate an IL-2-derived minimal promoter sequence and one or more of the following: NFAT (Nuclear Factor-Action-T) response elements of activated T cells; NFIL2D response elements, NF-κB / TCF response elements, NFAT / NFIL2B response elements, or NFIL2A / OCT response elements. The NFAT transcription factor is a key modulator of effector T cell state. NFAT is an early transcriptional checkpoint that progressively drives depletion. After TCR stimulation, NFAT is rapidly activated in T cells and forms a protein complex with AP-1, which is induced by appropriate co-stimulatory signaling, thereby regulating effector gene and T cell function. NFAT response elements can fusion 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). 5. Activation-Specific Promoter

[0260] In certain embodiments, the promoters are activation-specific promoters, such as the interleukin-2 (IL2) promoter and the programmed death (PD)-1 (CD279) promoter. Gene switch components may also be conditionally expressed upon immune cell activation by fusing binding sites for other nuclear factors of the pro-inflammatory signaling pathway, such as NF-KB, to a minimal promoter sequence (e.g., IL2).

[0261] In certain embodiments, the promoter includes an IL-2 core promoter. In some embodiments, at least one promoter includes an IL-2 minimal promoter. In another embodiment, at least one promoter includes an IL-2 enhancer and promoter variant. In yet another embodiment, at least one promoter includes an NF-κB binding site. In some embodiments, at least one promoter includes an (NF-κB)1-IL2 promoter variant. In some embodiments, at least one promoter includes an (NF-κB)3-IL2 promoter variant. In some embodiments, at least one promoter includes an (NF-κB)6-IL2 promoter variant. In some embodiments, at least one promoter includes a 1× activated T cell nuclear factor (NFAT) response element-IL2 promoter variant. In another embodiment, at least one promoter includes a 3× NFAT response element. In yet another embodiment, at least one promoter includes a 6× NFAT response element-IL2 promoter variant. In some embodiments, at least one promoter includes a human EF1A1 promoter variant. In some embodiments, at least one promoter includes a human EF1A1 promoter and enhancer. In some embodiments, at least one promoter includes a human UBC promoter. In some embodiments, at least one promoter includes six GAL4 inducible proximal factor-binding elements (PFBs). In some embodiments, at least one promoter includes a synthetic minimal promoter 1 (inducible promoter). Sequences for such promoters are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0262] In certain embodiments, the promoter may be 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, 1×NFAT response element-IL2 promoter variant, 3×NFAT response element-IL2 promoter variant, 6×NFAT response element-IL2 promoter variant, human EEF1A1 promoter variant, human EEF1A1 promoter and enhancer, human UBC promoter, and synthetic minimal promoter 1. 6. Constitutive promoter

[0263] In certain embodiments, the promoter is a constitutive promoter. Examples of such promoters include the monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters, such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. 7. Virus promoter

[0264] Exemplary promoters used in the vectors described herein include viral promoters operably combined with heterologous nucleic acid sequences encoding cytokines. Exemplary viral promoters may be derived from, but are not limited to, several known viruses, and include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAVs), and alphavirus vectors. Non-exclusive 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 intended herein include the cytomegalovirus (CMV) pre-early promoter, the CAG promoter (a combination of the CMV early enhancer element and the chicken beta-actin promoter), the monkey virus 40 (SV40) promoter, the cauliflower mosaic virus (CaMV) 35S RNA and 19S RNA promoters, the coat protein promoter for 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 variants thereof. 8. Other promoter elements

[0265] Additional promoter elements, such as enhancers (e.g., promoter enhancers), regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although some promoters have recently been shown to also contain functional elements downstream of the initiation site. Spacing between promoter elements is often flexible, and as a result, promoter function is conserved when elements are inverted or moved relative to each other. In thymidine kinase (TK) promoters, the spacing between promoter elements can increase to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0266] A synthetic promoter useful in the present invention may include an enhancer sequence. In one embodiment, the enhancer may be an mCMV enhancer sequence. In another embodiment, the mCMV enhancer sequence is about 500 to about 1,000 bp in length. In yet another embodiment, the enhancer sequence is about 700 bp in length. In yet another embodiment, the enhancer sequence includes 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 nucleic acid sequence of SEQ ID NO: 96 or a functional variant thereof, for example, SEQ ID NO: 96, or a conservatively substituted variant of SEQ ID NO: 96, or a non-conservatively substituted variant of SEQ ID NO: 96.

[0267] In another embodiment, the mCMV enhancer includes a transcription factor binding site. In yet another embodiment, the transcription factor binding site includes Sp1, Ebox, ETS, TRE, CREB, and GATA binding sites.

[0268] Response elements may also be included in the promoters described herein. Various response elements are known in the art. For example, to reduce the expression of a transgene during adenovirus production, which may negatively affect the overall production titer, a tetracycline response element (TRE, 2X TetO) may be located within the promoter element of a promoter, between the TATA box and the transcription start site. Thus, when a vector is produced in a cell line expressing a tetracycline (Tet) repressor, the expression of a transgene 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 interacts with the TRE element and blocks the initiation of transcription. Normal expression levels are observed when infecting producing cells that do not express a Tet repressor. Therefore, in one embodiment described herein, the synthetic promoter includes a TRE. The TRE contains a length of about 10 to about 100 bp. In another embodiment, the TRE is in the range of 10 bp to 100 bp in length. In one embodiment, the TRE includes a length of about 50 bp. In yet another embodiment, the TRE includes 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 nucleic acid sequence of SEQ ID NO: 98 or a conservedly substituted variant of SEQ ID NO: 98, or a non-conservatively substituted variant of SEQ ID NO: 98.

[0269] In certain embodiments, the promoter includes a transcription blocker, an enhancer sequence, and a response element. In some such embodiments, the promoter includes an mCMV enhancer and a TRE. C. Untranslated region

[0270] In some embodiments, the expression cassette may include an untranslated region (UTR) to regulate or enhance transgene expression. In one embodiment, the expression cassette may include an artificial untranslated region. 5'UTRs with splice units have been shown to enhance the expression of the transgene cassette. Therefore, in some embodiments, the cassette includes a 5'UTR with splice units. In a particular embodiment, the 5'UTR is engineered to include a synthetic splice site sequence extending from canine ATP2A2 intron 2 to the subsequent 5'UTR of the bovine CSN2 gene. In another embodiment, the 5'UTR includes 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 nucleic acid sequence of SEQ ID NO: 99 or a conservedly substituted variant of SEQ ID NO: 99, or a non-conservatively substituted variant of SEQ ID NO: 99. D. Terminal arrangement

[0271] In another embodiment, the expression cassette further comprises a stop sequence. The open reading frame of the cytokine transgene may be followed by the stop sequence. Like promoter sequences, the stop sequence may also contain various regulatory elements to ensure proper 3' transcription end processing. In one embodiment, the stop sequence comprises a partial human growth hormone (HGH) 3' untranslated region. In another embodiment, the stop sequence comprises a polyadenylation signal, including, but not limited to, an SV40 polyadenylation signal and / or an LTR polyadenylation signal. In yet another embodiment, the stop sequence comprises a human beta-actin (ACTb) transcription termination signal sequence. In yet another embodiment, the stop sequence comprises an HGH 3' untranslated region, a polyadenylation signal, and a human beta-actin transcription termination sequence. In another embodiment, the stop sequence includes 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 nucleic acid sequence of SEQ ID NO: 104 or a conservedly substituted variant of SEQ ID NO: 104 or a non-conservatively substituted variant of SEQ ID NO: 104. E. Polynucleotide Linker

[0272] Expression cassettes and constructs containing polynucleotide linkers that promote the expression of polynucleotides and the functionality of polypeptides described herein are also intended herein.

