Compositions and methods for treating HER2-positive metastatic breast cancer and other cancers

Abstract

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein wherein the vector genome comprises an AAV 5 'inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding an anti-Her2 antibody (i.e. Trastuzumab) operably linked to a regulatory control sequence, and an AAV 3' ITR, the anti-Her2 antibody comprising a heavy chain and a light chain, the regulatory control sequence directs expression of trastuzumab in a target cell. Also provided are a pharmaceutical composition comprising a formulation buffer comprising the rAAV as described herein, a nucleic acid molecule, a packaged host cell, an rAAV production system, and a method of treating HER2-positive brain metastatic cancer, optionally brain metastatic breast cancer.

Description

[0001] References to Electronic Sequence Listings

[0002] The electronic sequence listing submitted herein is named "UPN-22-9834PCT_Seq_List.xml", is 186,012 bytes in size, was created on March 30, 2023, and the contents of the electronic sequence listing (e.g., the sequences and text therein) are incorporated herein by reference in their entirety. Background Art

[0003] Brain metastasis is a common and devastating sequela of breast cancer for which treatment options are few and inadequate. 6-16% of breast cancer patients will develop central nervous system (CNS) metastases. From the time of diagnosis, these patients have a median one-year survival rate of 20%, and a median five-year survival rate of 1.3%. DiStefano A et al., Cancer. 1979;44:1913-1918; Takakura K et al., Metastatic tumors of the central nervous system. Tokyo: Igaku-Shoin, 1982; Hall WA et al., Long-term survival with metastatic cancer to the brain. Med Oncol. 2000 Nov;17(4):279-86; Pieńkowski T, Zielinski CC. Trastuzumab treatment in patients with breast cancer and metastatic CNS disease. Ann Oncol. 2010 May;21(5):917-24. Surgical resection of brain metastases is often not feasible, and chemotherapeutic agents are mostly excluded from the CNS by the blood-brain barrier (BBB) ​​[Nakayama A et al., Antitumor Activity of TAK-285, an Investigational, Non-Pgp Substrate HER2 / EGFR Kinase Inhibitor, in Cultured Tumor Cells, Mouse and Rat Xenograft Tumors, and in an HER2-Positive Brain Metastasis Model. J Cancer. 2013 Aug 16;4(7)]. Alternative therapies are needed for the treatment of brain metastases from breast cancer.

[0004] Breast cancers that overexpress the HER2 receptor tyrosine kinase are highly susceptible to metastasis to the CNS and account for 25-30% of all breast cancer cases [Bendell JC et al. Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma. Cancer. 2003 Jun 15;97(12):2972-7]. Trastuzumab is a first-line therapeutic immunoglobulin G (IgG) monoclonal antibody (mAb) directed against HER2; it has been reported to significantly improve survival in patients with HER2-positive disease [Lin NU et al., Brain metastases: the HER2 paradigm. Clin Cancer Res. 2007 Mar 15;13(6):1648-55; Palmieri D et al., Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain. Cancer Res. 2007 May 1;67(9):4190-8]. However, patients who benefit from trastuzumab often experience concurrent progression of CNS disease because the mAb cannot cross the BBB [Nakayama, supra]. Direct injection of trastuzumab into the CNS has been shown to be safe, and intrathecal administration of trastuzumab in patients with leptomeningeal carcinomatosis has been reported to extend overall survival from 2 months to 13.5 months [Zagouri F et al., Intrathecal administration of trastuzumab for the treatment of meningeal carcinomatosis in HER2-positive metastatic breast cancer: a systematic review and pooled analysis. Breast Cancer Res Treat. 2013 May;139(1):13-22]. Leptomeningeal carcinomatosis is associated with a compromised, rather than intact, blood-brain barrier. Park, EJ et al., J Controlled Release, 163 (2012), 277-284 reported that a combination of focused ultrasound bursts and circulating microbubbles can temporarily allow trastuzumab to cross the blood-brain barrier and the blood-tumor barrier.

[0005] Although current therapies have improved systemic disease control, the management of metastatic dissemination of human breast cancer to the CNS represents a significant therapeutic challenge. Summary of the invention

[0006] The present invention provides a therapeutic recombinant and replication-deficient adeno-associated virus (rAAV), comprising an adeno-associated virus (AAV) capsid containing a vector genome, wherein the vector genome comprises: (a) AAV-5′ inverted terminal repeat (ITR), (b) an expression cassette comprising a coding sequence for an anti-Her2 antibody, wherein the anti-Her2 antibody has a heavy chain and a light chain, and wherein the expression cassette comprises: (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to the anti-Her2 antibody heavy chain, (ii) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, wherein the nucleic acid sequence encodes the anti-Her2 antibody heavy chain, (iii) a furin cleavage site, (iv) a T2A element linker, (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to the anti-Her2 antibody light chain, and (vi) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO:5 NO:5 at least 95% identical sequence, the nucleic acid sequence encoding the anti-Her2 antibody light chain, and a regulatory control sequence operably linked to the sequence in the expression cassette, wherein the regulatory sequence comprises a ubiquitin C (UbC) promoter, an optional enhancer, an intron and a polyadenylation (polyA) sequence, and (c) AAV-3'ITR. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the anti-Her2 antibody heavy chain comprises SEQ ID NO:7, and the nucleic acid sequence encoding the leader peptide operably linked to the anti-Her2 antibody light chain comprises SEQ ID NO:9. In certain embodiments, the trastuzumab coding sequence comprises the nucleic acid sequence of SEQ ID NO:29 or a sequence at least 95% identical to SEQ ID NO:29. In certain embodiments, the expression cassette comprises a UbC promoter having a nucleic acid sequence of SEQ ID NO:24. In certain embodiments, the expression cassette comprises SV40polyA having a nucleic acid sequence of SEQ ID NO:23. In certain embodiments, the expression cassette comprises a chimeric intron having a nucleic acid sequence of SEQ ID NO: 22. In certain embodiments, the expression cassette comprises a nucleic acid sequence of SEQ ID NO: 26, or a nucleic acid sequence at least 99% identical to SEQ ID NO: 26. In certain embodiments, the vector genome comprises a nucleic acid sequence of SEQ ID NO: 25, or a nucleic acid sequence at least 99% identical to SEQ ID NO: 25.

[0007] In certain embodiments, the present invention provides a recombinant adeno-associated virus (rAAV), comprising an adeno-associated virus (AAV) capsid containing a vector genome, wherein the vector genome comprises: (a) AAV-5' inverted terminal repeat sequence (ITR), (b) an expression cassette, the expression cassette comprising a coding sequence for an anti-Her2 antibody, the anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising: (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to the anti-Her2 antibody heavy chain, (ii) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding the anti-Her2 antibody heavy chain, (iii) a furin cleavage site, (iv) a T2A element linker, (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to the anti-Her2 antibody light chain, (vi) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO:5 NO:5 at least 95% identical sequence, the nucleic acid sequence encodes an anti-Her2 antibody light chain, and a regulatory control sequence operably linked to the sequence in the expression cassette, wherein the regulatory sequence comprises one or more of the following: a promoter and a polyA sequence, the promoter is a chicken β-actin promoter or a CB7 hybrid promoter comprising a chimeric intron of a cytomegalovirus immediate early (CMV IE) enhancer, a chicken β-actin promoter, and a chimeric intron comprising a chicken β-actin intron, and the polyA sequence is an SV40 polyA sequence. In certain embodiments, the CB7 hybrid promoter has the nucleic acid sequence of SEQ ID NO:21. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO:28, a nucleic acid sequence at least 99% identical to SEQ ID NO:28, a nucleic acid sequence of SEQ ID NO:2, or a nucleic acid sequence at least 99% identical to SEQ ID NO:2. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 27, a sequence at least 99% identical to SEQ ID NO: 27, SEQ ID NO: 50, a sequence at least 99% identical to SEQ ID NO: 50, SEQ ID NO: 1, or a sequence at least 99% identical to SEQ ID NO: 1. In certain embodiments, the capsid is an AAVhu68 capsid, an AAV9 capsid, an AAVhu95 capsid, an AAVhu96, or an AAVrh91 capsid.

[0008] On the one hand, there is provided herein a composition and a pharmaceutical composition comprising rAAV and an aqueous suspension medium as described herein. In certain embodiments, the rAAV or its composition is used to treat brain metastatic cancer, wherein metastatic cancer is from a HER2-positive primary tumor, optionally for the treatment of brain metastatic breast cancer. In certain embodiments, a composition or a pharmaceutical composition is formulated for central nervous system (CNS) delivery, optionally wherein the CNS delivery is optionally intrathecal delivery, optionally by intraventricular (ICV) injection, intracerebellomedullary cisterna (ICM) injection, intraparenchymal injection, direct injection into a tumor or tumor bed, or by an Ommaya device. In certain embodiments, rAAV or its composition as described herein is used to prepare a drug for the treatment of HER2-positive brain metastatic cancer, optionally wherein the HER2-positive brain metastatic cancer is brain metastatic breast cancer. In certain embodiments, the composition can be delivered by a systemic route and / or delivered directly to a Her2-positive tumor (e.g., breast cancer or Her2-positive metastatic cancer) outside the CNS.

[0009] In another aspect, the present invention provides a recombinant nucleic acid molecule comprising: (a) AAV-5' inverted terminal repeat sequence (ITR), (b) an expression cassette, the expression cassette comprising: at least one open reading frame (ORF), the ORF comprising an anti-Her2 antibody heavy chain and an anti-Her2 antibody light chain, and a nucleic acid sequence that regulates the expression of the anti-Her2 antibody heavy chain and the anti-Her2 antibody light chain operably linked thereto, and (c) AAV-3'ITR, wherein the expression cassette comprises: (i) a promoter, the promoter is: (A) ubiquitin C (UbC), or (B) a CB7 hybrid promoter, the CB7 hybrid promoter comprising CMV IE enhancer, chicken β-actin promoter and chimeric intron, wherein the chimeric intron comprises a chicken β-actin splice donor and a rabbit β-globin splice acceptor, wherein the chicken β-actin splice donor comprises a chicken β-actin intron, and / or (ii) an intron, wherein the intron is a chimeric intron, and (iii) at least one ORF, wherein the at least one ORF comprises: a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3, wherein the nucleic acid sequence encodes an anti-Her2 heavy chain, (v) a furin cleavage site, (vi) a T2A linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 light chain, and (viii) a nucleic acid sequence comprising SEQ ID NO: NO:5, the nucleic acid sequence encodes an anti-Her2 light chain, (ix) an SV40 polyadenylation (polyA) sequence, and wherein the expression cassette further comprises a spacer sequence. In certain embodiments, the nucleic acid molecule comprises an expression cassette comprising the nucleic acid sequence of SEQ ID NO:26. In certain embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:25. In certain embodiments, the recombinant nucleic acid molecule is a plasmid.

[0010] In yet another aspect, provided herein is a packaging host cell comprising a recombinant nucleic acid molecule as described herein, the packaging host cell further comprising an AAV rep coding sequence operably linked to a sequence that expresses a rep protein in the packaging host cell, an AAV capsid coding sequence operably linked to a sequence that expresses an AAV capsid protein in the packaging host cell, and the helper virus functions necessary to allow the expression cassette and the AAV ITR to be packaged into the AAV capsid.

[0011] In another aspect, provided herein is a rAAV production system that can be used to produce rAAV as described herein, wherein the production system comprises a cell culture comprising: (a) a nucleic acid sequence encoding an AAV capsid protein; (b) a vector genome; and (c) sufficient AAV rep function and helper function to allow packaging of the vector genome into an AAV capsid.

[0012] In yet another aspect, a method for treating HER2-positive brain metastatic cancer is provided herein. In certain embodiments, a method for treating brain metastatic breast cancer is provided herein. The method comprises administering an effective amount of rAAV or a composition thereof, a pharmaceutical composition, or a suspension as described herein to a subject in need thereof. In certain embodiments, the suspension is formulated for intrathecal administration, intracerebellomedullary administration, or intraventricular administration. In certain embodiments, an anti-tumor regimen is provided herein, comprising administering rAAV, a composition thereof, or a pharmaceutical composition as described herein in combination with a biopharmaceutical, a small molecule, an anti-tumor agent, radiation, and / or a chemotherapeutic agent.

[0013] These and other aspects of the invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A A schematic representation of the designed study examining preclinical activity in mice is shown. Figure 1B Results demonstrating preclinical activity of engineered trastuzumab (antitumor activity against breast cancer brain metastases) are shown as a graph of the probability of mouse survival after tumor implantation.

[0015] Figure 2A Results of an ELISA are shown, plotting measurements of trastuzumab concentration (ng / mg protein) in perfused brain tissue samples collected following administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. Figure 2BResults of an ELISA are shown, plotting the measurement of trastuzumab concentrations (μg / mL) in serum samples collected following administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined constructs) and PBS. The mean peak serum concentration for patients receiving 500 mg (the highest dose) was 377 μg / mL.

[0016] Figure 3A Results of a biodistribution study are shown, where measurements of DNA concentration (GC / μg) in collected liver and brain tissues are plotted. Figure 3B Results of a biodistribution study are shown, where measurements of RNA concentration (copies / 100 ng of RNA) in collected liver and brain tissues are plotted.

[0017] Figure 4A Further shown are data measuring trastuzumab concentrations in serum samples collected on day 28 after administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (a construct previously examined) and PBS.

[0018] Figure 4B Further shown are data measuring trastuzumab concentrations in perfused brain tissue samples collected on day 28 after administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (a construct previously examined) and PBS.

[0019] Figure 4CFurther shown are data measuring the results of DNA biodistribution in brain and liver tissue samples collected on day 28 after administration of AAVrh91.CB-CI.IL2_V1_Trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_Trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_Trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.Trastuzumab.SV40 (a construct previously examined) and PBS.

[0020] Figure 5A Shown are the expression levels (μg / mL) of trastuzumab compared to capsid control and PBS as measured in serum samples at days -1, 7, 14 and 28 after administration of AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40.

[0021] Figure 5B Shown are the expression levels (μg / mL) of trastuzumab compared to capsid control and PBS as measured in brain tissue samples at days -1, 7, 14, and 28 after administration of AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40.

[0022] Figure 6 Vector biodistribution (GC / diploid cell) samples at days -1, 7, 14, and 28 after administration of AAVhu95.CB.CI.IL2_V1.Trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 are shown compared to capsid control and PBS.

[0023] Figure 7 Shown are quantitative results of tumor bioluminescence assessment of mouse xenografts (MDA-MB-453 (ER- / PR- / HER2+)) following treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype control.

[0024] Fig. 8A It is shown that ICV injection of AAV results in sustained expression of trastuzumab in Rag1 KO mice, as measured in serum samples by ELISA. Figure 8BIt is shown that ICV injection of AAV results in sustained expression of trastuzumab in Rag1 KO mice, as measured by ELISA in brain homogenate (perfused) samples.

[0025] Fig. 9 Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice (MDA-MB-453 (ER- / PR- / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 (disease remission) is shown.

[0026] Fig.10 Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice (BT-474 (ER+ / PR+ / HER2+) brain xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 (preventive and prophylactic treatment).

[0027] Fig.11A and 11B Representative Western blots are shown, demonstrating that the 2921st and 2922nd columns of AAVrh91.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 and AAVhu68.CMV.PI.Trastuzumab.SV40 (columns 2921 and 2922) Figure 2A and 2B ) after trastuzumab heavy chain ( Fig.11A ) and light chain ( Fig. 11B ) expression in brain lysates.

[0028] Fig. 12A A schematic representation of the experimental design of a study examining preclinical activity in mice is shown. Fig. 12B Shown are quantitative results of tumor bioluminescence assessment of mouse xenografts (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype control. Fig. 12C Kaplan-Meier survival analysis showing the survival probability of tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40

[0029] Fig.13A A schematic representation of the experimental design of a study examining preclinical activity in mice is shown. Fig. 13BTumor-bearing mice (MDA-MB-231 treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40) are shown. HER2 / 低 Kaplan-Meier survival analysis of the survival probability of patients with and without tumors. Fig. 13C Shown are HER2 expression levels in MDA-MB-231 cells, as measured by flow cytometry, after surface staining with an isotype control antibody. Fig.13D Shown are HER2 expression levels in MDA-MB-231 cells after surface staining with HER2 antibody, as measured by flow cytometry.

[0030] Fig.14A A schematic representation of the experimental design of a study examining preclinical activity in mice is shown.

[0031] Fig. 14B Shown are quantitative results of tumor burden assessment (bioluminescence assessment) of mouse xenografts (BT-474 (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to PBS and isotype control.

[0032] Fig. 14C Shown is a Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40.

[0033] Fig.15A Shown is a schematic representation of the experimental design for a study examining long-term transgene expression following single or double (re-dosing) administration of AAV.Trastuzumab in the MDA-MB-453 (ER- / PR- / HER2+) xenograft model.

[0034] Fig. 15B Shown are trastuzumab expression levels measured by ELISA using serum samples collected on day 98, plotted as μg / mL of trastuzumab in the MDA-MB-453 (ER- / PR- / HER2+) xenograft model.

[0035] Fig. 15C Shown are trastuzumab expression levels measured by ELISA using brain samples collected on day 98 (perfused brain homogenate), plotted as μg / mL of trastuzumab in the MDA-MB-453 (ER- / PR- / HER2+) xenograft model.

[0036] Fig.16 The expression level of trastuzumab in serum after administration of AAVhu95M199.CB7.CI.IL2_Trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Trastuzumab-coGW.SV40 into mice is shown.

[0037] Fig.17 Shown is the trastuzumab expression level in the brain (perfused) after administration of AAVhu95M199.CB7.CI.IL2_Trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Trastuzumab-coGW.SV40 in mice.

[0038] Fig.18A A schematic diagram of the experimental design for the study evaluating tumor burden is shown. Briefly, guide screws were implanted seven days before cell implantation (D-7) and MDA-MB-453 cells (2.5x10 5 cells / mouse), and on day 3 after cell implantation, mice were given 1x10 11 GC / mice were administered rAAV.Trastuzumab intracranially (ICV) and imaged at weeks 2, 4, and 6 after cell implantation.

[0039] Fig.18B Results of tumor burden assessments are shown, plotted as total flux (photons / second) at 2, 4, and 6 weeks post tumor cell implantation in mice administered AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. These results demonstrate the therapeutic efficacy of the rAAV.Trastuzumab vector.

[0040] Fig.18CThe Kaplan-Meier survival curves of mice after administration of AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 are shown.

[0041] Fig.19A A schematic diagram of the experimental design for the study evaluating antitumor activity is shown. Briefly, guide screws were implanted seven days before cell implantation (D-7), and E2 pellets (1.5×10 5 cells / mouse), and on day 3 after cell implantation, mice were given 1x10 11 GC / mice were administered rAAV.Trastuzumab intracranially (ICV) and imaged at week 2 after cell implantation.

[0042] Fig.19B Shown are the results of tumor burden assessment, which are plotted as total flux (photons / second) at 2, 4, and 6 weeks after tumor cell implantation in mice administered AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.

[0043] Fig.19C Shown are Kaplan-Meier survival curves of mice (with BT474 xenografts) after administration of AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.

[0044] Fig. 20 The results of the vector titration study are shown, which are plotted as measured by ELISA and mass spectrometry at 1×10 11 GC / mouse and 1x10 10GC / mouse. Trastuzumab expression levels measured in mice administered rAAV.Trastuzumab.

[0045] Fig.21A A schematic diagram of the experimental design for studies evaluating tumor challenge is shown.

[0046] Fig. 21B Tumor growth is shown, which is plotted as 1x10 11 Total flux (photons / second) measured at 2, 4, and 5 weeks after tumor implantation in mice dosed with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at GC / mouse.

[0047] Fig.22A A schematic diagram of the experimental design for studies evaluating tumor challenge is shown. Fig. 22B Tumor growth is shown, which is plotted as 1x10 10 Total flux (photons / second) measured at 2, 4, and 5 weeks after tumor implantation in mice dosed with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at GC / mouse.