[0273] In some cases, the linker may be a cleavable linker. The polynucleotide linker may be an oligomer. The polynucleotide linker may be a double-stranded, single-stranded, or a combination thereof DNA. In some cases, the linker may be RNA. The polynucleotide linker may be a double-stranded segment of DNA containing a desired restriction site, which can be added to create a terminal structure compatible with the polynucleotide-containing vectors described herein.

[0274] In some cases, polynucleotide linkers may be useful for modifying vectors containing polynucleotides as described herein. For example, vector modification involving a polynucleotide linker may involve alteration of multiple cloning sites or addition of polyhistidine tails. Polynucleotide linkers can also be used to adapt the ends of blunt-ended insert DNA for cloning into vectors cleaved with restriction enzymes that have adherent ends. The use of polynucleotide linkers may be more efficient than blunt-ended ligation to vectors and may provide a method for releasing the insert from the vector in downstream applications. The insert may be a polynucleotide sequence encoding a polypeptide useful for therapeutic applications.

[0275] In some embodiments, the polynucleotide linker may be ligated, in some cases, to a vector containing the polynucleotide described herein by a T4 ligase. To facilitate ligation, an excess of polynucleotide linker may be added to the composition containing the insert and vector. In some cases, the insert and vector are pre-treated before the linker is introduced. For example, pre-treatment with methylase can prevent unwanted cleavage of the insert DNA.

[0276] 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. 1. IRES array

[0277] In some embodiments, the polynucleotides described herein may be linked by “intrasequence ribosome entry sites” or “IRES” elements. IRESs can enable the simultaneous expression of multiple genes. For example, an IRES sequence can enable the production of multiple proteins from a single mRNA transcript. Ribosomes can bind to IRESs in a 5'-cap independent manner and initiate translation.

[0278] In expression cassettes containing IRES sequences, the first gene can be translated by cap-dependent ribosome scanning, a mechanism mediated by its own 5'-UTR, while the translation of subsequent genes can be achieved in a cap-independent manner by direct recruitment of ribosomes to the IRES. IRES sequences can enable eukaryotic ribosomes to bind and initiate translation without binding to the 5' cap end. IRES sequences can enable the expression of multiple genes from a single transcript (Mountford and Smith, Trends Genet. 11(5):179-84 (1995)).

[0279] In certain cases, the IRES region may be derived from the IRES sequences of viruses, such as picornaviruses, encephalomyocarditis viruses, and hepatitis C viruses. In other cases, the IRES sequence may be derived from encephalomyocarditis viruses. The terms "EMCV" or "encephalomyocarditis virus," as used herein, refer to any member of an isolate or strain of a encephalomyocarditis virus species of the genus Picornaviridae. Examples include the EMCV-R(Rueckert) virus and the Columbian SK virus. In some cases, cellular IRES elements, such as eukaryotic initiation factor 4G, immunoglobulin heavy chain binding protein, c-myc proto-oncogene, vascular endothelial growth factor, fibroblast growth factor-I, or any combination or modification thereof may be used. In some cases, cellular IRESs may have increased gene expression compared to viral IRESs.

[0280] IRES sequences from viruses, cells, or combinations thereof can be used in vectors. IRESs may be derived from encephalomyelitis virus (EMCV) or poliovirus (PV). In some cases, IRES elements may be derived from poliovirus (PV), encephalomyelitis virus (EMCV), foot-and-mouth disease virus (FMDV), porcine leukemia virus-1 (PTV-1), Aichi virus (AiV), Seneca Barley virus (SVV), hepatitis C virus (HCV), classical swine fever virus (CSFV), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-1 (HIV-I), Moloney's mouse leukemia virus (MoMLV), feline immunodeficiency virus (FIV), mouse mammary tumor virus (MMTV), latent human cytomegalovirus (pUL138), Epstein-Barr virus (EBNA-1), herpesvirus Marek's disease (MDV RLORF9), SV40 polycistronic 19S (SV40 The virus is selected from the group consisting of 19S), wheat aphid virus (RhPV), cricket paralysis virus (CrPV), Ectropis obliqua picorna-like virus (EoPV), Plautia stali enteric virus (PSIV), Triatoma virus (TrV), honeybee paralysis discistrovirus (IAPV, KBV), blackcurrant reversion virus (BRV), pelargonium variegation virus (PFBV), hibiscus chlorosis virus (HCRSV), rapeseed infective tobamovirus (CrTMV), potato leaf curl borerovirus (PLRV), tobacco etch virus (TEV), giardiavirus (GLV), leishmania RNA virus-I (LRV-1), and combinations or modifications thereof.

[0281] In some cases, IRES is selected from the group consisting of Apaf-1, XIAP, HIAP2 / c-IAP1, DAP5, Bcl-2, c-myc, CAT-I, INR, differentiated LEF-1, PDGF2, HIF-la, VEGF, FGF2, BiP, BAG-I, CIRP, p53, SHMTI, PITSLREp58, 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 contained between two open reading frames (ORFs), translation initiation can occur via a classical 5'-m7GpppN cap-dependent mechanism in the first ORF and a cap-independent mechanism in the second ORF downstream of the IRES element.

[0282] In some cases, an IRES sequence can be approximately 9 to 1,000 base pairs long. For example, an IRES sequence can be approximately 9 to 150 base pairs, or approximately 150 to 400 base pairs, or approximately 400 to 600 base pairs, or approximately 600 to 1,000 base pairs. In some embodiments, the IRES sequence is approximately 9, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 275, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 950, or 1,000 base pairs.

[0283] In some cases, the expression of downstream genes within a vector containing an IRES sequence may be reduced. For example, the expression of a gene following an IRES sequence may be reduced compared to the gene preceding the IRES sequence. This reduction in expression can range from 1% to 99.9% compared to the preceding gene, including 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% compared to the preceding gene. 2. Virus 2A sequence

[0284] In some embodiments, the polynucleotides described herein may be linked by a viral 2A element or sequence. The 2A element may have 5 to 100 base pairs and may be shorter than the IRES. In some cases, the 2A sequence may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 base pairs. The gene linked to 2A may be expressed in a single open reading frame, and "self-cleavage" may occur co-translationally between the last two amino acids of the GP at the C-terminus of the 2A polypeptide, resulting in equal amounts of co-expressed protein.

[0285] The viral 2A sequence can consist of approximately 20 amino acids. In some cases, the viral 2A sequence may contain the consensus motif Asp-Val / Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO: 122). The consensus motif sequence can act in a co-translational manner. For example, the normal formation of peptide bonds between glycine and proline residues may be prevented, which can lead to ribosome skipping and cleavage of nascent polypeptides. This effect can produce multiple genes at equimolar levels.