[0048] Fig.23A The results of tumor burden assessment as examined by imaging are shown, and the results are plotted as 1x10 10 GC / mouse and 1x10 11 Total flux (photons / second) in mice administered with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at doses of GC / mouse. Fig. 23B The results of Kaplan-Meier survival analysis are shown, and the results are plotted as 1x10 10 GC / mouse and 1x10 11 The survival probability of mice administered with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at a dose of GC / mouse.

[0049] Fig.24AShown are trastuzumab expression levels in serum after administration of rAAV.trastuzumab with and without IVIG pretreatment. Fig. 24B Shown are trastuzumab expression levels in the brain (perfused) at day 30 after administration of rAAV.trastuzumab with and without IVIG pretreatment.

[0050] Fig.25A Shown are trastuzumab expression levels in CSF as measured by ELISA on day 14 at days 0, 3, 7, 14, 21, and 36-37 following rAAV administration to NHPs of cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)), cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40), and negative controls. Fig.25B Shown are the levels of anti-drug antibodies as measured in CSF samples collected from NHPs at days 0, 3, 7, 14, 21, and 36-37 after administration of rAAV (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0051] Fig.26A Shown are trastuzumab expression levels in serum as measured by ELISA on days 0, 3, 7, 14, 21, and 36-37 after rAAV administration to NHPs of Cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)), Cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0052] Fig.26B Shown are the levels of anti-drug antibodies as measured in serum samples collected from NHPs at days 0, 3, 7, 14, 21, and 36-37 after administration of rAAV (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0053] Fig. 27 Quantification of trastuzumab protein in NHP brain tissue after ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0054] Fig.28A Quantification of trastuzumab protein in NHP spinal cord after ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0055] Fig.28B Quantification of trastuzumab protein in NHP dorsal root ganglia (DRG) after ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0056] Figures 29A to 29D The average number of reads by 10XGENOMICS (final sequencing depth - 883x10 6 Results of trastuzumab mRNA detection in NHP brain tissue (cerebellum and occipital cortex) using 100 reads / sample (100 reads / sample). No background detection of trastuzumab was observed in untreated monkeys. Fig.29A Shown are the results of trastuzumab mRNA detection in the cerebellum of NHP 18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.29B Shown are the results of trastuzumab mRNA detection in the cerebellum of NHP 20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.29C Shown are the results of trastuzumab mRNA detection in the occipital cortex of NHP 18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.29DShown are the results of trastuzumab mRNA detection in the occipital cortex of NHP 20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.

[0057] Fig. 30A and 30B The biodistribution of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) in various tissues of NHPs after ICM administration is shown. Fig. 30A Shown is the DNA biodistribution of AAV.Trastuzumab in various tissues of NHPs after ICM administration. Fig. 30B Shown is the RNA biodistribution of AAV.Trastuzumab in various tissues of NHPs after ICM administration.

[0058] Figures 31A-31H It was shown that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Fig.31A A schematic diagram of the AAV vector genome is shown. Fig.31B Figure 2 shows ICV delivery of trastuzumab-encoding AAV9 vector (1×10 11 Schematic diagram of the assessment of transgene expression in vivo after GC / mice). Fig.31C The results show that the AAV9.UbC.Trastuzumab vector (1×10 11 Longitudinal quantification of trastuzumab by ELISA in the sera of Rag1 KO mice treated ICV with either GC / mouse; n=10) or PBS (n=10). Fig.31D The results show that the AAV9.UbC.Trastuzumab vector (1×10 11 Longitudinal quantification of trastuzumab by ELISA in perfused brain tissue of Rag1 KO mice treated ICV with either GC / mouse; n=10) or PBS (n=10). Fig.31E shows DNA biodistribution, and Fig.31F Shown are RNA biodistribution analyses by qPCR of brain and liver tissues of Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab or PBS. Figure 31GWestern blot analysis of trastuzumab heavy and light chains in brain lysates of Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab (n=4) or PBS (n=2) is shown. Purified trastuzumab (10 ng / lane) and β-actin were used as positive and loading controls, respectively. Fig.31H Shown are brain homogenates of Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab (n=4) or PBS (n=2) to confirm the identity of trastuzumab by LC-MS. Data are shown as individual data points and mean±SEM.

[0059] Figures 32A to 32C The results show that the CNS transduction efficiency of AAV9.UbC.trastuzumab is not affected by pretreatment with intravenous immunoglobulin (IVIg) containing broadly neutralizing antibodies against AAV9. Adult female Rag1 KO mice were given ICV AAV9.UbC.trastuzumab (1×10 11 GC / mice; n=5) were pretreated with IVIg or control serum from C57BL6 / J donor mice 2 hours before. Mice treated with PBS ICV (n=3) were used as controls. Fig.32A shows the levels of trastuzumab in serum over time, and Fig.32B Shown are trastuzumab levels in perfused brain tissue (day 30) of ICV-treated mice measured by ELISA. Fig.32C Shown is DNA biodistribution analysis of AAV9.UbC.Trastuzumab vectors in brain, liver, and heart 30 days after vector administration by qPCR. Data are shown as individual data points and mean ± SEM. ***p≤0.0001; ns, not significant.

[0060] Figures 33A to 33E The results show that a single ICV administration of AAV9.UbC.trastuzumab resulted in tumor regression in a xenograft mouse model of HER2+ breast to brain metastases. Adult female Rag1 KO mice were injected ICV with 1×10 11 GC / animals AAV9.UbC.Trastuzumab or AAV9.Isotype control. Fig.33A Quantification of total photon flux by bioluminescent in vivo imaging system (IVIS) is shown, and Fig.33BShown are Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from the BT-474 cell line and treated ICV with AAV vectors (AAV9.Isotype control: n=9; or AAV9.UbC.Trastuzumab: n=12). Fig.33C Shown are representative bioluminescent IVIS images (week 4, left panel), total photon flux signal quantification (right panel), and Fig.33D Shown are Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from the MDA-MB-453 cell line after ICV treatment (AAV9.Isotype control: n=8; or AAV9.UbC.Trastuzumab: n=7). Fig.33E Shows Fig.33C and Fig.33D Long-term trastuzumab expression quantified by ELISA in perfused brain tissue at day 104 after ICV administration of AAV9.UbC.trastuzumab in a subset of mice shown (n=5). ***p<0.0001

[0061] Figures 34A to 34E Shown is the detection of trastuzumab in the CSF of female NHPs following intracisternal (ICM) administration of an AAV9 vector encoding trastuzumab. Fig.34A Experimental ICM delivery of 3 × 10 13 Schematic design of AAV9.UbC.Trastuzumab (n=2) vectors for GC / animal to inform the schedule of blood and CSF collection in life and the planned end of study time point. NHP#3ICM was injected with 3x10 13 GC and used as negative control. Fig.34D ) and serum ( Fig.34E ) and anti-drug antibodies against trastuzumab were quantified by ELISA, and the CSF ( Fig.34B ) and serum ( Fig.34C Detection of trastuzumab (a humanized IgG1 antibody) in patients with

[0062] Fig.35A , 35B and 36A to 36D show the use of 3×10 13 Whole CNS gene delivery and transgene transcription after ICM treatment of GC / animals with AAV9.UbC.Trastuzumab. Fig.35A Shown are absolute quantification of AAV vector GC in NHPs at days 36-37 after ICM administration of AAV9.UbC.Trastuzumab (n=2) vectors. Fig.35BShown are transcripts in selected tissues in NHPs by qPCR at days 36-37 after ICM administration of AAV9.UbC.Trastuzumab (n=2) vectors.

[0063] Figures 36A to 36D Shown are the results of single-cell nuclear RNA sequencing showing the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex of animals injected ICM with AAV9.UbC.trastuzumab vector.

[0064] Figures 37A to 37F Shown, 3×10 trastuzumab was delivered by ICM using AAV9.UbC. 13 GC / animals resulted in expression of transgenic protein in NHP CNS tissue at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. The experimental protocol is shown in Fig.34A middle. Fig.37A Results of LC-MS analysis for detection of a unique AAV-encoded trastuzumab-derived peptide (DTYIHWVR; SEQ ID NO: 56) in NHP brain homogenates are shown. Trastuzumab levels are presented as fold change relative to the input reference positive control (10 ng trastuzumab). Fig.37B Female Rag1 KO mice were shown to have received ICV administration of 1 × 10 10 Trastuzumab expression in perfused brain tissue 28 days after AAV9.isotype control (n=8) or AAV9.UbC.trastuzumab (n=8) in GC / animals as measured by ELISA. Fig.37C Trastuzumab protein expression in perfused brain regions was shown by determining tissue levels of human IgG by ELISA. Fig.37D Figure 3 shows the intracranial tumor cells implanted with reporter gene MDA MB 453 breast cancer cells, using 1×10 10 Quantification of total photon flux by IVIS imaging of adult female Rag1 KO mice ICV treated with AAV9.isotype control (n=8) or AAV9.UbC.trastuzumab (n=8) GC / animal. Fig.37E Shown is the expression of trastuzumab protein in the spinal cord, quantified by determining tissue levels of human IgG by ELISA. Fig.37F Figure 3 shows the intracranial tumor cells implanted with reporter gene MDA MB 453 breast cancer cells, using 1×10 10 Kaplan-Meier survival curves of adult female Rag1 KO mice ICV treated with AAV9.isotype control (n=8) or AAV9.UbC.trastuzumab (n=8) in GC / animal. ***p<0.0001 DETAILED DESCRIPTION

[0065] Provided herein is a recombinant replication-deficient adeno-associated virus (rAAV) vector having an AAV capsid and a vector genome packaged therein, the vector genome comprising at least one open reading frame, the at least one open reading frame comprising an engineered nucleic acid sequence encoding a heavy chain and a light chain of an anti-HER2 antibody (e.g., an anti-tumor immunoglobulin construct, trastuzumab), and a composition containing the rAAV vector, which is suitable for administration to treat anti-Her2 cancers including metastatic cancers. Provided are various methods of administration, including systemic administration, direct administration to primary tumors to secondary tumors (metastatic cancers) (e.g., intratumoral) and administration to the central nervous system (e.g., intrathecal administration), for the treatment of Her2-positive metastatic cancers, etc. Also provided are pharmaceutical compositions, formulations containing the pharmaceutical compositions, and particularly liquid aqueous suspensions. Also provided are the uses of these compositions. Also provided are methods of compositions useful for treating and / or preventing HER2-positive (HER2-human epidermal growth factor receptor 2; also referred to as Her2-positive, HER2+ or Her2+) metastatic cancers (e.g., brain cancer).

[0066] In certain embodiments, compositions and protocols described herein can be used to deliver anti-tumor immunoglobulin constructs to the central nervous system. Compositions comprising AAV-Ig described herein are very suitable for central nervous system (CNS) HER2-positive cancers (tumors), and particularly cancers located in the brain. The compositions and protocols can also be used to treat primary and / or secondary HER2-positive breast cancers, primary and / or secondary HER2-positive gastric cancers and / or primary and / or secondary HER2-positive gastric-gastroesophageal junction cancers and other HER-2-positive solid tumors and cancers.

[0067] As used herein, the term "CNS tumor" includes primary or metastatic cancers, which may be located in the brain (intracranial), meninges (connective tissue layer covering the brain and spinal cord) or spinal cord. Examples of primary CNS cancers may be gliomas (which may include glioblastomas (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas and epithelioma, and mixed gliomas), meningiomas, medulloblastomas, neuromas and primary CNS lymphomas (located in the brain, spinal cord or meninges), etc. Examples of metastatic cancers include metastatic cancers originating from other tissues or organs, such as breast cancer, lung cancer, lymphoma, leukemia, melanoma (skin cancer), colon cancer, kidney cancer, prostate cancer or other types that transfer to the brain.

[0068] As used herein, "anti-tumor" immunoglobulin constructs (including antibodies or antibody fragments as defined herein) encode polypeptide-based portions that bind to cell surface antigens or receptors located on cancer cells or solid tumors, and inhibit or prevent the growth and spread of malignant cells in tumors or non-solid tumors, and optionally reduce the size of tumors. Anti-tumor immunoglobulin polypeptides can exert their effects through a variety of mechanisms, such as inhibiting tumor cell growth by blocking growth factor receptors, cross-linking cell membrane antigens to deliver signals that control the cell cycle, blocking angiogenesis, blocking DNA repair after chemotherapy, or even inducing cell death. Alternatively, the anti-tumor immunoglobulin polypeptides can indirectly affect tumor growth by activating host immune effector functions (such as antibody-dependent and complement-mediated cytotoxicity). In one embodiment, the anti-tumor effects of the compositions and regimens described herein can be measured by the reduction of tumor size and / or by the improvement of progression-free survival rate compared to subjects not treated with or treated with other regimens.

[0069] As used herein, the term "immunoglobulin" includes antibodies, functional fragments thereof, and immunoadhesins. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, camelidized single domain antibodies, intracellular antibodies ("intrabodies"), recombinant antibodies, multispecific antibodies, antibody fragments, such as Fv, Fab, F(ab)2, F(ab)3, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibody (scFv), tandem / double scFv, Fc, pFc', scFvFc (or scFv-Fc), disulfide bonded Fv (dsfv), bispecific antibodies (bc-scFv), such as BiTE antibodies; camelid antibodies, surface remodeled antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as ), chimeric antibodies, chimeric antibodies comprising at least one human constant region, etc. "Antibody fragment" refers to at least a portion of an immunoglobulin variable region that binds to its target (eg, tumor cell, tumor cell receptor).

[0070] The rAAV vectors as described herein encode anti-HER2 antibody (e.g., trastuzumab) heavy and light chains. See, e.g., drugbank.ca / drugs / DB00072. The encoded trastuzumab heavy chain amino acid sequence is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 35; drugbank.ca / drugs / DB00072).In certain embodiments, the encoded trastuzumab heavy chain amino acid sequence is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 4; ie, without the terminal lysine (K)).

[0071] Trastuzumab light chain is a recombinant IgG1κ humanized monoclonal antibody that selectively binds to the extracellular domain of human epidermal growth factor receptor protein with high affinity (K d =5nM). The encoded amino acid sequence of the trastuzumab light chain is:

[0072] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:36 (drugbank_ca / drugs / DB00072), also SEQ ID NO:6)), which is incorporated herein by reference. See also 212-Pb-TCMC-trastuzumab [Areva Med, Bethesda, MD]. As described herein, a series of novel rAAV. anti-HER2-antibodies (rAAV. trastuzumab or AAV. trastuzumab) constructs have been developed that have demonstrated high yields, expression levels, and / or activity. In certain embodiments, the size of the antibody (protein) chains can be confirmed by Western blotting. In certain embodiments, the size of the antibody (protein) chains can be confirmed by mass spectrometry.

[0073] Provided herein are coding sequences, expression cassettes, and vector genomes encoding the trastuzumab heavy chain (HC) amino acid sequence of SEQ ID NO: 4 (amino acids 1 to 449 of SEQ ID NO: 35) and the light chain (LC) amino acid sequence of SEQ ID NO: 6 (also SEQ ID NO: 36), each of which is engineered to further have an exogenous leader sequence (also referred to as a "leader peptide" or "signal peptide" or "signal sequence") for each heavy chain and light chain (e.g., SEQ ID NO: 14 for a heavy chain amino acid sequence comprising a leader peptide, SEQ ID NO: 16 for a light chain amino acid sequence comprising a leader peptide). In certain constructs described herein, the leader sequence is derived from a human interleukin 2 (IL2 or IL-2) leader sequence. In certain embodiments, the IL2 of each heavy chain and light chain is the same. In certain embodiments, the IL2 is a modified IL2 of at least one of the heavy chain and light chain. Further, in certain constructs described in the working examples, the heavy chain and the light chain are separated by a T2A linker, which may result in one or more additional amino acids being added to the heavy chain [SEQ ID NO: 4 or SEQ ID NO: 14 (including a leader peptide)]. Further, in certain constructs described in the working examples, the heavy chain and the light chain are separated by a furin / T2A linker, which may result in one or more additional amino acids being added to the heavy chain [SEQ ID NO: 4 or SEQ ID NO: 14 (including a leader peptide)]. In one embodiment, a single arginine [R] is added to the heavy chain. However, in certain embodiments, another linker and / or a different system may be selected that may result in no additional amino acids or one or more additional amino acids [e.g., R, Lys (K), RK, RKR, RKRR (SEQ ID NO: 49), etc.]. In the construct encoding the anti-Her2 antibody trastuzumab product, the various names after the term trastuzumab, such as trastuzumab-coGW, trastuzumab-coX, trastuzumab-coGY, trastuzumab, refer to different nucleic acid coding sequences of the open reading frames of the heavy and light chains of the anti-Her2 antibody. It should be understood that after cleavage of the leader peptide and assembly of the anti-Her2 light and heavy chains (optionally, with mutant amino acids), the resulting anti-Her2 antibody product may be alternatively referred to herein as "anti-Her2 antibody" or trastuzumab, but may contain variant or mutant amino acid sequences compared to the amino acid sequences of SEQ ID NO: 35 (drugbank.ca / drugs / DB00072; trastuzumab heavy chain) and SEQ ID NO: 36 (drugbank_ca / drugs / DB00072; trastuzumab light chain).In certain embodiments, the anti-Her2 antibody may comprise one or more conservative, non-conservative amino acid substitutions, and insertions, truncations and / or deletions compared to the amino acid sequences of SEQ ID NO: 35 (drugbank.ca / drugs / DB00072; trastuzumab heavy chain) and SEQ ID NO: 36 (drugbank_ca / drugs / DB00072; trastuzumab light chain). In certain embodiments, the amino acid sequence of the anti-Her2 antibody comprises a truncation of a terminal lysine (K) at the C-terminus of the amino acid sequence compared to SEQ ID NO: 35.

[0074] In certain embodiments, the encoded amino acid sequence of trastuzumab has 724 amino acids, including trastuzumab heavy chain and light chain, separated by additional amino acids due to joints. For example, although each expression cassette in the expression cassette described herein encodes the same trastuzumab heavy chain and light chain, in one embodiment, an amino acid can be added in the last position of the heavy chain. In still other embodiments, two, three, four or more additional amino acids may be connected to the heavy chain. For example, in certain embodiments, the nucleic acid sequence encoding the trastuzumab heavy chain and light chain is separated by self-cleaving furin / T2A joints. A furin recognition site consisting of an arginine-lysine-arginine-arginine amino acid sequence can be used. Due to the furin-mediated cleavage mechanism, the trastuzumab expressed by the vector can contain an additional arginine (R) residue added to the last position of the heavy chain [SEQ ID NO:4 or SEQ ID NO:14 (comprising a leader peptide)]. In other embodiments, the trastuzumab expressed by the vector is modified to contain the dipeptide arginine-lysine at the heavy chain end, the tripeptide arginine-lysine-arginine at the heavy chain end or the polypeptide arginine-lysine-arginine-arginine at the heavy chain end. In certain embodiments, the trastuzumab immunoglobulin expressed by the vector is a heterogeneous mixture of two or more immunoglobulin products in these immunoglobulin products. Other furin protease cleavage sites (arginine-XX-arginine, or arginine-X-lysine or arginine-arginine) that can also generate C-terminal heterogeneity can be used. In other words, the trastuzumab immunoglobulin expressed by other vectors can be a heterogeneous population of immunoglobulins, wherein due to the C-terminal of the joint processing heavy chain, there are 0,1,2,3 or 4 amino acids.

[0075] In addition, the light chain and the heavy chain each contain a heterologous leader peptide that directs each of the immunoglobulin chains to the appropriate cellular compartment where the leader peptide is processed by the host cell machinery to leave the mature immunoglobulin chain, for example, the HC with the leader peptide comprises the amino acid sequence of SEQ ID NO: 14, and the LC with the leader peptide comprises the amino acid sequence of SEQ ID NO: 16 and allows the two chains to self-assemble into a recombinant anti-Her antibody in vivo.

[0076] In certain embodiments, in vivo self-assembly into recombinant trastuzumab antibodies can be confirmed by native gel electrophoresis (e.g., polyacrylamide gel electrophoresis (native PAGE)) and denaturing gel electrophoresis (i.e., SDS-PAGE). In certain embodiments, trastuzumab does not comprise HC or LC leader peptide sequences (HC amino acids of SEQ ID NO: 4 and LC amino acid sequences of SEQ ID NO: 6).

[0077] As used herein, the term "trastuzumab" refers to an immunoglobulin construct ("anti-Her2 antibody") comprising a heavy chain having an amino acid sequence of SEQ ID NO: 4, or a sequence that is about 95% to about 100% identical thereto, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.9% identical thereto, and values ​​therebetween, as determined by contiguous amino acid sequence (e.g., due to linker processing with -1, 0, 1, 2, 3 or 4 amino acids at the C-terminus of the heavy chain), and a light chain having SEQ ID NO: 4. NO:6 amino acid sequence or about 99% to about 100% identical with it, or at least 99.9% identical with it and the sequence of the value therebetween (for example, due to joint processing), as determined by continuous amino acid sequence, the continuous amino acid sequence provides at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or similar and / or identical or greater than 100% biological activity (for example, binding) or function of trastuzumab immunoglobulin product. In certain embodiments, greater than 100% of the in vitro produced (for example, in CHO cells) trastuzumab activity and / or function is achieved, for example, about 105%, about 110%, about 115%, about 120%, about 125%, about 130% or greater. This biological activity or function can be determined by any suitable method, for example, in vitro assays, animal models or by monitoring patients after treatment. In certain embodiments, AAV-expressed trastuzumab immunoglobulin heavy and light chains provide advantages over protein-based trastuzumab compositions derived from in vitro produced trastuzumab, including, for example, improved glycosylation, reduced deamidation, and / or improved stability.

[0078] In one embodiment, the engineered coding sequences for the heavy and light chains of trastuzumab are provided in SEQ ID NO: 17. In one embodiment, the engineered coding sequences for the heavy and light chains of trastuzumab are provided in SEQ ID NO: 29. In certain embodiments, provided herein is at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence at least 95% identical to SEQ ID NO: 3, the nucleic acid sequence encoding the trastuzumab heavy chain; a linker; a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: 5, the nucleic acid sequence encoding the trastuzumab light chain. In certain embodiments, provided herein is at least one ORF, the at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3, the nucleic acid sequence encoding a trastuzumab heavy chain; a linker; a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5, the nucleic acid sequence encoding a trastuzumab light chain. In certain embodiments, the linker between the heavy chain and the light chain is a Thosea asigna virus (T2A) linker. In certain embodiments, the T2A linker comprises a nucleic acid sequence of SEQ ID NO: 32 or a sequence at least 95% identical to SEQ ID NO: 32. In certain embodiments, the T2A linker comprises a nucleic acid sequence of SEQ ID NO: 32 or a sequence at least 95% identical to SEQ ID NO: 32, the nucleic acid sequence or the sequence encoding an amino acid sequence of SEQ ID NO: 52. In certain embodiments, the linker further comprises a furin cleavage at the 5' end of T2A, optionally connected by a flexible linker (e.g., a "GSG linker"). In certain embodiments, the furin cleavage sequence comprises a nucleic acid sequence of SEQ ID NO:31, or a sequence at least 95% identical to SEQ ID NO:31. In certain embodiments, the furin / T2A linker comprises a nucleic acid sequence of SEQ ID NO:33, or a sequence at least 95% identical to SEQ ID NO:33. In certain embodiments, the furin / T2A linker comprises a nucleic acid sequence of SEQ ID NO:33, or a sequence at least 95% identical to SEQ ID NO:33, which encodes an amino acid sequence of SEQ ID NO:53.In certain embodiments, at least one ORF is provided herein, comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3, encoding a trastuzumab heavy chain; a linker comprising furin cleavage and T2A, wherein the furin cleavage site is located at the 5' end of T2A, optionally connected by a flexible linker (e.g., a "GSG linker"); a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5, encoding a trastuzumab light chain. In certain embodiments, the trastuzumab heavy chain has an amino acid sequence of SEQ ID NO: 4 containing a leader sequence. In other embodiments, the trastuzumab light chain has an amino acid sequence of SEQ ID NO: 6 containing a leader sequence. For example, the leader sequence can be about 15 to about 25 amino acids, preferably about 20 amino acids. In certain embodiments, the processing of the heavy and light chains of trastuzumab is guided by a leader peptide derived from a human IL2 protein. In one embodiment, the leader sequence is an interleukin (IL) IL-2 leader sequence, which can be a wild-type human IL2, MYRMQLLSCIALSLALVTNS [SEQ ID NO: 8] or a mutated leader sequence, such as MYRMQLLLLIALSLALVTNS [SEQ ID NO: 10] or MRMQLLLLIALSLALVTNS [SEQ ID NO: 12]. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO: 7 or a sequence at least 95% identical to SEQ ID NO: 7. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO: 7 or a sequence at least 95% identical to SEQ ID NO: 7, and the nucleic acid sequence or the sequence encodes SEQ ID NO: 8. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO: 9. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO: 9, or a sequence at least 95% identical to SEQ ID NO: 9, which encodes SEQ ID NO: 10. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO: 11, or a sequence at least 95% identical to SEQ ID NO: 11. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO: 11, or a sequence at least 95% identical to SEQ ID NO: 11, which encodes SEQ ID NO: 12. In certain embodiments, processing of the heavy and light chains of trastuzumab is directed by the same leader peptide.In certain embodiments, the processing of the heavy and light chains of trastuzumab is guided by different leader peptides. In certain embodiments, the heavy chain of trastuzumab is guided by a leader peptide comprising an amino acid sequence of SEQ ID NO: 8. In certain embodiments, the heavy chain of trastuzumab is guided by a leader peptide comprising an amino acid sequence of SEQ ID NO: 8, which is encoded by a nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the light chain of trastuzumab is guided by a leader peptide comprising an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the light chain of trastuzumab is guided by a leader peptide comprising an amino acid sequence of SEQ ID NO: 10, which is encoded by a nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the light chain of trastuzumab is guided by a leader peptide comprising an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the light chain of trastuzumab is guided by a leader peptide comprising an amino acid sequence of SEQ ID NO: 12, which is encoded by a nucleic acid sequence of SEQ ID NO: 11. Other leader sequences, or other leader sequences that are exogenous to the heavy and light chains, may also be used.

[0079] In certain embodiments, at least one ORF is provided herein, comprising: a nucleic acid sequence comprising SEQ ID NO:7, encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, encoding a trastuzumab heavy chain; a linker comprising a furin cleavage site (SEQ ID NO:31), a "GSG" linker, and T2A (SEQ ID NO:32), wherein the furin cleavage site is located at the 5' end of T2A; a nucleic acid sequence comprising SEQ ID NO:9, encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, encoding a trastuzumab light chain.

[0080] In certain embodiments, provided herein is at least one ORF, comprising: a nucleic acid sequence comprising SEQ ID NO:7, encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, encoding a trastuzumab heavy chain; a linker comprising a furin cleavage site (SEQ ID NO:31), a "GSG" linker, and T2A (SEQ ID NO:32), wherein the furin cleavage site is located at the 5' end of T2A; a nucleic acid sequence comprising SEQ ID NO:11, encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, encoding a trastuzumab light chain.

[0081] As used herein, the term "biological sample" refers to any cell, biological fluid or tissue. Samples suitable for use in the present invention may include, but are not limited to, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may be further diluted with saline, buffer, or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional methods.

[0082] As used herein, "patient" or "subject" means male or female humans, dogs, and animal models used for clinical research. In one embodiment, the subject of these methods and compositions is a person diagnosed with HER2 positive (HER2+) brain metastatic cancer, optionally wherein HER2+ brain metastatic cancer is breast cancer, optionally wherein HER2+ brain metastatic cancer is gastrointestinal cancer. In certain embodiments, the human subject of these methods and compositions is a fetus, a newborn, an infant, a toddler, a preschooler, a pupil, a teenager, a young adult, or an adult.

[0083] With respect to the description of these inventions, in another embodiment, it is contemplated that each of the compositions described herein is useful in the methods of the invention. In addition, it is also contemplated that in another embodiment, each of the compositions described herein as being useful in the methods is itself an embodiment of the invention.

[0084] Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs and with reference to the public text, which provides a general guide for many of the terms used in this application to those skilled in the art.

[0085] Open reading frames and nucleic acid molecules

[0086] On the one hand, an engineered nucleic acid sequence (also referred to as trastuzumab-coGW) encoding an anti-Her2 antibody (i.e., trastuzumab immunoglobulin) is provided. Trastuzumab immunoglobulin comprises a heavy chain and a light chain. In one embodiment, the engineered nucleic acid sequence can be used to improve production, transcription, expression or safety in a subject. In another embodiment, the engineered sequence can be used to increase the efficacy of the resulting therapeutic composition or treatment. In further embodiments, the engineered sequence can be used to increase the efficacy of the expressed trastuzumab immunoglobulin, but a lower dose of the therapeutic agent for delivering the immunoglobulin can also be allowed to increase safety. In certain embodiments, a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a trastuzumab immunoglobulin is provided herein. In certain embodiments, the recombinant nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 17 or a sequence identical to SEQ ID NO: 17 at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% and values ​​therebetween. In certain embodiments, the recombinant nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 29, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and values ​​therebetween that are identical to SEQ ID NO: 29. In certain embodiments, the recombinant nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 29.

[0087] In one embodiment, the engineered coding sequences of the heavy and light chains of trastuzumab are provided in SEQ ID NO: 17. In certain embodiments, the engineered coding sequences of the heavy and light chains of trastuzumab comprise a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and values ​​therebetween identical to SEQ ID NO: 17. In one embodiment, the engineered coding sequences of the heavy and light chains of trastuzumab are provided in SEQ ID NO: 29. In certain embodiments, the engineered coding sequences of the heavy and light chains of trastuzumab comprise a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and values ​​therebetween identical to SEQ ID NO: 29. In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3, the nucleic acid sequence encoding a trastuzumab heavy chain; a linker; a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5, the nucleic acid sequence encoding a trastuzumab light chain. In certain embodiments, the nucleic acid sequence encoding the trastuzumab heavy chain comprises SEQ ID NO: 3 or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% identical to SEQ ID NO: 3, and values ​​included therein. In certain embodiments, the nucleic acid sequence encoding the trastuzumab light chain comprises SEQ ID NO: 3 or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% identical to SEQ ID NO: 5. In certain embodiments, provided herein is at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3, encoding a trastuzumab heavy chain comprising the amino acid sequence of SEQ ID NO: 4; a linker; a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5, encoding a trastuzumab light chain comprising the amino acid sequence of SEQ ID NO: 6.In certain embodiments, provided herein is at least one ORF, comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, encoding a trastuzumab heavy chain comprising the amino acid sequence of SEQ ID NO:4; a linker; a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO:5, encoding a trastuzumab light chain comprising the amino acid sequence of SEQ ID NO:6.

[0088] In certain embodiments, the linker between the heavy chain and the light chain is a T2A linker. In certain embodiments, the T2A linker comprises a nucleic acid sequence of SEQ ID NO: 32 or a sequence at least 95% identical to SEQ ID NO: 32. In certain embodiments, the linker further comprises a furin cleavage at the 5' end of T2A, optionally connected by a flexible linker (e.g., a "GSG linker"). In certain embodiments, the furin cleavage sequence comprises a nucleic acid sequence of SEQ ID NO: 31 or a sequence at least 95% identical to SEQ ID NO: 31. In certain embodiments, the furin / T2A linker comprises a nucleic acid sequence of SEQ ID NO: 33 or a sequence at least 95% identical to SEQ ID NO: 33.

[0089] In certain embodiments, the leader peptide is an interleukin (IL) IL-2 leader peptide. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the heavy chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO:7. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the heavy chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO:7, which encodes the amino acid sequence of SEQ ID NO:8 as the leader peptide of IL2. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the heavy chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO:9. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the heavy chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO:9, which encodes the amino acid sequence of SEQ ID NO:10 as a modified leader peptide of IL2. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the heavy chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO:11. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the heavy chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12 as a modified IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the light chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the light chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO: 7, which encodes the amino acid sequence of SEQ ID NO: 8 as an IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the light chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the light chain of trastuzumab comprises the nucleic acid sequence of SEQ ID NO: 9, which encodes the amino acid sequence of SEQ ID NO: 10 as a modified IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12 as a modified IL2 leader peptide.

[0090] In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab immunoglobulin comprises an IL2 leader peptide, a trastuzumab heavy chain, optionally a furin cleavage sequence, a T2A linker, an IL2 leader peptide, optionally a modified IL2 leader peptide, and a trastuzumab light chain at 5' to 3'. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab immunoglobulin comprises an IL2 leader peptide, a trastuzumab heavy chain, a furin cleavage sequence, a T2A linker, an IL2 leader peptide, optionally a modified IL2 leader peptide, and a trastuzumab light chain at 5' to 3'.

[0091] In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab heavy chain comprising an IL2 leader peptide comprises SEQ ID NO: 13. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab heavy chain comprising an IL2 leader peptide comprises a sequence at least 95% identical to SEQ ID NO: 13. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab heavy chain comprising an IL2 leader peptide comprises SEQ ID NO: 13 encoding the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab light chain comprising a modified IL2 leader peptide comprises SEQ ID NO: 15. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab light chain comprising a modified IL2 leader peptide comprises a sequence at least 95% identical to SEQ ID NO: 15. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab light chain comprising a modified IL2 leader peptide comprises SEQ ID NO: 15 encoding the amino acid sequence of SEQ ID NO: 16.

[0092] In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab immunoglobulin comprises the nucleic acid sequence of SEQ ID NO: 17, wherein the sequence comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab immunoglobulin comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 17, wherein the sequence comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab immunoglobulin comprises the nucleic acid sequence of SEQ ID NO: 29, wherein the sequence comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a furin cleavage sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab immunoglobulin comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 29, wherein the sequence comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a furin cleavage sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence.

[0093] In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence of SEQ ID NO:7, encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, encoding a trastuzumab heavy chain; a nucleic acid sequence encoding a linker comprising a nucleic acid sequence of SEQ ID NO:31 encoding a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding T2A; a nucleic acid sequence of SEQ ID NO:7, encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, encoding a trastuzumab light chain.

[0094] In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence of SEQ ID NO:7, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:8 as a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4 as a trastuzumab heavy chain; a nucleic acid sequence encoding a linker comprising a nucleic acid sequence of SEQ ID NO:31 encoding the amino acid sequence of SEQ ID NO:49 as a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding the amino acid sequence of SEQ ID NO:51 as T2A; a nucleic acid sequence of SEQ ID NO:7, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:8 as a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:6 as a trastuzumab light chain.

[0095] In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence of SEQ ID NO:7, encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, encoding a trastuzumab heavy chain; a nucleic acid sequence encoding a linker comprising a nucleic acid sequence of SEQ ID NO:31 encoding a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding T2A; a nucleic acid sequence of SEQ ID NO:9, encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, encoding a trastuzumab light chain.

[0096] In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence of SEQ ID NO:7, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:8 as a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4 as a trastuzumab heavy chain; a nucleic acid sequence encoding a linker comprising a nucleic acid sequence of SEQ ID NO:31 encoding the amino acid sequence of SEQ ID NO:49 as a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding the amino acid sequence of SEQ ID NO:51 as T2A; a nucleic acid sequence of SEQ ID NO:9, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:10 as a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:6 as a trastuzumab light chain. The amino acid sequence of NO:6.

[0097] In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence of SEQ ID NO:7, encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, encoding a trastuzumab heavy chain; a nucleic acid sequence encoding a linker comprising a nucleic acid sequence of SEQ ID NO:31 encoding a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding T2A; a nucleic acid sequence of SEQ ID NO:11, encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, encoding a trastuzumab light chain.

[0098] In certain embodiments, provided herein is at least one open reading frame (ORF), the at least one ORF comprising: a nucleic acid sequence of SEQ ID NO:7, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:8 as a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4 as a trastuzumab heavy chain; a nucleic acid sequence encoding a linker comprising a nucleic acid sequence of SEQ ID NO:31 encoding the amino acid sequence of SEQ ID NO:49 as a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding the amino acid sequence of SEQ ID NO:51 as T2A; a nucleic acid sequence of SEQ ID NO:11, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:12 as a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:49 as a trastuzumab light chain. The amino acid sequence of NO:6.

[0099] As described herein, "nucleic acid" can be RNA, DNA or a modification thereof, and can be single-stranded or double-stranded, and can be selected from, for example, the following groups: nucleic acids encoding proteins of interest, oligonucleotides, nucleic acid analogs, such as peptide-nucleic acids (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acids (LNA), etc. Nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of nucleotides of any type (e.g., peptide nucleic acid oligomers). It will be understood by those skilled in the art that functional variants of these nucleic acid molecules are also intended to be part of the present invention. Functional variants are nucleic acid sequences that can be directly translated using the standard genetic code to provide an amino acid sequence identical to an amino acid sequence translated from a parent nucleic acid molecule. Such nucleic acid sequences include, but are not limited to, for example, in certain embodiments, miRNA is a dorsal root ganglion (drg)-specific miRNA target sequence. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three, or preferably at least four tandem repeats of a dorsal root ganglion (drg)-specific miRNA target sequence. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven or preferably at least eight tandem repeats of dorsal root ganglion (drg) specific miRNA. See, for example, PCT / US19 / 67872, filed on December 20, 2019 and now published as WO 2020 / 132455, which is incorporated herein by reference. See also International Patent Application No. PCT / US21 / 32003, filed on May 12, 2021 and now published WO2021 / 231579A1, which is incorporated herein by reference. See also U.S. Provisional Patent Application No. 63 / 279,561, filed on November 15, 2021, which is incorporated herein by reference in its entirety.

[0100] In certain embodiments, recombinant nucleic acid molecules encoding trastuzumab immunoglobulins and other constructs encompassed by the present invention and useful for generating expression cassettes and vector genomes can be engineered for expression in yeast cells, insect cells, or mammalian cells such as human cells. Methods are known and have been described previously (e.g., WO 96 / 09378). If at least one non-preferred codon is replaced by a more preferred codon compared to a wild-type (WT) sequence, the sequence is considered to be engineered. Herein, a non-preferred codon is a codon used less frequently than another codon encoding the same amino acid in an organism, and a more preferred codon is a codon used more frequently than a non-preferred codon in an organism. The frequency of codon use of a specific organism can be found in a codon frequency table, such as kazusa.jp / codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons are replaced by more preferred codons. Preferably, the most commonly used codons in an organism are used for engineered sequences. The replacement of preferred codons generally results in higher expression. It will also be appreciated by those skilled in the art that, due to the degeneracy of the genetic code, many different nucleic acid molecules can encode the same polypeptide. It should also be understood that the technician can use conventional techniques to replace the nucleotides that do not affect the amino acid sequence encoded by the nucleic acid molecule to reflect the codon usage of any specific host organism in which the polypeptide will be expressed. Therefore, unless otherwise indicated, "nucleic acid sequence encoding an amino acid sequence" includes degenerate versions of each other and all nucleotide sequences encoding the same amino acid sequence. Nucleic acid sequences can be cloned using conventional molecular biological techniques, or generated de novo by DNA synthesis, which can be performed by service companies (such as GeneArt, GenScript, Life Technologies, Eurofins) that have businesses in the field of DNA synthesis and / or molecular cloning using conventional procedures.