[0286] 2A peptides can enable the translation of multiple proteins into polypeptides within a single open reading frame, which can then be cleaved into individual polypeptides via a ribosome skipping mechanism (Funston et al., J Gen. Viral. 89(Pt 2):389-96 (2008)). In some embodiments, 2A sequences may include F / T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, as well as any combination thereof.

[0287] In some cases, a vector may contain an IRES sequence and a 2A linker sequence. In other cases, the expression of multiple genes linked to a 2A peptide may be promoted by a spacer sequence (GSG) preceding the 2A peptide. In some cases, a construct may combine spacers, linkers, adapters, promoters, or combinations thereof. For example, a linker may have a spacer (SGSG (SEQ ID NO: 121) or GSG, or a Whitlow linker) with a different 2A peptide, and a Furin linker (RAKR (SEQ ID NO: 123)) cleavage site. The spacer may be I-Ceui. In some cases, the linker may be manipulated. For example, the linker may be designed to include chemical features such as hydrophobicity. In some cases, at least two linker sequences may produce the same protein. In other cases, multiple linkers may be used in a vector. For example, the gene of interest may be separated by at least two linkers. 3. Polynucleotide-encoded polypeptide linkers

[0288] In certain embodiments, the polynucleotides described herein may encode two or more polypeptides. In some of these embodiments, the polynucleotides may be separated by an intervening sequence that encodes an intervening linker polypeptide. As used herein, the term “intervening linker polypeptide” means an amino acid sequence that separates two or more polypeptides encoded by a polynucleotide and can be distinguished from the term “peptide linker,” which refers to an amino acid sequence optionally included in the polypeptide constructs disclosed herein for linking a transmembrane domain to a cell surface polypeptide (e.g., including truncated variants of native polypeptides).

[0289] In certain cases, the interlinker polypeptide is a cleavage-sensitive interlinker polypeptide. In some embodiments, the interlinker polypeptide is a cleavable linker or a ribosome-skipping linker. In some embodiments, the cleavable linker or ribosome-skipping linker sequence is selected from the group consisting of 2A, GSG-2A, GSG linker, SGSG linker (SEQ ID NO: 121), furinlink variants and their derivatives. In some embodiments, the 2A linker is a p2A linker, T2A linker, F2A linker, or E2A linker. In some embodiments, the polypeptide of interest is expressed as a fusion protein linked by a cleavage-sensitive interlinker polypeptide. In certain embodiments, the cleavage-sensitive interlinker polypeptide may be one or more of F / T2A, T2A, p2A, 2A, GSG-p2A, GSG linker, and furinlink variants. Linkers (polynucleotide and polypeptide sequences), such as those disclosed in PCT / US2016 / 061668 (International Publication No. 2017083750), were published on May 18, 2017.

[0290] In certain embodiments, the linker polypeptide comprises the sequence disclosed in Table 3.

[0291] [Table 3]

[0292] In certain cases, the linker polypeptide may contain the amino acid sequence "RAKR" (SEQ ID NO: 123). In certain cases, the furin-intervened linker polypeptide may be encoded by a polynucleotide sequence containing "CGTGCAAAGCGT" (SEQ ID NO: 125) or "AGAGCTAAGAGG" (SEQ ID NO: 126).

[0293] In some embodiments, the intervening linker polypeptide comprises a furin 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 (Val) (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, e.g., (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 0 to 15.

[0294] The linkers described herein can, in certain cases, improve biological activity, increase expression yield, and achieve a desired pharmacokinetic profile. The linkers may also include hydrazones, peptides, disulfides, or thioesters.

[0295] Flexible linkers can be applied when the joined domains require a certain degree of movement or interaction. Flexible linkers can consist of small, nonpolar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr). Flexible linkers can have sequences that are predominant from stretching Gly and Ser residues ("GS" linkers). An example of a flexible linker may have the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 127). By adjusting the copy number "n", the length of this exemplary GS linker can be optimized to achieve proper separation of functional domains or to maintain the required internal domain interactions. In addition to GS linkers, other flexible linkers can be utilized for recombinant fusion proteins. In some cases, flexible linkers may also be rich in 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 can be used to improve solubility.

[0296] Flexible linkers useful in this invention may be rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. Flexible linkers may be a suitable choice when certain movements or interactions are desired for the fusion protein domains. In addition, although flexible linkers cannot have a rigid structure, they can function as passive linkers to maintain distance between functional domains. The length of the flexible linker can be adjusted to enable proper folding of the fusion protein or to achieve its optimal biological activity.

[0297] In some cases, the interlinker polypeptides described herein are rigid linkers. Rigid linkers can be used to maintain fixed distances between polypeptide domains. Examples of rigid linkers include, to name a few, alpha-helix-forming linkers, Pro-rich sequences, (XP)n, X-Pro skeletons, and A(EAAAK)nA (n=2~5) (SEQ ID NO: 128). Rigid linkers can exhibit a relatively rigid structure in some cases by employing an α-helix structure or by containing multiple Pro residues.

[0298] In some embodiments, the intervening linker polypeptide may be non-cleavable. The non-cleavable linker can covalently join its functional domains together and act as a single molecule throughout the entire in vivo or ex vivo process.

[0299] In other embodiments, the intervening linker polypeptide may be cleavable. A cleavable linker may be introduced to release a free functional domain in vivo. A cleavable linker may be cleaved in the presence of a reducing agent, a protease, to name a few examples. For example, a cleavable linker can be produced by utilizing the reduction of a disulfide bond. In the case of a disulfide linker, a cleavage event by disulfide exchange with a thiol, such as glutathione, can result in cleavage. In other cases, in vivo cleavage of a linker in a recombinant fusion protein may also be carried out by a protease that can be expressed in vivo in specific cells or tissues under pathological conditions (e.g., cancer or inflammation), or by a protease that can be confined within a particular cellular compartment. In some cases, a cleavable linker may enable targeted cleavage. For example, the specificity of many proteases can provide slower linker cleavage in a confined compartment. The cleavable linker may also include a hydrazone, a peptide, a disulfide, or a thioester. For example, hydrazones can confer serum stability. In other cases, hydrazones can enable cleavage in the acidic compartment. The acidic compartment can have a pH of up to 7. The linker may also contain a thioether. The thioether may be non-reducing. The thioether can be designed for intracellular proteolysis.

[0300] A method is provided for obtaining improved expression of a polypeptide construct, comprising the steps of: providing a polynucleotide encoding the polypeptide construct, comprising a first functional polypeptide and a second functional polypeptide, wherein the first functional polypeptide and the second functional polypeptide are linked by a linker polypeptide comprising a sequence having at least 60% identity with sequence APVKQ (SEQ ID NO: 133); and expressing the polynucleotide in a host cell, wherein the expression step results in improved expression of the polypeptide construct compared to a corresponding polypeptide construct that does not have a linker polypeptide comprising a sequence having at least 60% identity with sequence APVKQ (SEQ ID NO: 133). 4. Operated linkers and designed linkers

[0301] In some embodiments, the polynucleotide linker may be manipulated or designed. The method for designing the linker may be computer-based. In some cases, the computer-based method may include graphic techniques. The computer-based method can be used to search for suitable peptides from a library of three-dimensional peptide structures derived from a database. For example, the Brookhaven Protein Data Bank (PDB) can be used to measure the spatial distances between selected amino acids in the linker. D. Packaging sequence

[0302] In addition to the expression cassette, the vector may further include a packaging sequence. As used herein, the term “packaging sequence” refers to a sequence located within the gorilla adenovirus genome that is required for the insertion of viral DNA into a 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). E. HPV initial region protein

[0303] Constitutive or inducible expression of the early (E) region protein of HPV provides a target for an effective HPV vaccine.

[0304] HPV genes (E1-E8) regulate viral expression and replication, while late (L) genes control the viral protein code (8-10). The functions of HPV early region proteins include: El and E2 have functions in viral replication / transcription (e.g., E2 regulates the expression of E6 and E7; El / E2 interaction is essential for viral replication); E4 and E5 have increased expression during the later stages of the viral replication cycle; and E6 and E7 act concurrently during replication (E6 is required for episomal genome maintenance, and E7 expands the compartment of epithelial cell activity in DNA replication).

[0305] An exemplary embodiment of the present invention is an HPV6 / 11 vaccine that delivers a multi-epitope antigen design containing key immunogenic peptides derived from HPV 6 and 11 epitopes, namely E2(HPV6), E4(HPV6), E6(HPV6 / 11), and E7(HPV6 / 11), where the HPV6-derived peptides have high sequence similarity to HPV11. F. Adenovirus Expression Cassette

[0306] In some cases where an adenovirus vector is used, the expression cassette may be located at the E1 region deletion junction or the E4 deletion junction. In certain embodiments, the expression cassette is located at the E1 region deletion junction.

[0307] In a particular embodiment, the expression cassette is cloned from right to left with respect to the adenovirus viral genome.

[0308] In a particular embodiment, an expression cassette cloned from right to left within the viral genome of an adenovirus comprises the nucleic acid sequence of SEQ ID NO: 116 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: 116, or a codon-degenerate variant of SEQ ID NO: 116, or a conservedly substituted variant of SEQ ID NO: 116, or a non-conservatively substituted variant of SEQ ID NO: 116). III. Antigenic Bioinformatics Workflow for HPV Vaccine Design

[0309] [ka] A. Identification of new HPV antigenic components

[0310] Based on the crucial roles that HPV E2 and E4 gene components play in essential HPV function, the locations of corresponding proteins, and in-silico predictions, E2 and E4-derived antigens were identified for HPV therapeutic vaccines. Non-oncogenic and viral inactivation gene modifications were also applied to eliminate viral and oncogenic biological activity from HPV proteins, for example, in HPV E2 and E6 proteins.

[0311] By applying genetic engineering, we achieved the production of rearranged protein sequences that maintain the immunological characteristics of the peptides while eliminating the oncogenic and viral amplification functions of E7 and E4, respectively.

[0312] Therefore, some of the innovative aspects of the designs illustrated in this specification include: (1) the use of gene constructs encoding fusion proteins containing four or more different HPV proteins; (2) the combination of amino acid point mutation and duplicate polypeptide sequence shuffling techniques to inactivate oncogenic and essential viral functions; (3) the incorporation of HPV proteins containing multiple antigenic components derived from HPV proteins highly expressed in host-infected cells; (4) the design of hybrid antigens known for the first time; (5) the combination of epitopes from high-cancer-risk and low-cancer-risk HPV strains; (6) the use of mixed and adjustable repeat linkers; (7) the use of rigid linkers to stabilize polypeptide subunits and prevent undesirable intramolecular interactions; (8) the use of cleavable linkers between epitopes; and (9) the dual use of linker sequences that themselves provide protein-protein linker(-) function as well as both antigen and epitope (i.e., antigenicity conferred by the linker sequence).

[0313] Antigenicity is the ability to stimulate antibody production or a cell-mediated immune response. The antigenicity of the final design sequences was predicted by Vaxjen software, an alignment-independent model for antigen recognition based on the main chemical properties of the amino acid sequence. The results show that five antigenic sequences are antigenic. See Table 4 (Virus and Tumor Antigenicity). (SEQ ID NO: 121) (SEQ ID NO: 123) In certain embodiments, two or more polypeptides encoded by the polynucleotides described herein may be separated by an intervening sequence encoding a linker polypeptide. In certain cases, the linker is a cleavage-sensitive linker. In some embodiments, the polypeptide of interest is expressed as a fusion protein linked by a cleavage-sensitive linker polypeptide. In certain embodiments, the cleavage-sensitive linker polypeptide may be one or two of Furinlink, fmdv, p2a, GSG-p2a, and / or fp2a, as described below. In some cases, the linker is APVKQGSG (SEQ ID NO: 124).

[0314] Allergens are generally small antigens that elicit an antibody response. Allergenicity was predicted by ALLERTOP, a bioinformatics-based allergen prediction software that uses machine learning methods for classification, regardless of whether the antigen is allergen or non-allergen. This includes logistic regression (LR), decision trees (DT), simple Bayes (NB), random forest (RF), multilayer perceptron (MLP), and k-nearest neighbors (kNN). The results show that five antigen sequences are non-allergenic. See Table 4 (Allergenicity).

[0315] Cross-reactivity or the onset of autoimmune side effects in various tissues poses a significant safety risk in adoptive immunotherapy. Sequence homology analysis was performed using BLAST search, a basic local alignment search tool, to evaluate whether these novel antigens exhibit cross-reactivity with the human proteome. No host cross-reactivity was identified for these five antigen sequences. See Table 4 (Host Cross-Reactivity). B. Software / Tools

[0316] Software tools used in implementing the designs described herein include, but are not limited to, the following: See ALLERTOP (AllerTOP v.2 -- a server for in silico prediction of allergens; J Mol Model. 2014 Jun; 20(6):2278. doi: 10.1007 / s00894-014-2278-5. Epub 2014 May 31). • ANN (See Reliable prediction of T-cell epitopes using neural networks with novel sequence representations; Protein Sci. 2003 May; 12(5):1007-17). • BLAST (a basic local alignment search tool; NCBI, National Center for Biotechnology Information, US National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA). ·CLUSTALW2 (EMBL-EBI, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK. +44(0)1223 49 44 44). • GENEIOUS VI 1.1.5 (See geneious.com / biopharma / ) • See the IEDB consensus (A consensus epitope prediction approach identifies the breadth of murine T(CD8+)-cell responses to vaccinia virus; Nat Biotechnol. 2006 Jul; 24(7):817-9. Epub 2006 Jun 11). See NETMHCPAN 4.0 (Gapped sequence alignment using artificial neural networks: application to the MHC class I system; Bioinformatics. 2016 Feb 15;32(4):511-7. doi: 10.1093 / bioinformatics / btv639. Epub 2015 Oct 29). • PHYRE2 (The Phyre2 web portal for protein modeling, prediction and analysis; 07 May 2015; see nature.com / articles / nprot.2015.053; doi.org / 10.1038 / nprot.2015.053). • PYMOL MOLECULAR GRAPHICS system V2.ll (See pymol.org / 2 / ; sourceforge.net / projects / pymol / support) • VAXJEN (VaxiJen: a server for prediction of protective antigens, tumor antigens and subunit vaccines; see BMC Bioinformatics. 2007 Jan 5;8:4) III. HPV antigen design and its variants

[0317] In exemplary embodiments, the polynucleotide encoding the fusion protein (e.g., 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., HPV6 and HPV11 proteins).