[0101] "Engineering" means that the nucleic acid sequence encoding the trastuzumab immunoglobulin described herein is assembled and placed in any suitable genetic element, such as naked DNA, bacteriophage, transposon, cosmid, episome, etc., and the trastuzumab immunoglobulin carried on it is transferred to a host cell, for example, for generating a non-viral delivery system (e.g., RNA-based system, naked DNA, etc.) or for generating a viral vector in a packaging host cell and / or for delivery to a host cell of a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods for preparing such engineered constructs are known to nucleic acid manipulation technicians and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0102] In the context of nucleic acid sequences, the terms "percentage (%) identity", "sequence identity", "percentage of sequence identity" or "percentage of identity" refer to residues in a subsequence of two sequences that are identical when aligned for correspondence, or identical or having a specified percentage of identical amino acid residues or nucleotides (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity within a specified region (e.g., any of the modified ORFs provided herein) when compared and aligned for maximum correspondence within a comparison window or specified region), as measured using the BLAST or BLAST2.0 sequence comparison algorithm using the default parameters described below or by manual alignment and visual inspection (see, e.g., NCBI website, etc.). It is expected that the length of the sequence identity comparison may exceed the full length of a genome, the full length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides. However, identity between smaller fragments may also be desired, for example at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides.

[0103] The percentage identity of the amino acid sequence over the full length of a protein, a polypeptide, about 32 amino acids, about 330 amino acids or a peptide fragment thereof or a corresponding nucleic acid sequence encoding sequence can be readily determined. The length of a suitable amino acid fragment can be at least about 8 amino acids and can be up to about 700 amino acids. Typically, when referring to "identity," "homology," or "similarity" between two different sequences, "identity," "homology," or "similarity" is determined with reference to an "alignment" sequence. An "alignment" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence.

[0104] Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as "Clustal X", "Clustal Omega", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used with the default settings, although those skilled in the art can change these settings as needed. Alternatively, one skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as provided by the reference algorithm and program. See, for example, J.D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13): 2682-2690 (1999).

[0105] A number of sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "Clustal Omega", "CAP Sequence Assembly", "BLAST", "MAP" and "MEME", which can be accessed through Web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility program has also been used. Many algorithms known in the art can be used to measure nucleotide sequence identity, including those contained in the above programs. As another example, the GCG 6.1 version of the program FastA TM Compare polynucleotide sequences. Fasta TMThe alignment and percent sequence identity of the best overlap region between the query sequence and the search sequence are provided. For example, the percent sequence identity between nucleic acid sequences can be calculated using Fasta as provided in GCG version 6.1 with its default parameters (font size 6 and NOPAM coefficients for the scoring matrix). TM Where determined, such procedures are incorporated herein by reference.

[0106] It should be understood that the compositions of trastuzumab immunoglobulins and trastuzumab immunoglobulin coding sequences described herein are intended to be applicable to other compositions, regimens, aspects, embodiments and methods described across this specification.

[0107] Expression cassette

[0108] A gene therapy vector is provided herein, comprising an expression cassette, the expression cassette comprising an engineered nucleic acid sequence, the engineered nucleic acid sequence comprising an anti-Her2 antibody (i.e., trastuzumab immunoglobulin) coding sequence, the anti-Her2 antibody comprising a heavy chain and a light chain operably connected to a regulatory sequence directing its expression. A recombinant nucleic acid molecule is also provided herein, comprising an expression cassette, the expression cassette comprising a nucleic acid sequence encoding a trastuzumab immunoglobulin as described herein. A recombinant nucleic acid molecule is also provided herein, the recombinant nucleic acid molecule comprising a vector genome, the vector genome comprising a nucleic acid sequence encoding a trastuzumab immunoglobulin as described herein.

[0109] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably connected thereto, the regulatory sequence guiding or regulating the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, "operably connected" sequences include both regulatory sequences adjacent or not adjacent to the nucleic acid sequence and regulatory sequences acting in cis or trans with the nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette can contain regulatory sequences upstream of the gene sequence (5'), such as one or more of a promoter, an enhancer, an intron, etc., and an enhancer, or one or more of the regulatory sequences downstream of the gene sequence (3'), such as a 3' untranslated region ('3UTR) comprising a polyadenylation site, and other elements. In certain embodiments, the regulatory sequence is operably connected to the nucleic acid sequence of the gene product, wherein the regulatory sequence is separated from the nucleic acid sequence of the gene product by the inserted nucleic acid sequence, i.e., 5'-untranslated region (5'UTR). In certain embodiments, the expression cassette comprises the nucleic acid sequence of one or more gene products. In certain embodiments, the expression cassette can be a monocistronic expression cassette or a bicistronic expression cassette. In other embodiments, the term "transgenic" refers to one or more DNA sequences from an exogenous source inserted into a target cell. Typically, this expression cassette for generating a viral vector contains the coding sequence of the gene product described herein, and the coding sequence is flanked by packaging signals and other expression control sequences of the viral genome, such as sequences described herein. In certain embodiments, the vector genome can contain two or more expression cassettes.

[0110] The term "exogenous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally exist in its location in a chromosome or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but it exists in a non-natural state, for example, a different copy number or under the control of a different regulatory element.

[0111] The term "heterologous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein originates from a different organism or a different species of the same organism than the host cell or subject in which the nucleic acid or protein is expressed. The term "heterologous" when used with reference to a protein or nucleic acid in a plasmid, expression cassette or vector indicates that the protein or nucleic acid is present with another sequence or subsequence that the protein or nucleic acid in question is not found in nature in the same relationship to each other.

[0112] In certain embodiments, the regulatory sequence comprises a promoter. In certain embodiments, the regulatory sequence comprises one or more introns, one or more enhancers, and a polyadenylation (polyA) signal sequence.

[0113] In one embodiment, the regulatory sequence comprises a promoter. In one embodiment, the promoter is a chicken β-actin (also referred to as chicken β-actin, CB or CBA) promoter. Various chicken β-actin promoters have been described alone or in combination with various enhancer elements (e.g., CB7 is a chicken β-actin promoter with a cytomegalovirus enhancer element; CAG promoter, which includes a promoter, the first exon and the first intron of chicken β-actin, and a splice acceptor of the rabbit β-globin gene); CBh promoter [SJ Gray et al., Human Gene Therapy (Hu Gene Ther), September 2011; 22 (9): 143-1 153]. In certain embodiments, the promoter is a cytomegalovirus (CMV) promoter. In certain embodiments, the CB promoter comprises the nucleic acid sequence of SEQ ID NO: 20.

[0114] In other embodiments, the promoter is a CB7 (also referred to as a hybrid CB7) promoter, the CB7 promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and a chicken β-actin promoter, optionally with a spacer sequence, optionally with a chimeric intron comprising a chicken β-actin intron and further comprising a chicken β-actin splice donor (including exon sequences, chicken β-actin introns) and a rabbit β-globin splice acceptor. In certain embodiments, the CMV IE enhancer comprises the nucleic acid sequence of SEQ ID NO: 19. In certain embodiments, the CB7 promoter comprises the nucleic acid sequence of SEQ ID NO: 21. See, e.g., cytomegalovirus (CMV) immediate early enhancer (260 bp, C4; GenBank#K03104.1). Chicken β-actin promoter (281 bp; CB; GenBank#X00182.1). In other embodiments, the promoter is an ubiquitin C (UbC) promoter. See, e.g., WO 2001 / 091800. See, e.g., GenBank Accession Nos. AF232305 (rat) and D63791 (human), respectively. In certain embodiments, the UbC promoter comprises the nucleic acid sequence of SEQ ID NO: 24. Still other promoters and / or enhancers may also be selected.In another embodiment, suitable promoters may include, but are not limited to, the following: elongation factor 1 alpha (EF1α) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23); synapsin 1 promoter (see, e.g., Kügler S et al., Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47); neuron-specific enolase (NSE) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23); synapsin 1 promoter (see, e.g., Kügler S et al., Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47); neuron-specific enolase (NSE) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23); J et al. Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb;145(2):613-9. Epub 2003 Oct 16); or the CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene. Mol Biotechnol. 2016 Jan;58(1):30-6. doi:10.1007 / s12033-015-9899-5).In still other embodiments, multiple enhancers and / or promoters may also be included.

[0115] In certain embodiments, additional or alternative promoter sequences may be included as part of the expression control sequence (regulatory sequence), for example positioned between the selected 5' ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters [see, e.g., WO 2011 / 126808 and WO 2013 / 04943], tissue-specific promoters or promoters responsive to physiological cues may be utilized in the vectors described herein. The promoter can be selected from different sources, such as human cytomegalovirus immediate-early (CMV IE) enhancer / promoter, SV40 early enhancer / promoter, JC polyoma virus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP) and chicken β-actin promoter.

[0116] In one embodiment, the expression cassette is designed to be expressed and secreted in a human subject. In one embodiment, the expression cassette is designed to be expressed in the central nervous system (CNS), including cerebrospinal fluid and brain. In other embodiments, the expression cassette can be used for expression in the CNS and systemically. Suitable promoters can be selected, including but not limited to constitutive promoters, tissue-specific promoters, or inducible / regulated promoters. An example of a constitutive promoter is the chicken beta-actin promoter. See also, CB7 above. Examples of tissue-specific promoters are well known for use in liver (albumin, Miyatake et al., (1997) Journal of Virology (J. Virol.), 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Therapy, 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Human Gene Therapy, 7:1503-14), neurons (such as neuron-specific enolase (NSE) promoter, Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15; neurofilament light chain gene, Piccioli et al., (1996) Human Gene Therapy, 7:1503-14), and neurons (such as neuron-specific enolase (NSE) promoter, Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15; neurofilament light chain gene, Piccioli et al., (1996) Human Gene Therapy, 7:1503-14). et al., (1991) Proc. Natl. Acad. Sci. USA, 88:56115; and neuron-specific vgf genes, Piccioli et al., (1995) Neuron, 15:373-84), and other tissues. Alternatively, a regulatable promoter can be selected. See, for example, WO 2011 / 126808B2, which is incorporated herein by reference.

[0117] In addition to the promoter, the vector may contain one or more other suitable transcription initiation sequences, transcription termination sequences, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, for example, WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product.

[0118] In one embodiment, the regulatory sequence further comprises an enhancer. In one embodiment, the regulatory sequence comprises an enhancer. In another embodiment, the regulatory sequence contains two or more expression enhancers. These enhancers may be identical or may be different. For example, an enhancer may include an α mic / bik enhancer or a CMV IE enhancer. Such an enhancer may be present in two copies positioned adjacent to each other. Alternatively, the double copies of the enhancer may be separated by one or more sequences.

[0119] In one embodiment, the regulatory sequence further comprises an intron. In another embodiment, the intron is a chicken beta-actin intron. In one embodiment, the intron is 875bp (GenBank#X00182.1). In one embodiment, the intron is a 973bp intron from a chicken beta-actin gene (GenBank#X00182.1). In certain embodiments, the chicken beta-actin intron comprises the nucleic acid sequence of SEQ ID NO:34. In certain embodiments, the intron is a chimeric intron (CI)-a hybrid intron consisting of a human beta-globin splice donor and an immunoglobulin G (IgG) splice acceptor element. In certain embodiments, the intron is a chimeric intron comprising a chicken beta-actin splice donor (including exon sequences), a chicken beta-actin intron, and a rabbit beta-globin splice acceptor. In certain embodiments, the intron is a chimeric intron comprising a chicken β-actin intron comprising the nucleic acid sequence of SEQ ID NO: 51. In another embodiment, the intron is a chimeric intron comprising a chicken β-actin intron comprising the nucleic acid sequence of SEQ ID NO: 51. In certain embodiments, the chimeric intron is a nucleic acid sequence comprising SEQ ID NO: 22. (Chimeric) introns. Other suitable introns include introns known in the art, which may be human β-globin introns and / or commercially available introns and introns described in WO 2011 / 126808.

[0120] In one embodiment, the regulatory sequence further comprises a polyadenylation signal (polyA). Examples of suitable polyA sequences include, for example, rabbit beta globin (RBG or rBG) poly A, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences can be selected to stabilize mRNA. In certain embodiments, polyA is rabbit beta globin poly A (rabbit beta globin polyA or rBG). See, for example, WO 2014 / 151341. In certain embodiments, human growth hormone (hGH) polyadenylation sequence, SV40 polyA, or synthetic polyA can be included in the expression cassette. In certain embodiments, SV40 polyA is selected. In certain embodiments, SV40 polyA comprises the nucleic acid sequence of SEQ ID NO:23.

[0121] In certain embodiments, the expression cassette comprises a trastuzumab coding sequence and may include other regulatory sequences thereof. The desired regulatory sequences are operably linked to the coding sequence in a manner that allows the trastuzumab coding sequence to be transcribed, translated and / or expressed in the target cell.

[0122] In certain embodiments, the target cell can be a central nervous system cell. In certain embodiments, the target cell is one or more of an excitatory neuron, an inhibitory neuron, a glial cell, a cortical cell, a frontal cortical cell, a cerebral cortical cell, a spinal cord cell. In certain embodiments, the target cell is the pia mater (LM) of the CNS. In certain embodiments, the target cell is the substance of the CNS.

[0123] In certain embodiments, the expression cassette comprises: (i) a promoter and / or promoter elements comprising an enhancer and a promoter, optionally with a spacer sequence therebetween, (ii) introns, (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3, encoding the trastuzumab heavy chain, (v) optionally a furin cleavage site, (vi) a T2A element linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO:5, encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence.

[0124] In certain embodiments, the expression cassette comprises: (i) a promoter and / or promoter elements comprising an enhancer and a promoter, optionally with a spacer sequence therebetween, (ii) introns, (iii) a Kozak sequence, (iv) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (v) a nucleic acid sequence comprising SEQ ID NO:3, encoding a trastuzumab heavy chain, (vi) a furin cleavage site, (vii) a T2A element linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (ix) a nucleic acid sequence comprising SEQ ID NO:5, encoding a trastuzumab light chain, and (x) an SV40 polyadenylation (polyA) sequence.

[0125] In certain embodiments, the expression cassette comprises: (i) a promoter element comprising a chicken β-actin promoter (CB) and an enhancer, (ii) an intron which is a chicken β-actin intron (CI), (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3, encoding the trastuzumab heavy chain, (v) a T2A linker element linker, (vi) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (vii) a nucleic acid sequence comprising SEQ ID NO:5, encoding the trastuzumab light chain, and (viii) an SV40 polyadenylation (polyA) sequence.

[0126] In certain embodiments, the expression cassette comprises: (i) a promoter element comprising a chicken β-actin promoter (CB) and an enhancer, (ii) an intron which is a chicken β-actin intron (CI), (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding the trastuzumab heavy chain, (v) a furin cleavage site, (vi) a T2A element linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO:5, the nucleic acid sequence encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence.

[0127] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter, the CB7 hybrid promoter comprising a CMV IE enhancer, a chicken β-actin promoter, and a chimeric intron, the chimeric intron comprising a chicken β-actin intron (CI), (ii) a Kozak sequence, (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence at least 95% identical to SEQ ID NO: 3, the nucleic acid sequence encoding a trastuzumab heavy chain, (v) optionally a furin cleavage site, (vi) a T2A element linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: NO:5 is at least 95% identical to the sequence encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence.

[0128] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter, the CB7 hybrid promoter comprising a CMV IE enhancer, a chicken β-actin promoter, and a chimeric intron, the chimeric intron comprising a chicken β-actin splice donor (including exon sequences) a chicken β-actin intron, and a rabbit b-globin splice acceptor, (ii) a Kozak sequence, (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence at least 95% identical to SEQ ID NO: 3, the nucleic acid sequence encoding a trastuzumab heavy chain, (v) a furin cleavage site, (vi) a T2A element linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: NO:5 is at least 95% identical to the sequence encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence.

[0129] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter comprising a nucleic acid sequence of SEQ ID NO:21, (ii) a Kozak sequence, (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide comprising a nucleic acid sequence of SEQ ID NO:7 operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding a trastuzumab heavy chain, (v) a furin cleavage site comprising a nucleic acid sequence of SEQ ID NO:31, (vi) a T2A element linker comprising a nucleic acid sequence of SEQ ID NO:32, (vii) a nucleic acid sequence encoding a leader peptide comprising a nucleic acid sequence of SEQ ID NO:9 operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO:5 NO:5 is at least 95% identical to a sequence encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence comprising the nucleic acid sequence of SEQ ID NO:23.

[0130] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter comprising a nucleic acid sequence of SEQ ID NO:21, (ii) a Kozak sequence, (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide comprising a nucleic acid sequence of SEQ ID NO:7 operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding a trastuzumab heavy chain, (v) a furin cleavage site followed by a T2A element linker (furin / T2A) connected by a "GSG" linker, comprising a nucleic acid sequence of SEQ ID NO:33, (vi) a nucleic acid sequence encoding a leader peptide comprising a nucleic acid sequence of SEQ ID NO:9 operably linked to a trastuzumab light chain, (vii) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO: NO:5 is at least 95% identical to a sequence encoding the trastuzumab light chain, and (vii) an SV40 polyadenylation (polyA) sequence comprising the nucleic acid sequence of SEQ ID NO:23.

[0131] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter, the CB7 hybrid promoter comprising a CMV IE enhancer and a chicken β-actin promoter and a chimeric intron, the chimeric intron comprising a chicken β-actin intron (CI), (ii) a Kozak sequence, (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding a trastuzumab heavy chain, (v) a furin cleavage site, (vi) a T2A element linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO: NO:5 is at least 95% identical to the sequence encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence.

[0132] In certain embodiments, the expression cassette comprises: (i) a promoter that is a ubiquitin C (UbC) promoter, (ii) an intron that is a chimeric intron comprising a Promega intron (PI), (iii) a Kozak sequence, (iv) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (v) a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence at least 95% identical to SEQ ID NO: 3, the nucleic acid sequence encoding the trastuzumab heavy chain, (vi) optionally a furin cleavage site, (vii) a T2A element linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (ix) a nucleic acid sequence comprising SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: 5, the nucleic acid sequence encoding the trastuzumab light chain, and (x) an SV40 polyadenylation (polyA) sequence

[0133] In certain embodiments, the expression cassette comprises: (i) a promoter that is a ubiquitin C (UbC) promoter, (ii) an intron that is a chimeric intron comprising a Promega intron (PI), (iii) a Kozak sequence, (iv) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (v) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding the trastuzumab heavy chain, (vi) a furin cleavage site, (vii) a T2A element linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (ix) a nucleic acid sequence comprising SEQ ID NO:5 or a sequence at least 95% identical to SEQ ID NO:5, the nucleic acid sequence encoding the trastuzumab light chain, and (x) an SV40 polyadenylation (polyA) sequence

[0134] In certain embodiments, the expression cassette comprises: (i) a promoter that is a ubiquitin C (UbC) promoter (SEQ ID NO:24), (ii) an intron that is a chimeric intron comprising a Promega intron (PI) (SEQ ID NO:22), (iii) a Kozak sequence, (iv) at least one ORF that comprises: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain (SEQ ID NO:7), (v) a nucleic acid sequence that comprises SEQ ID NO:3 or a sequence that is at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding a trastuzumab heavy chain, (vi) a furin cleavage site (SEQ ID NO:31), (vii) a T2A element linker (SEQ ID NO:32), (viii) a nucleic acid sequence that encodes a leader peptide operably linked to a trastuzumab light chain (SEQ ID NO:11), (ix) a nucleic acid sequence that comprises SEQ ID NO:5 or a sequence that is at least 95% identical to SEQ ID NO:3. NO:5 is at least 95% identical to a sequence encoding a trastuzumab light chain, and (x) an SV40 polyadenylation (polyA) sequence (SEQ ID NO:23).

[0135] In certain embodiments, the expression cassette comprises: (i) a promoter that is a ubiquitin C (UbC) promoter (SEQ ID NO:24), (ii) an intron that is a chimeric intron comprising a Promega intron (PI) (SEQ ID NO:22), (iii) a Kozak sequence, (iv) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain (SEQ ID NO:7), (v) a nucleic acid sequence comprising SEQ ID NO:3 or a sequence that is at least 95% identical to SEQ ID NO:3, the nucleic acid sequence encoding a trastuzumab heavy chain, (vi) a furin cleavage site that is linked to a T2A element linker (furin / T2A) via a "GSG" linker (SEQ ID NO:33), (vii) a nucleic acid sequence that encodes a leader peptide operably linked to a trastuzumab light chain (SEQ ID NO:11), (viii) a nucleic acid sequence that comprises SEQ ID NO: NO:5 or a sequence that is at least 95% identical to SEQ ID NO:5, said nucleic acid sequence encoding the trastuzumab light chain, and (ix) an SV40 polyadenylation (polyA) sequence (SEQ ID NO:23).