[0318] Examples of 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. Such combinations include, but are not limited to, combinations of HPV6 and / or HPV11 protein types and multiple copies or variants of a single HPV6 and / or HPV11 protein.

[0319] In some embodiments, the HPV6 E2 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E2 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, the HPV6 E2 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1. In some embodiments, the HPV6 E2 protein includes an amino acid sequence having 1 to 36 amino acid substitutions compared to SEQ ID NO: 1 (e.g., 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions). In some embodiments, the HPV6 E2 protein includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the HPV E2 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 105 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV E2 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 105.

[0320] In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3 or 7 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 or 7. In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 or 7. In some embodiments, the HPV6 E4 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 3 or 7. In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3. In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3. In some embodiments, the HPV6 E4 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 3. In some embodiments, the HPV6 E4 protein includes 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 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7. In some embodiments, the HPV6 E4 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7. In some embodiments, the HPV6 E4 protein contains an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 7. In some embodiments, the HPV6 E4 protein contains the amino acid sequence of SEQ ID NO: 3 or 7.In some embodiments, the HPV6 E4 protein contains the amino acid sequence of SEQ ID NO: 3. In some embodiments, the HPV6 E4 protein contains the amino acid sequence of SEQ ID NO: 7. In some embodiments, the HPV E4 protein contains amino acid sequences selected from SEQ ID NOs: 3, 7, 107, 111, and 172-179. In some embodiments, the HPV E4 protein contains amino acid sequences selected from SEQ ID NOs: 3 and 7.

[0321] In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11 or 40 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 or 40. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11 or 40. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 11 or 40. In some embodiments, the HPV6 E6 protein includes 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). In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 11. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40. In some embodiments, the HPV6 E6 protein contains an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 40. In some embodiments, the HPV6 E6 protein contains the amino acid sequence of SEQ ID NO: 11 or 40. In some embodiments, the HPV6 E6 protein contains the amino acid sequence of SEQ ID NO: 11. In some embodiments, the HPV6 E6 protein contains the amino acid sequence of SEQ ID NO: 40.In some embodiments, the HPV6 E6 protein includes 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 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110. In some embodiments, the HPV6 E6 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 110. In some embodiments, the HPV6 E6 protein includes the amino acid sequence of SEQ ID NO: 110. In some embodiments, the HPV6 E6 protein includes an amino acid sequence selected from SEQ ID NOs: 11, 40, 110, and 197-204. In some embodiments, the HPV6 E6 protein comprises an amino acid sequence selected from SEQ ID NOs: 11, 40, and 110.

[0322] In some embodiments, the HPV6 E7 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5 or 9 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV6 E7 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5 or 9. In some embodiments, the HPV6 E7 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5 or 9. In some embodiments, the HPV6 E7 protein includes an amino acid sequence having 1 to 10 amino acid substitutions compared to SEQ ID NO: 5 or 9 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution). In some embodiments, the HPV6 E7 protein includes 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 HPV6 E7 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5. In some embodiments, the HPV6 E7 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5. In some embodiments, the HPV6 E7 protein includes an amino acid sequence having 1 to 10 amino acid substitutions compared to SEQ ID NO: 5 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution). In some embodiments, the HPV6 E7 protein includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the HPV6 E7 protein contains 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 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9. In some embodiments, the HPV6 E7 protein contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9.In some embodiments, the HPV6 E7 protein includes an amino acid sequence having 1 to 10 amino acid substitutions (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 9. In some embodiments, the HPV6 E7 protein includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the HPV6 E7 protein includes the amino acid sequence of SEQ ID NO: 5 or 9. In some embodiments, the HPV6 E7 protein includes 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 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 109. In some embodiments, the HPV6 E7 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 109. In some embodiments, the HPV6 E7 protein includes an amino acid sequence having 1 to 10 amino acid substitutions (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 109. In some embodiments, the HPV6 E7 protein includes the amino acid sequence of SEQ ID NO: 109. In some embodiments, the HPV6 E7 protein includes an amino acid sequence selected from SEQ ID NOs: 5, 9, 109, and 189-196. In some embodiments, the HPV6 E7 protein includes an amino acid sequence selected from SEQ ID NOs: 5, 9, and 109.

[0323] Examples of 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 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV11 E6 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42. In some embodiments, the HPV11 E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 42. In some embodiments, the HPV11 E6 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 42. In some embodiments, the HPV11 E6 protein includes the amino acid sequence of SEQ ID NO: 42. In some embodiments, the HPV11 E6 protein contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 108 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV11 E6 protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 108. In some embodiments, the HPV11 E6 protein contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 108. In some embodiments, the HPV11 E6 protein contains an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 108. In some embodiments, the HPV11 E6 protein contains the amino acid sequence of SEQ ID NO: 108. In some embodiments, the HPV11 E6 protein contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 112 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV11 E6 protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 112.In some embodiments, the HPV11 E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 112. In some embodiments, the HPV11 E6 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 112. In some embodiments, the HPV11 E6 protein includes the amino acid sequence of SEQ ID NO: 112. In some embodiments, the HPV11 E6 protein includes an amino acid sequence selected from SEQ ID NOs: 42, 108, 112, 180-188, and 213-220. In some embodiments, the HPV11 E6 protein includes an amino acid sequence selected from SEQ ID NOs: 42, 108, and 112.

[0324] In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45 or 106 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45 or 106. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 45 or 106. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 45 or 106. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 45. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 45. In some embodiments, the HPV11 E7 protein includes the amino acid sequence of SEQ ID NO: 45. In some embodiments, the HPV11 E7 protein contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 106 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV11 E7 protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 106. In some embodiments, the HPV11 E7 protein contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 106.In some embodiments, the HPV11 E7 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 106. In some embodiments, the HPV11 E7 protein includes the amino acid sequence of SEQ ID NO: 106. In some embodiments, the HPV11 E7 protein includes 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 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 114. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 114. In some embodiments, the HPV11 E7 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 114. In some embodiments, the HPV11 E7 protein includes the amino acid sequence of SEQ ID NO: 114. In some embodiments, the HPV11 E7 protein includes an amino acid sequence selected from SEQ ID NOs: 45, 106, 114, 164-171, and 229-236. In some embodiments, the HPV11 E7 protein includes an amino acid sequence selected from SEQ ID NOs: 45, 106, and 114.

[0325] A consensus sequence between the HPV6 E2 protein sequence and the HPV11 E2 protein sequence, or between fragments thereof, can be determined and can be called the HPV E2 protein. In some embodiments, the HPV E2 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 105 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV E2 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 105. In some embodiments, the HPV E2 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 105. In some embodiments, the HPV E2 protein includes an amino acid sequence having 1 to 36 amino acid substitutions compared to SEQ ID NO: 105 (e.g., 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions). In some embodiments, the HPV E2 protein comprises an amino acid sequence selected from SEQ ID NOs. 105 and 154-163.