[0136] In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 2 (CB.CI.IL2.V1.Trastuzumab-coGW.SV40) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 2 (CB.CI.IL2.V1.Trastuzumab-coGW.SV40) or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 26 (UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 26 (UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40), or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 28 (CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 28 (CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40), or a sequence at least 99% identical thereto.

[0137] It should be understood that the compositions in the expression cassettes described herein are intended to be applicable to other compositions, schemes, aspects, embodiments and methods described in this specification.

[0138] Recombinant adeno-associated virus (rAAV)

[0139] Provided herein is a recombinant adeno-associated virus (rAAV), comprising an engineered nucleic acid sequence encoding a trastuzumab immunoglobulin (e.g., rAAV.trastuzumab-coGW). Provided herein is a recombinant adeno-associated virus (rAAV) that can be used to treat HER2-positive brain metastatic cancer. rAAV comprises: (a) an AAV capsid; and (b) a vector genome packaged in the AAV capsid of (a). Suitably, the selected AAV capsid targets the cells to be treated. In certain embodiments, the capsid is from clade F. However, in certain embodiments, another AAV capsid source, i.e., clade A, can be selected. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAVrh91 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAVhu96 capsid. The vector genome comprises an AAV 5' inverted terminal repeat (ITR), an expression cassette comprising at least one open reading frame (ORF), and an AAV 3' ITR, wherein the ORF comprises a trastuzumab heavy chain and a trastuzumab light chain and a nucleic acid sequence regulating the expression of the trastuzumab heavy chain and the light chain operably linked thereto.

[0140] As used herein, the term "vector genome" refers to a nucleic acid molecule that is packaged in a viral capsid (e.g., an AAV capsid) and can be delivered to a host cell or a patient's cell. In certain embodiments, the vector genome comprises terminal repeats (e.g., AAV inverted terminal repeats (ITRs)) required for packaging the vector genome into the capsid at the 5' and 3' ends, and contains an expression cassette therebetween, the expression cassette comprising a trastuzumab immunoglobulin gene (e.g., trastuzumab-coGW) operably linked to a sequence directing its expression as described herein.

[0141] The AAV sequences of the vector typically contain cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, e.g., BJ Carter, Handbook of Parvoviruses, P. Tijsser, ed., CRC Press, pp. 155-168 (1990)). The length of the ITR sequence is about 145 base pairs (bp). Preferably, substantially the entire sequence encoding the ITR is used in the molecule, although a certain degree of minor modification of these sequences is permitted. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70:520 532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing a transgene in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, ITR is from an AAV different from the AAV supplying capsid. In one embodiment, the ITR sequence is from AAV2. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are missing. In certain embodiments, the vector genome (e.g., plasmid) includes a shortened AAV2 ITR of 130 base pairs, in which the external A element is missing. During the amplification of the vector DNA using the internal A element as a template and packaging into the capsid to form viral particles, the shortened ITR may be restored to a wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3'ITR are used. However, ITRs from other AAV sources can be selected. In the case where the source of ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as a pseudotype. However, other configurations of these elements may be suitable.

[0142] In one embodiment, the rAAV.trastuzumab-coGW vector has an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises at least one element heterologous to the AAV capsid. In one embodiment, the vector genome contains from 5' to 3': (a) AAV 5'ITR; (b) optionally an enhancer; (c) a promoter; (d) an intron; (e) a leader sequence and a trastuzumab heavy chain coding sequence; (f) optionally a furin cleavage sequence; (g) a T2A linker; (h) a leader sequence and a trastuzumab light chain coding sequence; (i) a polyA signal; and (j) AAV3'ITR.

[0143] In one embodiment, the vector genome contains from 5' to 3': (a) AAV 5' ITR; (b) CB7 promoter, wherein the CB7 promoter comprises a CMV IE enhancer, a chicken β-actin promoter and a chimeric intron, wherein the chimeric intron comprises a chicken β-actin splice donor (including exon sequences), a chicken β-actin intron and a rabbit b-globin splice acceptor, optionally with a spacer sequence; (c) a Kozak sequence; (d) a leader sequence and a trastuzumab heavy chain coding sequence; (e) a furin cleavage sequence; (f) a T2A linker; (g) a leader sequence and a trastuzumab light chain coding sequence; (h) an SV40 polyA signal; and (i) AAV 3' ITR. In one embodiment, the vector genome contains from 5′ to 3′: (a) AAV 5'ITR; (b) CB7 promoter, wherein the CB7 promoter comprises a CMV IE enhancer, a chicken β-actin promoter and a chimeric intron, wherein the chimeric intron comprises a chicken β-actin splice donor (including exon sequences), a chicken β-actin intron and a rabbit b-globin splice acceptor, optionally with a spacer sequence; (c) a Kozak sequence; (d) a leader sequence and a trastuzumab heavy chain coding sequence; (e) a T2A linker; (f) a leader sequence and a trastuzumab light chain coding sequence; (f) an SV40 polyA signal; and (h) AAV3'ITR.

[0144] In one embodiment, the vector genome contains from 5' to 3': (a) AAV 5'ITR; (b) chicken β-actin promoter; (c) chicken β-actin intron (CI); (d) leader sequence and trastuzumab heavy chain coding sequence; (e) furin cleavage sequence; (f) T2A linker; (g) leader sequence and trastuzumab light chain coding sequence; (h) SV40 polyA signal; and (i) AAV3'ITR. In one embodiment, the vector genome contains from 5' to 3': (a) AAV 5'ITR; (b) chicken β-actin promoter; (c) chicken β-actin intron (CI); (d) leader sequence and trastuzumab heavy chain coding sequence; (e) T2A linker; (f) leader sequence and trastuzumab light chain coding sequence; (g) SV40 polyA signal; and (h) AAV3'ITR.

[0145] In one embodiment, the vector genome contains from 5' to 3': (a) AAV 5' ITR; (b) ubiquitin C (UbC) promoter; (c) chimeric (Promega) intron (PI); (d) Kozak sequence; (e) leader sequence and trastuzumab heavy chain coding sequence; (f) furin cleavage sequence; (g) T2A linker; (h) leader sequence and trastuzumab light chain coding sequence; (i) SV40 polyA signal; and (j) AAV 3' ITR. In one embodiment, the vector genome contains from 5' to 3': (a) AAV5'ITR; (b) ubiquitin C (UbC) promoter; (c) chimeric (Promega) intron (PI); (d) Kozak sequence; (e) leader sequence and trastuzumab heavy chain coding sequence; (f) T2A linker; (g) leader sequence and trastuzumab light chain coding sequence; (h) SV40 polyA signal; and (i) AAV3'ITR.

[0146] In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 1 (5'-ITR.CB.CI.IL2.V1.Trastuzumab-coGW.SV40.3'-ITR) or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 1 (5'-ITR.CB.CI.IL2.V1.Trastuzumab-coGW.SV40.3'-ITR) or a sequence that is at least 99% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 25 (5'-ITR.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR) or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 25 (5'-ITR.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR) or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 27 (5'-ITR.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 27 (5'-ITR.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR) or a sequence at least 99% identical thereto.

[0147] In certain embodiments, the AAV capsid is a clade F AAV capsid, wherein the clade F AAV capsid is selected from AAVhu68 capsid [see, e.g., US2020 / 0056159; PCT / US21 / 55436; SEQ ID NOs: 37 and 38 for nucleic acid sequence; SEQ ID NO: 39 for amino acid sequence], AAVhu95 capsid [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 2021, International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022; SEQ ID NOs: 43 and 44 (hu95 nucleic acid sequence)], and SEQ ID NO: 45 (hu95 amino acid sequence), or AAVhu96 capsid [see, e.g., U.S. Provisional Application No. 63 / 251,599 filed on October 2, 2021 and International Patent Application No. PCT / US2022 / 077315 filed on September 30, 2022; SEQ ID NOs: 46 and 47 (hu96 nucleic acid sequence) and SEQ ID NO: 48 (hu96 amino acid sequence)], AAV9 capsid (SEQ ID NO: 54 for nucleic acid sequence; SEQ ID NO: 55 for amino acid sequence). In certain embodiments, the AAV capsid is a clade A capsid, such as AAVrh91 capsid (nucleotide sequences of SEQ ID NOs: 40 and 41; amino acid sequence of SEQ ID NO: 42). See PCT / US20 / 030266, filed April 29, 2020, now published WO2020 / 223231, which are incorporated herein by reference, and International Application No. PCT / US21 / 45945, filed August 13, 2021, which is incorporated herein by reference.

[0148] In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAV9 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAVhu96 capsid.

[0149] In certain embodiments, the AAV capsid for the compositions and methods described herein is selected based on target cells. In certain embodiments, the AAV capsid transduces CNS cells and / or PNS cells. In certain embodiments, other AAV capsids can be selected. The AAV capsid can be selected from cy02 capsid, rh43 capsid, AAV8 capsid, rh01 capsid, AAV9 capsid, rh8 capsid, rh10 capsid, bb01 capsid, hu37 capsid, rh02 capsid, rh20 capsid, rh39 capsid, rh64 capsid, AAV6 capsid, AAV1 capsid, hu44 capsid, hu48 capsid, cy05 capsid, hu11 capsid, hu32 capsid, pi2 capsid or its variant. In certain embodiments, the AAV capsid is a clade F capsid, such as AAV9 capsid, AAVhu68 capsid, hu31 capsid, hu32 capsid or its variant. See, for example, WO 2005 / 033321, WO 2018 / 160582, and US2015 / 0079038, published on April 14, 2015, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV capsid is a non-branch F capsid, such as a clade A, B, C, D, or E capsid. In certain embodiments, the non-branch F capsid is AAV1 or a variant thereof. In certain embodiments, the AAV capsid transduces target cells other than nervous system cells. In certain embodiments, the AAV capsid is a clade A capsid (e.g., AAV1, AAV6, AAVrh91), a clade B capsid (e.g., AAV 2), a clade C capsid (e.g., hu53), a clade D capsid (e.g., AAV7), or a clade E capsid (e.g., rh10).

[0150] The AAV capsid is an assembly of a heterogeneous population of vp1, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein, the term "heterogeneous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population composed of non-identical elements, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0151] As used herein, the term "heterogeneous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population consisting of non-identical elements (e.g., having vp1, vp2, or vp3 (also referred to as VP1, VP2, VP3 or Vp1, Vp2, Vp3) monomers (proteins) with different modified amino acid sequences). The term "heterogeneous population" used in conjunction with vp1, vp2, and vp3 proteins (alternatively referred to as isotypes) refers to differences in the amino acid sequences of vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications from predicted amino acid residues. These subpopulations include at least certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, wherein the deamidation causes amino acid changes and other optional modifications.

[0152] In certain embodiments, AAV capsids are provided having a heterogeneous population of AAV capsid isotypes (i.e., VP1, VP2, VP3) containing multiple highly deamidated "NG" positions. In certain embodiments, the highly deamidated positions are in the positions identified below with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that the reference "NG" is ablated and the mutant "NG" is engineered into another position.

[0153] As used herein, the terms "target cell" and "target tissue" may refer to any cell or tissue intended to be transduced by a subject's AAV vector. The terms may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart.

[0154] Additionally, provided herein is a recombinant nucleic acid molecule comprising: (a) an AAV 5' inverted terminal repeat (ITR), (b) an expression cassette comprising: at least one open reading frame (ORF), the ORF comprising a trastuzumab heavy chain and a trastuzumab light chain, and a nucleic acid sequence regulating the expression of the trastuzumab heavy chain and the trastuzumab light chain operably linked thereto, and (c) an AAV 3' ITR, wherein the expression cassette comprises: (i) a promoter, the promoter being a ubiquitin C (UbC) promoter, and (ii) an intron, the intron being a chimeric intron (e.g., available from Promega), and (iii) at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO:3, the nucleic acid sequence encoding the trastuzumab heavy chain, (v) a furin cleavage site, (vi) a T2A linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO:5, the nucleic acid sequence encoding the trastuzumab light chain, (ix) an SV40 polyadenylation (polyA) sequence, and wherein the expression cassette further comprises a spacer sequence. In certain embodiments, the recombinant nucleic acid molecule comprises an expression cassette comprising the nucleic acid sequence of SEQ ID NO:26. In certain embodiments, the recombinant nucleic acid molecule comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO:25. In certain embodiments, the recombinant nucleic acid molecule is used in a rAAV production system as described herein.

[0155] Additionally, a rAAV production system that can be used to produce rAAV as described herein is provided herein. The production system comprises a cell culture, the cell culture comprising: (a) a nucleic acid sequence encoding an AAV capsid protein; (b) a vector genome; and (c) sufficient AAV rep functions and auxiliary functions to allow the vector genome to be packaged into an AAV capsid. In certain embodiments, the vector genome is SEQ ID NO: 1. In certain embodiments, the vector genome is SEQ ID NO: 25. In certain embodiments, the vector genome is SEQ ID NO: 27. In certain embodiments, the cell culture is a bacterial cell culture. In certain embodiments, the cell culture is a mammalian cell culture. In certain embodiments, the cell culture is a human embryonic kidney 293 (HEK293) cell culture. In certain embodiments, the cell culture is a suspension cell culture. In certain embodiments, AAV rep is from a different AAV. In certain embodiments, wherein AAV rep is from AAV2. In certain embodiments, the AAV rep coding sequence and the cap gene are on the same nucleic acid molecule, wherein a spacer is optionally present between the rep sequence and the cap gene.

[0156] For use in producing AAV viral vectors (e.g., recombinant (r) AAV), the vector genome can be carried on any suitable vector (e.g., plasmid) delivered to a packaging host cell. The plasmids that can be used in the present invention can be engineered so that they are suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, and other cells. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art.

[0157] In certain embodiments, the recombinant nucleic acid molecule is a plasmid. In certain embodiments, the recombinant nucleic acid molecule (e.g., plasmid) can be used for rAAV production. In certain embodiments, the recombinant nucleic acid molecule (e.g., plasmid) that can be used for rAAV production comprises a vector genome, and the vector genome comprises 5'-ITR.CB.CI.IL2.V1.trastuzumab-coGW.SV40.3'-ITR. In certain embodiments, the recombinant nucleic acid molecule (e.g., plasmid) that can be used for rAAV production comprises a vector genome, and the vector genome comprises the nucleic acid sequence of SEQ ID NO:1. In certain embodiments, the recombinant nucleic acid molecule (e.g., plasmid) that can be used for rAAV production comprises a vector genome, and the vector genome comprises 5'-ITR.UbC.PI.IL2_furin protease_V1.trastuzumab-coGW.SV40.3'-ITR. In certain embodiments, the recombinant nucleic acid molecule (e.g., plasmid) that can be used for rAAV production comprises a vector genome, and the vector genome comprises the nucleic acid sequence of SEQ ID NO:25. In certain embodiments, the recombinant nucleic acid molecules (e.g., plasmids) that can be used for rAAV production include a vector genome that includes 5'-ITR.CB7.CI.IL2_Furin protease_V1.Trastuzumab-coGW.SV40.3'-ITR. In certain embodiments, the recombinant nucleic acid molecules (e.g., plasmids) that can be used for rAAV production include a vector genome that includes a nucleic acid sequence of SEQ IDNO:27.

[0158] Methods for generating and isolating AAV suitable for use as a vector are known in the art. See, for example, Grieger and Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10: 717-733; and references cited below, each of which is incorporated herein by reference in its entirety. As used herein, a gene therapy vector refers to an rAAV suitable for treating a patient as described herein. In order to package a gene into a virion, ITR is the only AAV component in cis required in the construct identical to the nucleic acid molecule containing the gene. The cap and rep genes can be supplied in trans.

[0159] In one embodiment, the expression cassette described herein is engineered to a gene element (e.g., a shuttle plasmid), and the immunoglobulin construct sequence carried thereon is transferred to a packaging host cell to produce a viral vector. In one embodiment, the selected gene element can be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be prepared. The method for preparing such constructs is known to nucleic acid manipulation technicians and includes genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, edited by Green and Sambrook, Cold Spring Harbor Press (2012) in Cold Spring Harbor, New York.

[0160] The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid lacking the desired genomic sequence packaged therein. These may also be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette, or contain only partially packaged genomic sequence insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.

[0161] Recombinant adeno-associated viruses (AAV) described herein can be generated using known techniques. See, for example, WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; US 7588772 B2. Such methods involve culturing host cells containing nucleic acid sequences encoding AAV capsid proteins; functional rep genes; expression cassettes consisting of at least AAV inverted terminal repeats (ITRs) and transgenes; and sufficient auxiliary functions to allow the expression cassettes to be packaged into AAV capsid proteins. Methods for generating capsids, their coding sequences, and methods for producing rAAV viral vectors have been described. See, for example, Gao et al., Proceedings of the National Academy of Sciences of the United States of America 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1.

[0162] In one embodiment, a production cell culture is provided for producing a recombinant AAV having a capsid selected from AAVhu68, AAVrh91, AAVhu95 or AAVhu96. Such a cell culture contains a nucleic acid that expresses an AAVhu68 capsid protein in a host cell (e.g., SEQ ID NO: 37 or SEQ ID NO: 38); a nucleic acid molecule suitable for packaging into an AAVhu68 capsid, for example, a vector genome containing an AAV ITR and a non-AAV nucleic acid sequence encoding a gene operably linked to a regulatory sequence that directs expression of the gene in a host cell; and AAV rep functions and adenovirus helper functions sufficient to allow packaging of the vector genome into a recombinant AAVhu68 or AAVrh91 capsid (e.g., SEQ ID NO: 40 or SEQ ID NO: 41), an AAVhu95 capsid (SEQ ID NO: 43 or SEQ ID NO: 44), an AAVhu96 capsid (SEQ ID NO: 47 or SEQ ID NO: 48). In one embodiment, the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells and other cells) or insect cells (e.g., Spodopterafrugiperda (Sf9) cells). In certain embodiments, the baculovirus provides the necessary helper functions for packaging the vector genome into the recombinant AAVhu68, AAVrh91, AAVhu95 or AAVhu96 capsid.

[0163] Optionally, rep function is provided by an AAV other than AAV2 which is selected as the source of complementary ITRs.

[0164] In one embodiment, cells are manufactured in suitable cell culture (e.g., HEK 293 or Sf9) or suspension. The method for manufacturing the gene therapy vector described herein includes methods well known in the art, such as generating plasmid DNA, generating vectors and purifying vectors for generating gene therapy vectors. In certain embodiments, the gene therapy vector is an AAV vector, and the generated plasmid is an AAV cis plasmid encoding an AAV vector genome and a gene of interest, an AAV trans plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process may include method steps, such as starting cell culture, performing cell passage, inoculating cells, transfecting cells with plasmid DNA, exchanging the medium after transfection with a serum-free medium, and collecting cells and culture medium containing the vector. The collected cells and culture medium containing the vector are referred to as crude cell collections herein. In another system, the gene therapy vector is introduced into insect cells by infecting with a baculovirus-based vector. For a review of these production systems, see generally, e.g., Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods for making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each of which are incorporated herein by reference in their entirety: U.S. Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0165] Thereafter, the crude cell collection can be a method step of the subject matter, such as concentrating the vector collection, diafiltration of the vector collection, microfluidization of the vector collection, nuclease digestion of the vector collection, filtering the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare a large amount of vector. Affinity chromatography purification is performed, followed by anion exchange resin chromatography to purify the vector drug product and remove empty capsids. These methods are described in more detail in WO2017 / 160360, entitled "Scalable Purification Method for AAV9," filed on December 9, 2016, which is incorporated herein by reference. WO2017 / 100676, filed on December 9, 2016, for purification methods for AAV8, and WO2017 / 100704, filed on December 9, 2016, and entitled "Scalable Purification Method for AAVrh10", filed on December 11, 2015, for purification methods for rh10, and WO2017 / 100674, filed on December 9, 2016, and entitled "Scalable Purification Method for AAV1", filed on December 11, 2015, for purification methods for AAV1 are all incorporated herein by reference. Other suitable methods may be selected.