[0326] A consensus sequence between the HPV6 E4 protein sequence and the HPV11 E4 protein sequence, or between fragments thereof, can be determined and may be called the HPV E4 protein, which may be included in any of the polynucleotide or fusion proteins described herein. In some embodiments, the HPV E4 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 107 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV E4 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 107. In some embodiments, the HPV E4 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 107. In some embodiments, the HPV E4 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 107. In some embodiments, the HPV6 E4 protein includes the amino acid sequence of SEQ ID NO: 107. In some embodiments, the HPV E4 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 111 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV E4 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 111. In some embodiments, the HPV E4 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 111. In some embodiments, the HPV E4 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 111. In some embodiments, the HPV6 E4 protein includes the amino acid sequence of SEQ ID NO: 111. In some embodiments, the HPV6 E4 protein includes an amino acid sequence selected from SEQ ID NOs: 107, 111, 172-179, and 205-212. In some embodiments, the HPV6 E4 protein comprises an amino acid sequence selected from SEQ ID NOs: 107 and 111.

[0327] A consensus sequence between the HPV6 E6 protein sequence and the HPV11 E6 protein sequence, or between fragments thereof, can be determined and may be called the HPV E6 protein, which may be included in any of the polynucleotide or fusion proteins disclosed herein. In some embodiments, the HPV E6 protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 115 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). In some embodiments, the HPV E6 protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. In some embodiments, the HPV E6 protein includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115. In some embodiments, the HPV E6 protein includes an amino acid sequence having 1 to 5 amino acid substitutions (e.g., 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution) compared to SEQ ID NO: 115. In some embodiments, the HPV E6 protein includes 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 NOs: 115 and 237-246.

[0328] A consensus sequence between the HPV6 E7 protein sequence and the HPV11 E7 protein sequence, or between fragments thereof, can be determined and may be called the HPV E7 protein, which may be included in any of the polynucleotide or fusion proteins disclosed herein. In some embodiments, the HPV E7 protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 113 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity). Exemplary variants include the amino acid sequences of SEQ ID NOs: 221–228. In some embodiments, the HPV E7 protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 113. In some embodiments, the HPV E7 protein comprises an amino acid sequence having at least 95% sequence identity with 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) compared to SEQ ID NO: 113. In some embodiments, the HPV E7 protein includes the amino acid sequence of SEQ ID NO: 113. In some embodiments, the HPV E7 protein includes amino acid sequences selected from SEQ ID NO: 113 and 221-228.

[0329] In some embodiments, the fusion protein comprises one or more copies of HPV6 and / or HPV11 proteins. For example, the fusion protein comprises one or more copies of HPV6 E4 protein, HPV6 E6 protein, HPV6 E7 protein, HPV11 E6 protein, or HPV11 E7 protein. In some embodiments, the fusion protein comprises an HPV6 E4 protein containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3 (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity), and an HPV6 E4 protein containing 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 fusion protein includes 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 includes 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 includes 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 includes an HPV6 E6 protein comprising 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 comprising 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 includes an HPV6 E6 protein comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11, and an HPV6 E6 protein comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40.In some embodiments, the fusion protein includes an HPV6 E6 protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11, and an HPV6 E6 protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40. In some embodiments, the fusion protein includes an HPV6 E6 protein containing an amino acid sequence of SEQ ID NO: 11, and an HPV6 E6 protein containing an amino acid sequence of SEQ ID NO: 40. In some embodiments, the fusion protein includes an HPV6 E7 protein containing 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), and an HPV6 E7 protein containing 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 fusion protein includes 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 includes an HPV6 E7 protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5, and an HPV6 E7 protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9. In some embodiments, the fusion protein includes an HPV6 E7 protein containing the amino acid sequence of SEQ ID NO: 5, and an HPV6 E7 protein containing the amino acid sequence of SEQ ID NO: 9.

[0330] In exemplary embodiments, the polypeptide construct or fusion protein encoded by the polynucleotide of the present invention has / contains 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 SEQ ID NO: 66, 68, 70, 72, or 74, or a conservatively substituted variant of SEQ ID NO: 66, 68, 70, 72, or 74). In some embodiments, the fusion protein contains an amino acid sequence having at least 80% identity with SEQ ID NO: 68 (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 66 (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 70 (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 72 (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity). In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 74 (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity).

[0331] In some embodiments, the fusion protein includes an amino acid sequence having at least 80% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 85% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 90% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 95% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 96% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 97% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 98% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes an amino acid sequence having at least 99% identity with SEQ ID NO: 68. In some embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 68.

[0332] In exemplary embodiments, the polypeptide construct of the present invention comprises the sequence of SEQ ID NO: 68 or a functional variant thereof (for example, 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 with SEQ ID NO: 68, or a conservatively substituted variant of SEQ ID NO: 68, or a non-conservatively substituted variant of SEQ ID NO: 68).

[0333] In certain embodiments, the polypeptide construct of the present invention has a functional variant of SEQ ID NO: 68 that, when compared to SEQ ID NO: 68, has a similar or enhanced binding affinity to HPV6 / 11-related proteins and / or produces a similar or enhanced immunogenic response. Such variants can be readily determined by sequence alignment software such as ClustalW.

[0334] In certain embodiments, the polypeptide construct of the present invention comprises one of SEQ ID NOs: 105-115 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 one of SEQ ID NOs: 105-115, or a conservatively substituted variant of one of SEQ ID NOs: 105-115, or a non-conservatively substituted variant of one of SEQ ID NOs: 105-115). In exemplary embodiments, the polypeptide construct of the present invention comprises each of SEQ ID NOs: 105-115.

[0335] In some embodiments, the polynucleotide construct variant may contain one or more conservatively substituted variants of the antigen region having 105-115. For example, any hydrophilic amino acid which may be substituted with any other hydrophilic amino acid, any aliphatic amino acid which may be substituted with any other aliphatic amino acid, any basic amino acid which may be substituted with any other basic amino acid, any aromatic amino acid which may be substituted with any other aromatic amino acid, and any combination thereof. Exemplary polynucleotide construct variants having conservatively substituted mutations include, but are not limited to, SEQ ID NOs: 143 and 144.

[0336] In some embodiments, polynucleotide construct variants may contain one or more amino acid additions or deletions. For example, one or more amino acids may be added to or removed from any of the antigenic regions of the polynucleotide constructs described herein. Exemplary polynucleotide construct variants having amino acid additions and / or deletions include, but are not limited to, SEQ ID NOs: 144-148.

[0337] In certain embodiments, the polypeptide construct variant has the same HPV6 / 11 antigen regions as SEQ ID NO: 68, but these antigen regions of SEQ ID NO: 68 are shuffled in a different order than the order of the antigen regions in SEQ ID NO: 68. In certain embodiments, the variant includes the 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) antigen regions of SEQ ID NO: 68, but these antigen regions are shuffled compared to the order of the antigen regions in SEQ ID NO: 68. Exemplary polypeptide construct variant sequences include, but are not limited to, SEQ ID NO: 134 (antigen region order: 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 order: 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 order: HPV E7, HPV11 E7, HPV E6, HPV E4, HPV11 E6, HPV6 E7, HPV6 E6, HPV E4, HPV11 E6, HPV E2, and HPV11 E7).

[0338] The polypeptide construct contains approximately 2 to approximately 20 antigenic regions (e.g., approximately 5 to approximately 15, approximately 10 to approximately 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, or 20 or more antigenic regions. In certain 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 compared to SEQ ID NO: 68. A polypeptide construct variant containing fewer antigenic regions compared to SEQ ID NO: 68 may lack any of the antigenic regions of SEQ ID NO: 68. Exemplary polypeptide construct variants containing fewer antigenic regions compared to SEQ ID NO: 68 include, but are not limited to, SEQ ID NO: 137 (antigen region order: 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 order: 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 order: HPV E7, HPV11 E7, HPV E6, HPV11 E6, HPV6 E7, HPV6 E6, HPV11 E6, HPV E2, HPV11 E7). In certain embodiments, the polypeptide construct variant has more 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 more antigenic regions compared to SEQ ID NO: 68. A polypeptide construct variant containing more antigenic regions compared to SEQ ID NO: 68 may have one or more antigenic regions attached to any portion of the antigenic region section of the polypeptide construct (e.g., N-terminus, C-terminus, between other antigenic regions, interspersed with other antigenic regions, and combinations thereof).Exemplary polypeptide construct variants containing more antigenic regions compared to SEQ ID NO: 68 include SEQ ID NO: 140 (antigen region order: 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 order: HPV E6 E7, 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 order: HPV E2, HPV11 E7, HPV E4, HPV11 E6, HPV6 E7, HPV E4, HPV6 E6, HPV E4, HPV11 Examples include HPV E6, HPV E7, HPV11 E7, HPV E7, and HPV E6, but are not limited to these.

[0339] In some embodiments, the antigen region or fusion protein may also include any of the linker peptides described herein (e.g., rigid linker polypeptides, flexible linker polypeptides, or a combination thereof).

[0340] In some embodiments, the antigen region or fusion protein may also include one of the agonist peptides described herein (also known 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). Examples of 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 HPV E7 agonist peptide), HPV11 agonist peptides (e.g., HPV11 E6 agonist peptide and HPV11 E7 agonist peptide), and HPV16 agonist peptides. In some embodiments, the agonist peptide, if included, comprises the amino acid sequence of HPV16 E6 agonist peptide, e.g., SEQ ID NO: 48 or SEQ ID NO: 50. In some embodiments, the agonist peptide, if included, comprises the amino acid sequence of HPV16 E7 agonist peptide, e.g., SEQ ID NO: 52 or SEQ ID NO: 54. IV. Vaccines

[0341] This disclosure provides vaccines comprising polynucleotides encoding fusion proteins described herein. In some embodiments, the vaccine comprises polynucleotides encoding fusion proteins comprising HPV6 proteins selected from HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, and HPV6 E7 protein; and HPV11 proteins selected from HPV11 E6 protein and HPV11 E7 protein. The vaccines of this disclosure can be used to prevent and / or treat HPV infection and HPV-related diseases.

[0342] In some embodiments, the vaccine comprises a polynucleotide encoding a fusion protein comprising HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV6 E7 protein, HPV11 E6 protein, and HPV11 E7 protein. 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 an amino acid sequence of SEQ ID NO: 68 or a conservatively substituted variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% identity with SEQ ID NOs. 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.

[0343] The vaccine polynucleotide may be operably ligated to elements that facilitate the expression of the fusion protein, such as a promoter, a 5' untranslated region (UTR), a transcription start site (TSS), a 3'UTR, a tetracycline response element, and / or a Kozak region. In some embodiments, the promoter is operably ligated to a promoter-enhancer region. The vaccines of this disclosure can be administered by any preferred 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.

[0344] In some embodiments, the vaccine comprises a vector containing a polynucleotide encoding a fusion protein. The vector may 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, retrovirus vector, lentivirus vector, vaccinia virus vector, or herpesvirus vector. In some embodiments, the vector is an adenovirus vector, such as a gorilla adenovirus vector.

[0345] In some embodiments, the vaccine comprises a polypeptide encoded by a polynucleotide. The polypeptide can be produced by any preferred method, e.g., expression in host cells, expression in a cell-free system, or chemosynthesis. The polypeptide can be purified by any preferred method, e.g., affinity chromatography, ion exchange chromatography, size exclusion chromatography, or high-performance liquid chromatography (HPLC). The polypeptide can be formulated using a pharmaceutically acceptable carrier for administration as a vaccine.

[0346] In some embodiments, the vaccine comprises a composition containing a polynucleotide, a vector, or a polypeptide. The composition may further 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.

[0347] In some embodiments, the vaccine comprises cells comprising a polynucleotide, vector, polypeptide, or composition. The cells may be any cell, e.g., bacterial cells, yeast cells, insect cells, or mammalian cells. In some embodiments, the cells may be immune cells, e.g., dendritic cells, macrophages, monocytes, B cells, T cells, or natural killer (NK) cells. In some embodiments, the cells may be dendritic cells, e.g., human dendritic cells. In some embodiments, the cells may be T cells, e.g., CD4+ T cells or CD8+ T cells. The T cells may be naive T cells, effector T cells, or memory T cells. The T cells may be regulatory T cells (Treg) or gamma delta T cells. The cells may be autologous or allogeneic to the subject to be vaccinated. The cells may be modified to express a fusion protein by any suitable method, e.g., transfection, transduction, or electroporation. The cells may be administered as a vaccine by any suitable route, e.g., intravenous, intradermal, or subcutaneous administration. The use of immune cells such as dendritic cells or T cells as vaccines can enhance the immune response to HPV antigens and improve vaccine efficacy. V. Treatment Method

[0348] The present invention relates, in part, to a method for treating a disease or disorder in a subject requiring such treatment, comprising the step of administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to the subject. In certain embodiments, the method comprises the step of administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to a subject having anogenital wart, lower genital neoplasms (e.g., intraepithelial neoplasms of the cervix, vagina, and vulva), cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer. In certain embodiments, the method comprises the step of administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to a subject having a malignant disease caused by HPV 6 / 11.

[0349] The present invention also relates, in part, to a method for priming a T cell response to HPV-infected (e.g., HPV 6 / 11+) cells in a subject requiring it (e.g., a subject having RRP), the method comprising the step of administering the vector of the present invention to the subject. In certain embodiments, the method comprises administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to a subject having a malignant disease caused by HPV 6 / 11.

[0350] The present invention also relates to inducing an anti-HPV immune response in subjects in need, such as individuals with RRP or other HPV-related diseases or disorders, including those related to HPV6 or HPV11. Inducing this immune response may involve increasing the recruitment, quantity, 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 administration of the described polynucleotides, vectors, fusion proteins, or compositions. In some cases, inducing an anti-HPV immune response involves administering a therapeutically effective amount of any of the polynucleotides, vectors, fusion proteins, or compositions thereof described herein to a subject. In some embodiments, inducing an anti-HPV immune response involves administering a therapeutically effective amount of any of the vectors described herein (e.g., a vector containing SEQ ID NO: 68) to a subject. In some embodiments, the therapeutically effective amount of a vector is about 1 × 10⁻⁶ 11 and approximately 5 × 10 11 Includes particle units (PU).