[0166] To calculate the content of empty and full particles, the VP3 band volume of the selected sample (e.g., in the examples herein, a preparation purified by an iodixanol gradient, where the number of genome copies (GC) = number of particles) is plotted relative to the loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test product peak. The number of particles per 20 μL loaded (pt) is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL obtains empty pt / mL. Empty pt / mL is divided by pt / mL and x100 to obtain the percentage of empty particles.

[0167] In general, methods for determining empty capsids and AAV vector particles with packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6: 1322-1330; Sommer et al., Molec. Ther. (2003) 7: 122-128. To test denatured capsids, the method includes subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis (composed of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% triacetate in a buffer), then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). Then, an anti-AAV capsid antibody is used as a primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., Journal of Virology (2000) 74: 9281-9293). Then use secondary antibody, described secondary antibody is combined with primary antibody and contains a kind of device for detecting the combination with primary antibody, more preferably contains the anti-IgG antibody of the detection molecule covalently bound thereto, most preferably is the sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase.A kind of method for detecting combination is used to semi-quantitatively determine the combination between primary antibody and secondary antibody, preferably can detect the detection method of radioisotope emission, electromagnetic radiation or colorimetric change, most preferably is chemiluminescent detection kit.For example, for SDS-PAGE, sample can be extracted from column fraction and heated in SDS-PAGE loading buffer containing reducing agent (for example, DTT), and capsid protein is resolved on prefabricated gradient polyacrylamide gel (for example, Novex).Silver staining can be carried out according to the manufacturer's instructions using SilverXpress (Invitrogen, CA) or other suitable staining methods (that is, SYPRO ruby ​​or Coomassie staining).In one embodiment, the concentration of AAV vector genome (vg) in column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, primers and TaqMan TMFluorescent probes are further diluted and amplified samples. The number of cycles (threshold cycles, Ct) required for each sample to reach a defined fluorescence level is measured on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in a Q-PCR reaction. The value of the cycle threshold (Ct) obtained from the sample is used to determine the vector genome titer by normalizing it relative to the Ct value of the plasmid standard curve. Endpoint determination based on digital PCR can also be used.

[0168] On the one hand, an optimized q-PCR method is used, which utilizes a broad spectrum serine protease, such as proteinase K (as commercially available from Qiagen). More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, and then heat inactivated. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer can be concentrated 2 times or more. Typically, proteinase K treatment is about 0.2 mg / mL, but can vary between 0.1 g / mL and about 1 mg / mL. The treatment step is usually carried out at about 55°C for about 15 minutes, but can be carried out at a lower temperature (e.g., about 37°C to about 50°C) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60°C) for a shorter period of time (e.g., about 5 minutes to 10 minutes). Similarly, heat inactivation is typically performed at about 95°C for about 15 minutes, but the temperature can be lowered (e.g., from about 70°C to about 90°C) and the time can be extended (e.g., from about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

[0169] Additionally or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining the titer of single-stranded and self-complementary AAV vector genomes by ddPCR have been described. See, for example, M. Lock et al., Hu Gene Therapy Methods / Hum Gene Ther Methods. April 2014; 25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0170] In certain embodiments, the manufacturing process of rAAV as described herein relates to the methods described in U.S. Provisional Patent Application No. 63 / 371,597 filed on August 16, 2022 and U.S. Provisional Patent Application No. 63 / 371,592 filed on August 16, 2022, which are incorporated herein by reference in their entirety.

[0171] Briefly, a method for separating rAAVhu68 (or AAVrh91, AAVhu95 or AAVhu96) particles having a packaged genomic sequence from a genome-defective AAVhu68 (or AAVrh91 or AAVhu95 or AAVhu96) intermediate involves subjecting a suspension comprising recombinant AAVhu68 (or AAVrh91) viral particles and AAVhu68 (or AAVrh91 or AAVhu95 or AAVhu96) capsid intermediates to high performance liquid chromatography, wherein the AAVhu68 (or AAVrh91 or AAVhu95 or AAVhu96) viral particles and the AAVhu68 intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2 (or about 9.8 for AAVrh91) and subjected to a salt gradient while monitoring the ultraviolet absorbance of the eluent at about 260 nanometers (nm) and about 280 nm. Although not optimal for rAAVhu68 and AAVrh91, the pH can range from about 10 to 10.4. In this method, when the ratio of A260 / A280 reaches an inflection point, AAV intact capsids are collected from the eluted fractions. In one example, for the affinity chromatography step, the diafiltered product can be applied to an affinity resin (Life Technologies) that effectively captures AAV serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are effectively captured.

[0172] rAAV.trastuzumab-coGW (e.g., rAAV.UbC.PI.IL2_furin_V1.trastuzumab-coGW.SV40, rAAV.UbC.PI.IL2.V1.trastuzumab-coGW.SV40, rAAV.CB7.CI.IL2_furin_V1.trastuzumab-coGW.SV40, or rAAV.CB7.CI.IL2.V1.trastuzumab-coGW.SV40) is suspended in a suitable physiologically compatible composition (e.g., buffered saline). This composition can be stored frozen, subsequently thawed, and optionally diluted with a suitable diluent. Alternatively, the vector can be prepared as a composition suitable for delivery to a patient without the need for freezing and thawing steps.

[0173] As used herein, the term "clade" as it relates to AAV groups refers to groups of AAVs that are phylogenetically related to one another as determined using a Neighbor-Joining algorithm based on an alignment of AAV vpl amino acid sequences with a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no greater than 0.05. Neighbor-Joining algorithms have been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York) (2000). Computer programs that can be used to implement this algorithm are provided. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of the AAV vpl capsid protein, one skilled in the art can readily determine whether a selected AAV is contained within one of the clades identified herein, within another clade, or outside of these clades. See, e.g., G Gao et al., Journal of Virology, 2004 Jun;78(10):6381-6388, which identifies clades A, B, C, D, E, and F, and provides nucleic acid sequences of novel AAVs, GenBank Accession Nos. AY530553 to AY530629. See also WO 2005 / 033321.

[0174] The abbreviation "sc" refers to self-complementarity. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, instead of waiting for the cell-mediated second strand synthesis, the two complementary halves of scAAV will associate to form a double-stranded DNA (dsDNA) ready for immediate replication and transcription. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", "Gene Therapy", (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0175] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial virus particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, it cannot generate progeny virus particles, but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome can be engineered to be "gutless" - containing only the genes of interest, which are flanked by the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for gene therapy because replication and infection by progeny virus particles will not occur unless the viral enzymes required for replication are present.

[0176] As used herein, the terms "recombinant rAAV", "rAAV" and "artificial AAV", which are used interchangeably, mean but are not limited to AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprises a nucleic acid heterologous to AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such artificial capsids can be generated by any suitable technique using a combination of selected AAV sequences (e.g., fragments of vp1 capsid protein) and heterologous sequences, which can be obtained from different selected AAVs, non-contiguous portions of the same AAV, from non-AAV viral sources, or from non-viral sources. Artificial AAV can be but are not limited to pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Pseudotyped vectors can be used in the present invention, in which the capsid of one AAV is replaced by a heterologous capsid protein. In one embodiment, AAV2 / 5 and AAV2 / 8 are exemplary pseudotyped vectors. Selected genetic elements can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion delivery. The method for preparing such constructs is known to nucleic acid manipulation technicians and includes genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (2012).

[0177] In many cases, rAAV particles are known to be resistant to DNA enzymes. However, in addition to this endonuclease (DNA enzyme), other endonucleases and exonucleases can also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded DNA and / or double-stranded DNA and RNA. Such steps can contain a single nuclease or a mixture of nucleases for different targets, and can be endonucleases or exonucleases.

[0178] The term "nuclease-resistant" indicates that the AAV capsid has been fully assembled around an expression cassette designed to deliver genes to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present during the production process.

[0179] As used herein, the term "heterogeneous" or any grammatical variations thereof when used to refer to vp capsid proteins refers to a population consisting of non-identical elements, such as having vp1, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.

[0180] The term "heterogeneous" used in conjunction with the vp1, vp2, and vp3 proteins (alternatively referred to as isotypes) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications from predicted amino acid residues. These subpopulations include at least certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, wherein the deamidation causes the amino acid changes and other optional modifications.

[0181] As used herein, unless otherwise specified, a "subgroup" of vp proteins refers to a group of vp proteins that have at least one defined common property and consist of at least one group member to less than all members of a reference group. For example, unless otherwise specified, a "subgroup" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid. Unless otherwise specified, a "subgroup" of vp3 proteins can be one (1) vp3 protein that is less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subgroup of vp proteins; vp2 proteins can be a separate subgroup of vp proteins, and vp3 is still another subgroup of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can contain subgroups with different modifications, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., at asparagine-glycine pairs.

[0182] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and with reference to the public texts, which provide those skilled in the art with a general guide to many of the terms used in this application.

[0183] Pharmaceutical composition

[0184] In one aspect, provided herein is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer.

[0185] In one embodiment, about 1x10 9 Genome copies (GC) / mL is about 1x10 14 In a further embodiment, the rAAV is prepared at about 3x10 9 GC / mL to about 3x10 13 In another embodiment, the rAAV is prepared at about 1×10 9GC / mL to about 1x10 13 GC / mL rAAV is prepared. In one embodiment, at least 1x10 11 GC / mL preparation of rAAV.

[0186] Also provided herein is a composition comprising a rAAV as described herein and an aqueous suspension medium. In certain embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration. On the one hand, the composition contains at least one rAAV stock solution and optional carriers, excipients, and / or preservatives.

[0187] As used herein, the "stock" of rAAV refers to a population of rAAV. Although its capsid protein has heterogeneity due to deamidation, the rAAV in the stock is expected to share the same vector genome. The stock may include rAAV with a capsid having, for example, a unique heterogeneous deamidation pattern of a selected AAV capsid protein and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of a production system. Various production systems may be selected, including but not limited to the production systems described herein.

[0188] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic agents and absorption slow-release agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such culture media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles (such as liposomes, nanocapsules, nanoparticles, lipid nanoparticles (LNP), microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce the compositions of the present invention into suitable host cells. Specifically, the vector genome delivered by the rAAV vector can be formulated for delivery or encapsulation in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc.

[0189] In one embodiment, the composition includes a final formulation suitable for delivery to a subject, such as an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be transported as a concentrate that is diluted to be administered to a subject. In other embodiments, the composition can be lyophilized and reconstituted when administered.

[0190] A suitable surfactant or combination of surfactants may be selected from non-toxic nonionic surfactants.

[0191] In one embodiment, the composition includes a carrier, a diluent, an excipient and / or an adjuvant. In view of the indications for which the transfer virus is directed, a person skilled in the art can easily select a suitable carrier. For example, a suitable carrier includes saline, which can be formulated with a variety of buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The buffer / carrier should include components that prevent rAAV from adhering to the infusion tubing but do not interfere with the binding activity of rAAV in vivo. Suitable surfactants or combinations of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated in a primary hydroxyl group is selected, such as Poloxamer 188 (also known as Poloxamer 188). F68 [BASF], F68, F68, P188), which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylyl glyceride), polyoxy-oleyl ethers, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol, may be selected. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three numbers: the first two digits x100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x10 gives the percentage of the polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

[0192] In certain embodiments, the rAAV.trastuzumab-coGW-containing composition is delivered at a pH in the range of 6 to 8, or 7.2 to 7.8, or 7.5 to 8. For intrathecal delivery, a pH above 7.5 may be desired, e.g., 7.5 to 8, or 7.8. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desired.

[0193] In certain embodiments, the formulation may contain a buffered saline solution that does not contain sodium bicarbonate. Such formulations may contain a buffered saline solution that contains water containing one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof, such as Harvard's buffer. In one embodiment, the buffer is PBS. In another embodiment, the buffer is artificial cerebrospinal fluid (aCSF), such as Eliott's formulation buffer; or Harvard apparatus perfusion fluid (artificial CSF with the following final ion concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155). The aqueous solution may further contain P188, a poloxamer commercially available from BASF, formerly known as F68 is sold. The pH of the aqueous solution may be 7.2.

[0194] In another embodiment, the formulation may contain a buffered saline solution comprising 1 mM sodium phosphate (Na3PO4), 150 mM sodium chloride (NaCl), 3 mM potassium chloride (KCl), 1.4 mM calcium chloride (CaCl2), 0.8 mM magnesium chloride (MgCl2), and 0.001% poloxamer (e.g., ) 188. See, e.g., harvardapparatus.com / harvard-apparatus-perfusion-fluid.html. In certain embodiments, Harvard buffer is preferred because better pH stability is observed when Harvard buffer is used.

[0195] In certain embodiments, the formulation buffer is artificial CSF with Pluronic F68. In other embodiments, the formulation may contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0196] Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients in addition to rAAV and one or more carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0197] The composition according to the present invention may include a pharmaceutically acceptable carrier, as defined above. Suitably, the composition described herein includes an effective amount of one or more AAVs, which are suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient, which is designed to be delivered to a subject by injection, an osmotic pump, an intrathecal catheter or delivered by another device or approach. In certain embodiments, the ommaya reservoir is used for delivery. In one example, the composition is formulated for intrathecal delivery. In one example, the composition is formulated for intravenous (iv) delivery.

[0198] In one embodiment, a therapeutically effective amount of the carrier is included in a pharmaceutical composition. The choice of carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0199] As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject during the course of treatment or the dose or amount delivered in a single unit (or multiple units or divided doses) administration.

[0200] In certain embodiments, the composition can be formulated in a dosage unit to contain about 1×10 rAAV per gram of brain mass. 9 GC to about 1x10 per gram (g) of brain mass 13 In another embodiment, the dosage is 1×10 per gram of brain mass. 10 GC to about 1x10 per gram of brain mass 13 GC. In a specific embodiment, the dose of the vector administered to the patient is at least about 1.0 x 10 9 GC / g, about 1.5x10 9 GC / g, about 2.0x10 9 GC / g, about 2.5x10 9 GC / g, about 3.0x10 9 GC / g, about 3.5x10 9 GC / g, about 4.0x10 9 GC / g, about 4.5x10 9 GC / g, about 5.0x10 9 GC / g, about 5.5x10 9 GC / g, about 6.0x10 9 GC / g, about 6.5x10 9GC / g, about 7.0x10 9 GC / g, about 7.5x10 9 GC / g, about 8.0x10 9 GC / g, about 8.5x10 9 GC / g, about 9.0x10 9 GC / g, about 9.5x10 9 GC / g, about 1.0x10 10 GC / g, about 1.5x10 10 GC / g, about 2.0x10 10 GC / g, about 2.5x10 10 GC / g, about 3.0x10 10 GC / g, about 3.5x10 10 GC / g, about 4.0x10 10 GC / g, about 4.5x10 10 GC / g, about 5.0x10 10 GC / g, about 5.5x10 10 GC / g, about 6.0x10 10 GC / g, about 6.5x10 10 GC / g, about 7.0x10 10 GC / g, about 7.5x10 10 GC / g, about 8.0x10 10 GC / g, about 8.5x10 10 GC / g, about 9.0x10 10 GC / g, about 9.5x10 10 GC / g, about 1.0x10 11 GC / g, about 1.5x10 11 GC / g, about 2.0x10 11 GC / g, about 2.5x10 11 GC / g, about 3.0x10 11 GC / g, about 3.5x10 11 GC / g, about 4.0x10 11 GC / g, about 4.5x10 11 GC / g, about 5.0x10 11 GC / g, about 5.5x10 11 GC / g, about 6.0x10 11 GC / g, about 6.5x10 11 GC / g, about 7.0x10 11 GC / g, about 7.5x10 11 GC / g, about 8.0x10 11 GC / g, about 8.5x10 11GC / g, about 9.0x10 11 GC / g, about 9.5x10 11 GC / g, about 1.0x10 12 GC / g, about 1.5x10 12 GC / g, about 2.0x10 12 GC / g, about 2.5x10 12 GC / g, about 3.0x10 12 GC / g, about 3.5x10 12 GC / g, about 4.0x10 12 GC / g, about 4.5x10 12 GC / g, about 5.0x10 12 GC / g, about 5.5x10 12 GC / g, about 6.0x10 12 GC / g, about 6.5x10 12 GC / g, about 7.0x10 12 GC / g, about 7.5x10 12 GC / g, about 8.0x10 12 GC / g, about 8.5x10 12 GC / g, about 9.0x10 12 GC / g, about 9.5x10 12 GC / g, about 1.0x10 13 GC / g, about 1.5x10 13 GC / g, about 2.0x10 13 GC / g, about 2.5x10 13 GC / g, about 3.0x10 13 GC / g, about 3.5x10 13 GC / g, about 4.0x10 13 GC / g, about 4.5x10 13 GC / g, about 5.0x10 13 GC / g, about 5.5x10 13 GC / g, about 6.0x10 13 GC / g, about 6.5x10 13 GC / g, about 7.0x10 13 GC / g, about 7.5x10 13 GC / g, about 8.0x10 13 GC / g, about 8.5x10 13 GC / g, about 9.0x10 13 GC / g, about 9.5x10 13 GC / g or about 1.0x10 14 GC / g brain mass.

[0201] In addition, the replication-defective virus composition can be formulated in a dosage unit so as to contain the replication-defective virus in an amount of about 1.0 x 10 9 GC to about 1.0x10 16 GC range (to treat subjects with an average body weight of 70 kg), including all integer or fractional amounts within the range, and for human patients, preferably 1.0 x 10 12 GC to 1.0x10 14 GC. In one embodiment, the composition is formulated to contain at least 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 or 9x10 9 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 or 9x10 10 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 or 9x10 11 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 or 9x10 12GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1x10 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 or 9x10 13 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1x10 14 , 2x10 14 , 3x10 14 , 4x1014, 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 or 9x10 14 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1x10 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 or 9x10 15 GC, including all integers or fractional amounts within the range. In one embodiment, for human use, the dosage range may be 1x10 10 About 1x10 12 GC, including all integer or fractional amounts within the stated range.

[0202] In certain embodiments, the composition is delivered by intrathecal, optionally by intracisternal (ICM) injection. In certain embodiments, the composition is delivered by intraparenchymal administration. In certain embodiments, the composition is delivered by Ommaya reservoir delivery system. In certain embodiments, the composition is delivered by direct injection into a tumor or tumor bed.

[0203] In one embodiment, a pharmaceutical composition comprising an rAAV as described herein is present at about 1 x 10 9 GC to about 1x10 per gram of brain mass 13 Dosage administration of GC.

[0204] The rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient.

[0205] It should be understood that the compositions within the pharmaceutical compositions described herein are intended to be applicable to other compositions, regimens, aspects, embodiments and methods described across this specification.

[0206] Application and solutions

[0207] In one aspect, the method provided herein is a method of treating a human subject diagnosed with HER2-positive cancer. The method comprises administering to the subject a suspension of a rAAV vector as described herein. In one embodiment, the method comprises administering to a subject with HER2-positive brain metastatic cancer a dose of about 1 x 10 9 GC to about 1x10 per gram of brain mass 14 In another embodiment, the method comprises administering to a subject a dose of about 1 x 10 9 GC / kg body weight is about 1x10 14 GC / kg body weight of a suspension of rAAV as described herein in formulation buffer. Suitable dosages for systemic and / or intratumoral administration can be determined by one skilled in the art.

[0208] In some embodiments, the method comprises treating a subject diagnosed with HER2-positive cancer belonging to refractory and / or resistant cancer. In some embodiments, the method comprises treating a subject diagnosed with HER2-positive breast cancer belonging to refractory and / or resistant cancer. In some embodiments, the method comprises treating a subject diagnosed with HER2-positive brain metastatic breast cancer belonging to refractory and / or resistant cancer. In some embodiments, the method comprises treating a subject diagnosed with HER2-positive brain metastatic breast cancer belonging to refractory and / or resistant cancer. In some embodiments, the method comprises treating a subject diagnosed with trastuzumab-resistant HER2-positive cancer. In some embodiments, the method comprises treating a subject with HER2-positive brain metastatic breast cancer. In some embodiments, the method comprises treating a subject diagnosed with trastuzumab-resistant HER2-positive breast cancer (e.g., estrogen receptor (ER) positive, progesterone receptor (PR) positive, and Her2 positive). In certain embodiments, the method comprises treating a subject diagnosed with ER-negative, PR-negative, and Her2-positive cancer. In certain embodiments, the method comprises treating a subject diagnosed with HER2 / low tumors.

[0209] As used herein, "refractory cancer" and / or "resistant cancer" refers to a cancer that is refractory or resistant to one or more cancer therapies, such as cancer chemotherapy (cytotoxic chemotherapy). In certain embodiments, refractory and / or resistant cancers cannot be treated by surgical intervention. In certain embodiments, refractory and / or resistant cancers initially do not respond to chemotherapy or radiotherapy treatments. In certain embodiments, refractory and / or resistant cancers do not respond to cancer therapeutic agents over time.

[0210] As used herein, "trastuzumab resistance" refers to a cancer that is refractory or resistant to trastuzumab treatment. In certain embodiments, "refractory" or "resistant" means that cancer (i.e., HER2 positive) is unresponsive to trastuzumab after a standard course of treatment, for example, even after trastuzumab treatment, cancer continues to progress. In certain embodiments, trastuzumab-resistant cancers themselves are resistant to trastuzumab treatment. In certain embodiments, trastuzumab-resistant cancers acquire resistance, wherein cancer cells initially respond to treatment, but after a period of time, no longer respond to trastuzumab treatment (i.e., refractory to treatment). In certain embodiments, resistance is developed in later treatment, wherein HER2 positive tumors are unresponsive or resistant to trastuzumab therapy.

[0211] When assessed in vitro, in certain embodiments, the trastuzumab-resistant cancer cells are derived from parental cells that are sensitive to trastuzumab and are treated with a composition and / or solution comprising trastuzumab as a prior treatment or as a means of exerting selective pressure. See also Pohlman, PR et al., Resistance to Trastuzumab in Breast Cancer, 2009, Clinical Cancer Research 15(24):7479-7491; Vu, T. and Claret, FX, Trastuzumab: updated mechanisms of action and resistance in breast cancer, 2012, Front. Oncol. 2(62); Rimawi, MF et al., Resistance to Anti-HER2 Therapies in Breast Cancer, 2015, American Society of Clinical Oncology Educational Book 35 (May 14, 2015) e157-e164, which are incorporated herein by reference.

[0212] In some embodiments, the method comprises administering to the subject a dose of 1 x 10 11 Up to 1x10 14 GC / kg of a suspension of rAAV as described herein in a formulation buffer. In certain embodiments, the method comprises intravenously administering a dose range of about 1×10 12 The genome copies (GC) / kg of rAAV is about 1x10 14 GC / kg of rAAV. In certain embodiments, the dose is about 1x10 13 GC / kg is about 1x10 14 GC rAAV or about 3x10 13 GC / kg. In certain embodiments, it is contemplated that about 3x10 12 GC / kg is about 1x10 14 The dose of GC / kg includes about 3.0x10 13 GC / kg and about 1.0x10 13 A dose of GC / kg was delivered by intravenous administration.

[0213] In one embodiment, a therapeutically effective amount of rAAV described herein is delivered to a subject. As used herein, a "therapeutically effective amount" refers to the amount of a composition comprising a nucleic acid sequence encoding a trastuzumab immunoglobulin, which delivers and expresses an amount of immunoglobulin sufficient to achieve efficacy in target cells. The dosage is adjusted to balance the therapeutic benefit with any side effects, and such dosages can vary depending on the therapeutic application using the recombinant vector. The expression level of the transgenic product can be monitored to determine the dosage frequency of the resulting viral vector, preferably an AAV vector containing a transgenic. Optionally, a dosage regimen similar to the dosage regimen described for therapeutic purposes can be used for immunization using the composition described herein.

[0214] Suitable, conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., brain, CSF, heart), intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intraventricular, intrathecal, ICM, lumbar puncture, and other parenteral routes of administration. Routes of administration may be combined if desired.

[0215] Suitable volume for delivering these dosages and concentrations can be determined by those skilled in the art.For example, the volume of about 1 μ L to 150 mL can be selected for CNS delivery, wherein for adults, a larger volume is selected.Usually, for newborn infants, suitable volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL can be selected.For young children, the volume of about 0.5 mL to about 20 mL can be selected.For children, the volume of up to about 30 mL can be selected.For prepubertal teenagers and adolescents, the volume of up to about 50 mL can be selected.In still other embodiments, the patient can accept the intrathecal administration of the volume selected as about 5 mL to about 15 mL or about 7.5 mL to about 10 mL.Other suitable volumes and dosages for CNS delivery, intratumoral delivery and / or for systemic delivery (e.g., IV) can be determined.The dosage will be adjusted to balance the therapeutic benefit and any side effects, and such dosage can be varied according to the therapeutic application using a recombinant vector.

[0216] In one embodiment, the method comprises administering rAAV as described herein to a subject in need. The recombinant vector described above can be administered or delivered to a host cell according to the disclosed method. The rAAV preferably suspended in a physiologically compatible carrier can be administered to a subject, a human or a non-human mammal patient. In certain embodiments, for administration to human patients, rAAV is appropriately suspended in an aqueous solution containing saline, a surfactant and a physiologically compatible salt or a mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7 or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH within this range may be desired for intrathecal delivery; and for intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. However, other pHs within the widest range and these sub-ranges may be selected for other delivery routes.

[0217] Suitably, the pharmaceutical compositions and uses thereof as described herein comprise delivery to a subject by injection, an osmotic pump, an intrathecal catheter, or for delivery by another device or route. In one example, the composition is formulated for intrathecal delivery.

[0218] As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a drug administration route by injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery can include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital / intracisternal and / or C1-2 puncture. For example, the material can be introduced by lumbar puncture to diffuse throughout the subarachnoid space. In another example, it can be injected into the cerebellomedullary cistern. In certain embodiments, rAAV, vectors or compositions as described herein are administered to subjects in need by intrathecal administration. In certain embodiments, intrathecal administration is performed as described in U.S. Patent Publication No. 2018 / 0339065A1, published on November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, CNS administration is performed using an Ommaya reservoir (also referred to as an Ommaya device or Ommaya system).

[0219] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to administration of a drug directly into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically by suboccipital puncture or by injection directly into the cisterna magna or by a permanently placed cannula.

[0220] As used herein, the terms "intraparenchymal," "dentate nucleus," or IDN refer to a route of administration of the composition directly into the dentate nucleus. IDN allows for targeting of the dentate nucleus and / or cerebellum. In certain embodiments, IDN administration is performed using The procedure was performed using a neuronavigation system (MRI Interventions, Inc., Memphis, TN) and ventricular catheterization, which allows for MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

[0221] Suitably, the pharmaceutical compositions and uses thereof as described herein comprise delivery of a tumor bed to a subject by surgical and non-surgical techniques. See also Gilbert, A. and Machluf, M., Nano to micro delivery systems: targeting angiogenesis in brain tumors, J Angiogenes Res. 2010 Oct 8; 2(1):20; Balyasnikova, IV. et al., Intranasal Delivery of Mesenchymal Stem Cells Significantly Extends Survival of Irradiated Mice with Experimental Brain Tumors, Molecular Therapy, 2014, 22(1):P140-P148, Epub 2014 Jan 1, which are incorporated herein by reference in their entirety. In certain embodiments, the pharmaceutical compositions and uses thereof as described herein comprise direct delivery to a tumor or tumor bed of the CNS. In certain embodiments, the pharmaceutical compositions and uses thereof as described herein comprise delivery by a systemic route. In certain embodiments, the pharmaceutical compositions and uses thereof as described herein comprise direct delivery to a Her2-positive tumor (e.g., breast cancer or Her2-positive metastatic cancer or gastric-gastroesophageal junction cancer) located outside the CNS. See also Ma, H. et al., Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral (AAV) vector, 2002, Gene Therapy, 9:2-11; Liang, C.et al., Local Expression of Secondary Lymphoid Tissue Chemokine Delivered by Adeno-Associated Virus within the Tumor Bed Stimulates Strong Anti-Liver Tumor Immunity, Journal of Virology, 2007, 81(17): 9502-9511; Xu, X. et al., Adeno-associated virus (AAV)-based gene therapy for glioblastoma, Cancer Cell International, 2021, 21: 76, e-published Jan 26, 2021; Reul, J. et al., Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors, Methods Vectors, Methods (Oncology Frontiers), 2019, 9:52, Epub 2019 Feb 14; Sheth, RA. et al., Assessment of Image-Guided Intratumoral Delivery of Immunotherapeutics in Patients With Cancer, JAMA Network Open. 2020; 3(7):e207911, Epub 2020 Jul 29; Tchou, J. et al., Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer, Cancer Immunol Res. Res), 2017, 5(12): 1152-1161, electronic version November 6, 2017, all of which are incorporated herein by reference.

[0222] In other aspects, suitably, the compositions of the invention are designed such that the rAAV vector carries a nucleic acid expression cassette encoding a trastuzumab immunoglobulin construct and a regulatory sequence directing its trastuzumab immunoglobulin expression in selected cells. After the vector is administered to the CNS, the vector delivers the expression cassette to the CNS and expresses the protein immunoglobulin construct in vivo. The use of the compositions described herein in anti-tumor methods is described, as well as the use of these compositions in anti-tumor regimens that may optionally involve the delivery of one or more other anti-tumor agents or other active agents.

[0223] As described above, a composition may contain a single type of AAV vector as described herein, which contains an expression cassette for delivering an anti-tumor trastuzumab immunoglobulin construct in vivo. Alternatively, a composition may contain two or more different AAV vectors, each of which has a different expression cassette packaged therein. For example, the two or more different AAVs may have different expression cassettes for expressing immunoglobulin polypeptides, which are assembled in vivo to form a single functional immunoglobulin construct. In another example, the two or more AAVs may have different expression cassettes that express immunoglobulin polypeptides directed against different targets, such as two providing two functional immunoglobulin constructs (e.g., an anti-Her2 immunoglobulin construct and a second anti-tumor immunoglobulin construct).

[0224] The regimen described herein, in addition to one or more of the combinations described herein, further comprises one or more combinations with anti-tumor biological drugs, anti-tumor small molecule drugs, chemotherapeutic agents, immunopotentiators, radiation, surgery, etc. The biological drugs described herein are based on peptides, polypeptides, proteins, enzymes, nucleic acid molecules, vectors (including viral vectors), etc.

[0225] In one embodiment, the method further comprises that the subject receives immunosuppressive co-therapy. The immunosuppressant for such co-therapy includes but is not limited to glucocorticoids, corticosteroids, antimetabolites, T cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs) and cytostatic agents, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies or agents active against immunophilins. Immunosuppressants may include nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25) or CD3 directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) binding agents.

[0226] In certain embodiments, immunosuppressive therapy can be started on the 0th day, the 1st day, the 2nd day, the 7th day or more days before or after applying gene therapy. Such therapy can be related to the administration of two or more drugs (for example, prednisone, mycophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)) in the same day. One or more of these drugs can be continued to be used with the same dosage or adjusted dosage after gene therapy is applied. As required, such therapy can last for about 1 week (7 days), about 60 days or longer. In certain embodiments, the scheme without tacrolimus is selected.

[0227] In one embodiment, the method further comprises administering anti-AAV neutralizing antibodies (NAbs) to the subject to reduce peripheral transduction and mitigate the potential risk of trastuzumab-induced cardiotoxicity. In certain embodiments, the method further comprises detecting the presence of systemic AAV NAbs before treatment with anti-AAV NAbs, wherein the anti-AAV NAb level exceeds the predetermined level for rAAV capsids (or serum cross-reactive capsids) and does not require pretreatment. Such levels can be, for example, more than about 1:10, about 1:20, about 1:50, about 1:100, about 1:250, or higher or lower levels. In certain embodiments, the method further comprises administering human anti-AAV polyclonal antibodies (e.g., plasma-derived, mixed human immunoglobulins (IVIG)), anti-AAV monoclonal antibodies, or anti-AAV antibody mixtures to the patient intravenously about 1 day to about 2 hours before treatment with rAAV-trastuzumab (e.g., rAAV.trastuzumab-coGW).

[0228] In certain embodiments, a combination regimen is provided to prevent off-target delivery of rAAV, the regimen comprising: (a) pre-treating the patient by systemically administering a composition comprising an anti-AAV capsid neutralizing antibody directed against the AAV capsid in a recombinant AAV vector, and (b) administering rAAV as described herein (e.g., rAAV.trastuzumab-coGW) to the central nervous system (CNS). See also U.S. Provisional Patent Application No. 63 / 328,227, filed April 6, 2022, which is incorporated herein by reference in its entirety.

[0229] As used herein, "neutralizing antibodies" or "NAbs" specifically bind to viral capsids or envelopes, and interfere with the infectivity of a virus or a recombinant viral vector with a viral capsid or envelope, thereby preventing the recombinant viral vector from delivering an effective amount of a gene product encoded by an expression cassette in its vector genome. Neutralizing antibodies in patient serum can be assessed using various methods. The terms method and assay can be used interchangeably. As used herein, the terms "neutralization assay" and "serum virus neutralization assay" refer to serological tests for the presence of systemic antibodies that can prevent the infectivity of a virus. Such assays can also qualitatively or quantitatively distinguish the binding ability (e.g., magnitude) or efficiency of an antibody neutralizing target. Immunological assays can include enzyme immunoassays (EIA), radioimmunoassays (RIA) using radioisotopes, fluorescent immunoassays (FIA) using fluorescent materials, chemiluminescent immunoassays (CLIA) using chemiluminescent materials, and counting immunoassays (CIA) using particle counting techniques, other improved assays, such as Western blots, immunohistochemistry (IHC) and agglutination. One of the most common enzyme immunoassays is enzyme-linked immunosorbent assays (ELISA).

[0230] Examples of suitable methods include those described by, e.g., R Calcedo et al., Journal Infectious Diseases, 2009, 199: 381-290; GUO et al., "Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay", Molecular Therapy: Methods & Clinical Development, Vol. 13, June 2019, T. Ito et al., "A convenient enzyme-linked immunosorbent assay for rapid screening of anti-adeno-associated virus neutralizing antibodies", Ann Clin Biochem, 2009; 46: 508-510; US2018 / 0356394A2 (Voyager Therapeutics, Inc., New York, NY, USA). Additionally, commercial kits exist (see, e.g., Athena Diagnostics, Invitrogen, ThermoFisher.com; Covance).

[0231] The neutralizing ability of antibody is usually measured by the expression of reporter genes such as luciferase or GFP. In order to determine and compare the activity of neutralizing antibodies, in one of the neutralization assays described herein, the antibody of the test should show 50% or higher neutralizing activity. In certain embodiments, neutralizing ability is determined by measuring the activity of reporter gene product (e.g., luciferase, GFP). The neutralizing ability of antibody to specific viral vector can be at least 50%, for example at least 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0232] As used herein, the term "NAb titer" is a measure of how many neutralizing antibodies (e.g., anti-AAV NAbs) are produced, which neutralize the physiological effects of the epitope (e.g., AAV) to which they are targeted. Anti-AAV NAb titers can be measured as described in, for example, Calcedo, R et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009. 199 (3): pp. 381-390, which is incorporated herein by reference.

[0233] Still other co-therapeutics may also include, for example, anti-IgG enzymes, which have been described as being useful for depleting anti-AAV antibodies (and thus may allow administration to patients testing above a threshold level of antibodies to a selected AAV capsid), and / or delivery of anti-FcRN antibodies and / or one or more of the following: a) a steroid or a combination of steroids and / or (b) an IgG cleaving enzyme, (c) an Fc-IgE binding inhibitor; (d) an Fc-IgM binding inhibitor; (e) an Fc-IgA binding inhibitor; and / or (f) gamma interferon. Anti-FcRN antibodies, including, for example, rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all formed HanAll BioPhrma Co. Ltd), Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx SE), orilanolimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co. Ltd. In certain embodiments, a combination of anti-FcRN antibodies is administered. In certain embodiments, an anti-FcRN antibody is administered in combination with a suitable anti-FcRn ligand (ie, a peptide or protein construct that binds to human FcRn to inhibit IgG binding).

[0234] In one embodiment, a combination regimen for treating Her2 positive tumors or Her2 positive metastatic tumor patients is provided, wherein the regimen includes administering a combination of a carrier described herein with a ligand that inhibits human FcRn binding and a pre-existing patient neutralizing antibody (e.g., IgG). In certain embodiments, the patient may not understand any therapeutic treatment with the carrier, and may have pre-existing immunity due to previous infection with a wild-type virus. In other embodiments, the patient may have neutralizing antibodies due to previous treatment or vaccination. In certain embodiments, the patient may have 1:1 to 1:20, or more than 1:2, more than 1:5, more than 1:10, more than 1:20, more than 1:50, more than 1:100, more than 1:200, more than 1:300 or higher neutralizing antibodies. In certain embodiments, the patient has 1:1 to 1:200, or 1:5 to 1:100, or 1:2 to 1:20, or 1:5 to 1:50 or 1:5 to 1:20 range of neutralizing antibodies. In certain embodiments, the patient receives a single anti-FcRn ligand (e.g., an anti-FcRn antibody) as the sole agent that modulates FcRn-IgG binding and allows for efficient vector delivery. In other embodiments, the patient may receive a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor down-regulator (e.g., interferon gamma), an IgG enzyme, or other suitable component). Such a combination may be particularly desirable for patients with particularly high neutralizing antibody levels (e.g., greater than 1:200).

[0235] In certain embodiments, an anti-FcRn ligand (e.g., antibody) is administered to a patient with neutralizing antibodies before a selected viral vector, and is optionally administered simultaneously with a selected viral vector. In certain embodiments, it may be desirable to continue to express an anti-FcRn ligand after administration of a gene therapy vector in the short term (on a transient basis), for example, until the viral vector is cleared from the patient's body. In certain embodiments, it may be desirable to continue to express an anti-FcRn ligand. Optionally, in this embodiment, the ligand can be delivered by a viral vector, for example, in a viral vector expressing a therapeutic transgene. However, when the therapeutic gene delivered is an antibody or an antibody construct or another construct comprising an IgG chain, this embodiment is undesirable. In such embodiments, when an antibody construct with an IgG chain is delivered to a patient with pre-existing immunity by a viral vector, an anti-FcRn ligand is delivered or administered transiently so that the amount of anti-FcRn ligands in the circulation is removed from the serum before an effective level of vector-mediated transgenic product is expressed.

[0236] In certain embodiments, the FcRn ligand is delivered one to seven days before the administration of the vector (e.g., rAAV). In certain embodiments, the FcRn ligand is delivered every day. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct) is delivered on the same day of the administration of the vector. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct) is delivered at least one day to four weeks after rAAV administration. In certain embodiments, the ligand is delivered after rAAV administration for four weeks to six months. In certain embodiments, the ligand is administered by a route of administration different from that of rAAV. In certain embodiments, the ligand is administered orally, intravenously, or intraperitoneally. See also International Patent Application No. PCT / US2021 / 037575 of WO 2021 / 257668 A1, filed on June 16, 2021 and now published, which is incorporated herein by reference in its entirety.