[0351] In a particular embodiment, the disease or disorder to be treated is RRP, and the route of administration is subcutaneous.

[0352] In certain embodiments, the method of the present invention prevents disease progression with a lower PU dose than prior methods known in the art. For example, in some embodiments, the method is 5e 9In some embodiments, the method prevents disease protection by a PU dose of a vector, composition, or vaccine. In other embodiments, the method prevents disease protection by a 5e10 PU dose of a vector, composition, or vaccine. In some embodiments, the method of the present invention prevents disease progression by administering a smaller amount of therapeutic composition than previous methods known in the art. For example, in some embodiments, the method prevents disease protection by administering only a single dose of a vector, composition, or vaccine.

[0353] In certain embodiments, the subject to be treated is a mammal, such as a primate. In some embodiments, the subject to be treated is a human.

[0354] The method may include the administration of polynucleotides, polypeptides, vectors, compositions, vaccines, or cells in therapeutically effective amounts to treat a disease or disorder. The method may include the administration of polynucleotides, polypeptides, vectors, compositions, vaccines, or cells in therapeutically effective amounts to increase the activity of the T cell response to a specific HPV protein or antigen (e.g., HPV6 / 11 specific protein or antigen). The method may include the administration of polynucleotides, polypeptides, vectors, compositions, vaccines, or cells in therapeutically effective amounts to treat RRP. The method may include the administration of polynucleotides, polypeptides, vectors, compositions, vaccines, or cells in therapeutically effective amounts to reduce the target's Derkay score.

[0355] 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 anti-inflammatory agent encoded, the vector used for administration, the type of cell and / or vaccine, and the route of administration.

[0356] In certain embodiments, the vector, composition, or vaccine is administered in multiple doses. In certain embodiments, the amount of medication in one dose is approximately 0.1 × 10⁻⁶ 9 ~About 10×10 12 Particle unit, 0.1 × 10⁻⁶ 9~Approx. 1.0×10 12 Particle size, approximately 0.1 × 10⁻⁶ 9 ~About 10×10 11 Particle size, approximately 0.1 x 10⁻¹⁶ 9 ~about 1.0x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 9 ~about 0.5x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 9 ~approximately 0.1 x 10 11 Particle size, approximately 1.0 x 10⁻¹⁶ 10 ~about 10x10 11 Particle size, approximately 1.0 x 10⁻¹⁶ 10 ~approximately 0.1 x 10 11 Particle size, approximately 0.1 x 10⁻¹⁶ 11 ~about 10x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~approximately 9x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~about 8x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~approximately 7x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~about 6x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 12 ~about 10x10 12 Particle size, approximately 0.5 x 10⁻¹⁶ 12 ~about 1.0x10 12 Particle size, approximately 1.0 x 10⁻¹⁶ 11 ~approximately 0.1 x 10 12 Particle size, approximately 0.1 x 10⁻¹⁶ 12 ~about 10x10 12 Particle unit, approximately 1 x 10⁻⁶ 10 Particle unit, approximately 5 x 10 10 Particle unit, approximately 5 x 10 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 9 x 10 11 Particle unit, approximately 10x10 11 Particle unit, approximately 1 x 10⁻⁶ 12 Particle unit, approximately 2 x 10⁻¹⁶ 12 Particle unit, approximately 3 x 10⁻¹⁶ 12 Particle unit, approximately 4 x 10 12 Particle unit, approximately 5 x 10 12Particle units, about 6x10 12 Particle units, about 7x10 12 Particle units, about 8x10 12 Particle units, about 9×10 12 Particle units, or about 10×10 12 Particle units may be included.

[0357] In certain embodiments, the dosage is about 1.0×10 5 ~ about 1.0×10 10 Plaque-forming units (PFU), e.g., about 0.5×10 5 ~ about 0.5×10 10 PFU, about 0.1x10 5 x0.1x10 10 PFU, about 1x10 6 ~ about 1x10 9 PFU, about 0.5x10 6 ~ about 0.5x10 9 PFU, about 0.1x10 6 ~ about 0.1x10 9 PFU, about 1x10 7 ~ about 1x10 8 PFU, about 0.5x10 7 ~ about 0.5x10 8 PFU, about 0.1x10 7 ~ about 0.1x10 8 P...

Claims

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

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

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

1.

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

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

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

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

42.

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

45.

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

7.

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

40.

17. The polynucleotide according to claim 1, wherein 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.

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

68.

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

68.

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

68.

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

68.

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

68.

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

68.

24. The polynucleotide according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 68 or a conservatively substituted variant thereof.

25. The polynucleotide according to claim 1, wherein the fusion protein comprises the amino acid sequence of Sequence ID No.

68.

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

66.

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

70.

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

72.

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

74.

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

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

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

33. The polynucleotide according to claim 1, operably ligated 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.

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

35. A vector comprising a polynucleotide according to any one of claims 1 to 34.

36. The vector according to claim 35, which is a plasmid, a viral vector, or a nonviral vector.

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

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

40. The vector according to claim 37, wherein the adenovirus vector is a gorilla adenovirus vector.

41. The vector according to 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 requiring such response, comprising the step of administering a therapeutically effective amount of the vector according to claim 30 to the subject.

43. The aforementioned effective therapeutic dose is approximately 1 × 10 １１ and approximately 5 x 10 １１ The method according to claim 42, comprising particle units (PU).

44. A method for treating an HPV-related disease or disorder in a subject requiring such treatment, comprising the step of administering a therapeutically effective amount of the vector according to claim 35 to the subject.

45. The method according to claim 44, wherein the HPV-related disease or disorder is HPV-6 related disease or disorder or HPV-11 related disease or disorder.

46. The method according to claim 44, wherein the HPV-related disease or disorder is HPV-related cancer.

47. The method according to claim 44, wherein the HPV-related disease or disorder is recurrent respiratory papillomatosis (RRP), anogenital warts, lower genital neoplasms, cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer.

48. The method according to claim 44, wherein the HPV-related disease or disorder is RRP.

49. The aforementioned effective therapeutic dose is approximately 1 × 10 １１ and approximately 5 x 10 １１ The method according to claim 44, comprising particle units (PU).

50. The method according to claim 44, further comprising the step of administering an additional therapy.

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

52. The method according to claim 44, further comprising a weight loss procedure.

53. A fusion protein encoded by a polynucleotide according to any one of claims 1 to 34.

54. A composition comprising a polynucleotide according to any one of claims 1 to 34.

55. The composition according to claim 49 for use in the treatment of a disease or disorder in a subject requiring it.

56. Use of a polynucleotide according to any one of claims 1 to 34 in the manufacture of a pharmaceutical for use in the treatment of a disease or disorder in a subject requiring such use.

57. A kit comprising a polynucleotide according to any one of claims 1 to 34.

58. A vaccine comprising a polynucleotide according to any one of claims 1 to 34.

59. The vaccine according to claim 58, for use in the treatment of a disease or disorder in a subject requiring it.

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