[0237] In one embodiment, the compositions described herein are used in a method for delaying tumor growth, wherein the tumor is HER2+ brain metastatic cancer. In one embodiment, the compositions described herein are used in a method for delaying tumor growth, wherein the tumor is HER2+ brain metastatic breast cancer. In one embodiment, the compositions described herein are used in a method for delaying tumor growth, wherein the tumor is HER2+ brain metastatic gastrointestinal cancer. In still another embodiment, the compositions described herein can be used to reduce the tumor size of a subject. In another embodiment, the compositions described herein can be used to reduce the number of cancer cells in non-solid tumor cancers. In another embodiment, the compositions described herein can be used for prevention. In one embodiment, the compositions described herein are used in a method for preventing metastasis in a patient with HER2+ tumors, wherein the patient has a risk of metastasis. In one embodiment, the compositions described herein are used in a method for preventing brain metastasis in a patient with advanced HER2+ tumors, wherein the patient has a risk of brain metastasis. In one embodiment, the compositions described herein are used to prevent brain metastasis in a patient with d HER2+ tumors after stereotactic radiosurgery, i.e., a method for preventing recurrence in a patient with HER2+ breast cancer brain metastasis. In another embodiment, the compositions provided herein are used in a method for improving the overall survival and / or progression-free survival of a patient. In certain embodiments, the compositions described herein are used in a method of treatment to delay progression of brain metastases in patients who have achieved disease stabilization by standard of care therapy. In certain embodiments, the compositions described herein are used in a method of treatment to control brain metastases and alleviate symptoms in patients who have progressed. Optionally, the AAV compositions as described herein are administered in the absence of additional exogenous pharmacological or chemical agents or other physical disruptions of the blood-brain barrier.

[0238] In combination therapy, the immunoglobulin constructs delivered by AAV described herein are administered before, during or after starting treatment with another agent and in any combination thereof, i.e., before and during, before and after, during and after, or before, during and after starting anti-tumor therapy. For example, AAV can be administered between 1 day and 30 days, preferably between 3 days and 20 days, and more preferably between 5 days and 12 days before starting radiotherapy. In another embodiment of the invention, chemotherapy is administered simultaneously with AAV-mediated immunoglobulin (antibody) therapy, or more preferably after AAV-mediated immunoglobulin (antibody) therapy. In still other embodiments, the compositions of the present invention can be combined with other biological agents, such as recombinant monoclonal antibody drugs, antibody-drug conjugates, etc. Further, the combination of immunoglobulin constructs delivered by different AAVs as discussed above can be used in such treatment regimens.

[0239] Any suitable method or route may be used to administer the compositions containing AAV as described herein, and optionally co-administer anti-tumor agents and / or antagonists of other receptors. The anti-tumor agent regimen used in accordance with the present invention includes any regimen that is considered most suitable for treating the patient's tumor condition. Different malignancies may require the use of specific anti-tumor antibodies and specific anti-tumor drugs, which will be determined based on the patient to patient situation. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration. The dose of the antagonist administered depends on many factors, including, for example, the type of antagonist, the type and severity of the tumor being treated, and the route of administration of the antagonist.

[0240] Reagent test kit

[0241] In certain embodiments, a kit is provided, the kit comprising a concentrated carrier suspended in a formulation (optionally frozen), an optional dilution buffer, and the device and components required for intrathecal, intraventricular or intracisternal administration. In another embodiment, the kit may additionally or alternatively include components for intravenous delivery. In one embodiment, the kit provides enough buffers to allow injection. Such buffers can allow the dilution of the concentrated carrier to be about 1:1 to 1:5 or more. In other embodiments, a higher or lower amount of buffer or sterile water is included to allow the treating clinician to perform dose titration and other adjustments. In still other embodiments, one or more components of the device are included in the kit. Suitable dilution buffers, such as saline, phosphate buffered saline (PBS) or glycerol / PBS are available.

[0242] It is to be understood that the compositions in the kits described herein are intended to be applicable to other compositions, regimens, aspects, embodiments and methods described across this specification.

[0243] Devices and methods for delivering pharmaceutical compositions

[0244] On the one hand, the vector, rAAV or its composition provided herein can be administered intrathecally by the method and / or device described in WO2017 / 136500 and WO 2018 / 160582 provided in this section, and the document is incorporated herein by reference. Alternatively, other devices and methods can be selected. In certain embodiments, the method includes a step of injecting into the cerebellomedullary cistern of the patient by a spinal needle under the occipital bone guided by CT. As used herein, the term computed tomography (CT) refers to radiography, wherein a three-dimensional image of a body structure is constructed by a computer by a series of planar cross-sectional images made along an axis. In certain embodiments, the device is described in No. 2018-0339065A1, U.S. Patent Publication No. 2018-0339065A1, published on November 29, 2019, which is incorporated herein by reference as a whole. In certain embodiments, the vector, rAAV or its composition provided herein can be administered using an Ommaya reservoir.

[0245] It should be understood that the compositions in the devices described herein are intended to be applicable to other compositions, schemes, aspects, embodiments and methods described in this specification.

[0246] As used herein, the phrase "relieve symptoms", "improve symptoms" or any grammatical variant thereof refers to reversing Her2+ metastatic cancer in brain-related symptoms. In one embodiment, the mitigation or improvement refers to the total number of symptoms of the patient after administering the described composition or using the described method, which is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use. In another embodiment, the mitigation or improvement refers to the severity or progression of symptoms after administering the described composition or using the described method, which is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use.

[0247] As used herein, "conservative amino acid substitution" or "conservative amino acid substitution" refers to an amino acid change, substitution or replacement with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity and size), which is well known to practitioners in the art. See also, for example, FRENCH et al. What is a conservative substitution? Journal of Molecular Evolution, March 1983, Vol. 19, No. 2, pp. 171-175 and YAMPOLSKY et al. The Exchangeability of Amino Acids in Proteins, Genetics. August 2005; 170(4): 1459-1472, each of which is incorporated herein by reference in its entirety.

[0248] As used herein, the term "administering" or any grammatical variations thereof refers to delivering a composition described herein to a subject.

[0249] The words "comprise / comprises / comprising" are to be interpreted as inclusive rather than exclusive. The words "consist / consisting" and variations thereof are to be interpreted as exclusive rather than inclusive. Although various embodiments in the specification are presented using "comprising" language, in other cases, it is also intended to use "consisting of" or "consisting essentially of" language to include and describe related embodiments. As used throughout this specification and claims, the terms "comprise", "comprises", "including" and variations thereof include other components, elements, integers, steps, etc. In contrast, the term "consisting of" and variations thereof do not include other components, elements, integers, steps, etc.

[0250] It should be noted that the term "a" or "an" refers to one or more. Thus, the terms "a or an", "one or more", and "at least one" are used interchangeably herein.

[0251] As used herein, unless otherwise indicated, the term "about" or "~" refers to a variation of ±10% relative to a reference integer and values ​​therebetween. For example, "about" 500 μM includes ±50 (i.e., 450-550, including integers therebetween). For other values, particularly when referring to percentages (e.g., 90% of the taste), the term "about" includes all values ​​within the range, including integers and fractions.

[0252] As used herein, unless otherwise indicated, the term "about" when used to modify a numerical value means a variation of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9 and ±10 or values ​​therebetween) relative to a given reference.

[0253] In some cases, the term "E+#" or the term "e+#" is used to refer to an exponent. For example, "5E10" or "5e10" is 5x10 10 . These terms can be used interchangeably.

[0254] With respect to the description of various embodiments herein, in another embodiment, it is contemplated that each of the compositions described herein is useful in the methods of the present invention. In addition, it is also contemplated that in another embodiment, each of the compositions described herein as being useful in the methods is itself an embodiment of the present invention.

[0255] Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs and with reference to the public text, which provides a general guide for many of the terms used in this application to those skilled in the art.

[0256] Examples

[0257] The following examples are provided to illustrate certain aspects of the claimed invention. The invention is not limited to these examples.

[0258] Breast cancer is the most common cause of brain metastasis in women, affecting 15-30% in all patients. Although systemic Her2 targeted therapy has achieved success, up to half of the patients with Her2-positive (HER2-positive, HER2+ or her2+) primary breast cancer develop breast cancer brain metastasis (BCBM). The subtype converted from Her2-primary breast tumor accounts for another 15% in Her2+BCBM, thereby making the total Her2 positive rate in the BCBM environment reach nearly 65% ​​in the case. HER2-positive BCBM can show as pia mater (LM) or substance. LM lesions directly enter the cerebrospinal fluid (CSF). LM disease accounts for 8% of the total cases of HER2-positive BCBM. Although Her2 targeted therapy is directly delivered to the central nervous system (CNS) and has shown promising clinical results, long-term feasibility is at least partially subject to the need for repeated administration to overcome the half-life of the drug in the cerebrospinal fluid relative to the limitation of shorter (e.g., respectively about 1 day relative to 5 days) in the systemic circulation.

[0259] Trastuzumab, also known as Herceptin, is a humanized monoclonal antibody (mAb) for HER2, developed by Genentech and commercialized by Genentech / Roche. Trastuzumab is intended to treat HER2+ breast cancer and metastatic breast cancer, and is used in combination with chemotherapy or as a single agent (after having received chemotherapy). Trastuzumab plus chemotherapy increases both the progression-free survival and overall survival of patients with metastatic breast cancer. Since trastuzumab cannot cross the blood-brain barrier, systemic administration of trastuzumab is not intended to treat BCBM.

[0260] In order to achieve sustained expression of trastuzumab (Herceptin) in the CNS without the need for frequent dosing, a gene therapy method was developed to treat BCBM, which includes a single local administration of an AAV vector encoding trastuzumab. Previous work has shown that intracerebroventricular (ICV) injection of AAV expressing trastuzumab doubles the life expectancy of Rag1 knockout (KO) mice carrying BT-474 breast cancer brain tumors. Rag1 KO mice that received AAV-trastuzumab before implanting BT-474 tumor cells into the brain showed a dose-dependent delay in tumorigenesis. See also, International Patent Application No. PCT / US2015 / 027491, filed on April 24, 2015 and published on October 29, 2015 as WO 2015 / 164723A1.

[0261] In the current study, extensive gene therapy vector engineering and in vivo evaluation were performed. Following a single ICV administration of the engineered vector to Rag1 KO mice, trastuzumab concentrations in the brain parenchyma ranged from 10 to 165 ng / mg brain protein by day 28.

[0262] Previous studies utilizing CNS delivery of gene therapy vectors have shown that vectors leak into the systemic circulation, which results in detectable transgene expression in the blood after peripheral transduction. In order to reduce the potential risk of trastuzumab-induced cardiotoxicity, the use of intravenously administered, plasma-derived, pooled human immunoglobulin (IVIG) as a source of anti-AAV neutralizing antibodies was investigated to reduce peripheral transduction. Rag1 KO mice that received IVIG two hours prior to ICV administration of AAV-trastuzumab showed similar levels of trastuzumab in the brain, while showing a 10-fold reduction in serum trastuzumab concentrations, compared to controls that received naive mouse serum. Vector biodistribution analysis indicated that the reduction in serum trastuzumab levels was due to effective blockade of liver transduction after IVIG pretreatment.

[0263] These data suggest that administration of AAV vectors to the CNS to achieve sustained expression of trastuzumab can effectively treat Her2+ BCBM. Additionally, patients with existing neutralizing antibodies to the AAV vector capsid do not need to be excluded in future clinical trials.

[0264] Example 1: Generation of rAAV containing engineered trastuzumab

[0265] In the study herein, various engineered sequences encoding trastuzumab were generated and compared. Using triple transfection technology, rAAV was generated using the following: (1) a cis plasmid encoding AAV2 rep protein and AAVhu68 VP1cap gene (alternatively, AAVrh91, AAVhu95 or AAV9), (2) a cis plasmid containing adenoviral helper genes not provided by a packaging cell line expressing adenovirus E1a, and (3) a trans plasmid containing a vector genome for packaging in an AAV capsid. See, for example, US2020 / 0056159. The trans plasmid is designed to contain a vector genome comprising an engineered trastuzumab sequence (the sequence comprising an IL-2 signal peptide and a T2A element), optionally comprising a furin cleavage sequence. The vector genome includes (among others): (i) (SEQ ID NO: 1); (ii) (SEQ ID NO: 25); (iii) (SEQ ID NO: 27); or (iv) (SEQ ID NO: 50).

[0266] The vector genome contains AAV 5' inverted terminal repeats (ITRs) and AAV 3'ITRs at the most 5' and 3' ends, respectively. ITRs flank the sequence of the expression cassette packaged into the AAV capsid, and the sequence of the expression cassette has a sequence encoding trastuzumab. The expression cassette further comprises a regulatory sequence operably linked to the fusion protein coding sequence, the regulatory sequence comprising a promoter (chicken β-actin promoter, a CB7 hybrid promoter containing a CMV IE enhancer, a chicken β-actin promoter and a chimeric intron (containing a chicken β-actin intron) or a ubiquitin C (UbC) promoter), an intron (chicken β-actin intron or a chimeric intron containing a Promega intron), SV40 polyA.

[0267] AAV vector technology allows to fully express engineered trastuzumab transgene in the brain using various promoters as indicated. Expressing trastuzumab by AAV vector provides an effective one-time gene therapy method for preventing and / or treating HER2+BCBM. The current treatment strategy for HER2+BCBM uses a combination of different HER2 regulators (anti-HER2 monoclonal antibodies and kinase inhibitors) and chemotherapeutics. In addition, intracranial delivery of AAV-trastuzumab can prevent or significantly delay the appearance of tumor lesions in HER2+ breast cancer patients at high risk of developing BCBM. Alone or in combination with the systemic HER2 kinase inhibitors of the care standard, CNS delivery of AAV-trastuzumab (engineered trastuzumab of the coding sequence) can be used as an alternative method to achieve disease remission or stability, because it can avoid treatment scraping, and cause the long-term persistent expression of trastuzumab.

[0268] Example 2: rAAV Evaluation in Mice

[0269] introduce

[0270] The human epidermal growth factor receptor (EGFR) tyrosine kinase family consists of four closely related membrane-bound cell surface receptors: EGFR, HER2, HER3, and HER4. Ligand-dependent homodimerization or heterodimerization of these receptors leads to phosphorylation of the intracellular tyrosine kinase domain and activates signaling pathways that promote cell proliferation, migration, and differentiation. Among the EGFR family proteins, HER2 is the only constitutively active orphan receptor with no known ligands; HER2 also has the strongest catalytic activity and is the preferred dimerization partner for all other members of the EGFR family. HER2 expression is required for normal development of mammalian embryos, and abnormal HER2 signaling is a known oncogenic driver. Amplification / overexpression of HER2 promotes tumorigenesis by overactivating oncogenic RAS-MAPK and PI3 / AKT / mTOR signaling pathways. HER2 has been found to be amplified / overexpressed in several tumors (e.g., breast, gastric, esophageal, and ovarian cancers) and is often associated with an aggressive tumor phenotype and a higher risk of recurrence.

[0271] HER2 positivity is observed in 15-20% of all invasive breast cancer cases. Up to half of patients with HER2+ breast cancer will go on to develop breast cancer brain metastases (BCBM). Subtype switching due to HER2- primary breast tumors accounts for an additional 15% of HER2+ BCBM, bringing the total HER2 positivity rate in the BCBM setting to nearly 65% ​​of cases. A regimen of humanized monoclonal antibodies against HER2 to target advanced HER2+ breast cancer has been associated with a significant extension of survival (1.5 years in 2001, compared to about 5 years today); however, despite the latest treatments (trastuzumab + tucatinib (HER2 tyrosine kinase inhibitor) + capecitabine), the median overall survival of patients diagnosed with HER2+ BCBM is only 29.1 months. This difference can be explained at least in part by the presence of an intact blood-brain barrier that blocks large molecular weight biologics (such as trastuzumab) from entering the brain parenchyma (the ratio of trastuzumab levels in serum to trastuzumab levels in cerebrospinal fluid (CSF) is 420:1). Due to subtherapeutic drug concentrations in the brain, HER2+ breast cancer patients receiving systemic antibody therapy are at increased risk of developing BCBM.

[0272] Alternatively, HER2 targeted therapy is directly delivered to the central nervous system (CNS) and has shown promising clinical results. A case report presents that a 34-year-old patient with HER2+BCBM, after repeated intraventricular injection of trastuzumab treatment, brain and dura mater metastatic lesions (>6 months) are stable. Another case report has shown that a 39-year-old patient with HER2+ breast cancer and leptomeningeal cancer spreads after repeated intrathecal infusion of trastuzumab, and overall health (about 11 months) significantly improves [Bousquet, G. et al., Intrathecal Trastuzumab Halts Progression of CNS Metastases in Breast Cancer, Journal of Clinical Oncology, 2016, 34 (16): e151-e154, electronic version June 1, 2016]. However, the long-term feasibility of this approach is limited by the need for repeated drug administration to overcome its significantly shorter half-life in the CSF versus the systemic circulation (approximately 1 day versus 5 days, respectively).

[0273] Trastuzumab expression study

[0274] In order to achieve sustained CNS expression of trastuzumab, a gene therapy method for treating HER2+BCBM was developed, which includes a single local administration of an adeno-associated virus (AAV) vector encoding trastuzumab (engineered coding sequence, i.e., trastuzumab-coGW). In order to evaluate the expression efficiency of trastuzumab in the CNS, 16 new AAV-trastuzumab vectors were engineered. All vector iterations were packaged into AAV rhesus monkey 91 (AAVrh91) capsids and injected into adult female Rag1 knockout (KO) mice (N=4 to 5 mice / vector group) aged 7-8 weeks via the intracerebroventricular (ICV) route for subsequent gene expression studies. Rag1 KO mice injected with phosphate-buffered saline (PBS) were used as a vehicle sham control group (N=5 mice). The Rag1 KO mouse model was selected for this purpose, namely because it is a preferred model for tumor stimulation studies to confirm HER2 targeting using a human-derived xenograft tumor model. Sub-M blood collections were performed on days -1, 7, 14, 21, and 28. On day 28, euthanasia was performed by systemic perfusion with PBS.

[0275] To quantify trastuzumab expression, 1x10 cells / mL of each AAVrh91-trastuzumab vector or PBS were used. 11 Serum and brain homogenates (at endpoint) from ICV-treated systemically perfused mice with 100 genomic copies (GC) / mouse were studied by ELISA (enzyme-linked immunosorbent assay) analysis over a 28-day time course (Table 1 below).

[0276] Table 1.

[0277]

[0278] Figure 2A Results of an ELISA are shown, plotting measurements of trastuzumab concentration (ng / mg protein) in perfused brain tissue samples collected following administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. Figure 2BResults of an ELISA are shown, plotting the measurement of trastuzumab concentrations (μg / mL) in serum samples collected following administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined constructs) and PBS. The mean peak serum concentration for patients receiving 500 mg (the highest dose) was 377 μg / mL. Figure 4A Further shown are data measuring trastuzumab concentrations in serum samples collected on day 28 after administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (a construct previously examined) and PBS. Figure 4B Further shown are data measuring trastuzumab concentrations in perfused brain tissue samples collected on day 28 after administration of AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (a construct previously examined) and PBS.

[0279] Additionally, Fig. 8A It is shown that ICV injection of AAV results in sustained expression of trastuzumab in Rag11 KO mice, as measured in serum samples by ELISA. Figure 8B It is shown that ICV injection of AAV results in sustained expression of trastuzumab in Rag1 KO mice, as measured by ELISA in brain homogenate (perfused) samples.

[0280] Additionally, vector biodistribution within the brain and liver was assessed to help determine the extent of brain and peripheral transduction following ICV administration (Table 2 below).

[0281] Table 2.

[0282]

[0283] Figure 3A and3B A summary view of the data in Table 1 is shown. Figure 3A Results of a biodistribution study are shown, where measurements of DNA concentration (GC / μg) in collected liver and brain tissues are plotted. Figure 3B Results of a biodistribution study are shown, where measurements of RNA concentration (copies / 100 ng of RNA) in collected liver and brain tissues are plotted. Figure 4C Further shown are data from measurements of DNA biodistribution in brain and liver tissue samples collected at day 28 following administration of AAVrh91.CB-CI.IL2_V1_Trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_Trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_Trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.Trastuzumab.SV40 (previously examined construct) and PBS. All vectors tested were well tolerated in mice and did not raise safety concerns.

[0284] After a single ICV delivery of all 16 AAVrh91-trastuzumab vectors, trastuzumab was detected in the serum and brain of mice by ELISA ( Figure 2A and 2B In the CB promoter cohort, the trastuzumab-coGW construct encoding the human engineered trastuzumab sequence (AAVrh91.CB7.CI.IL2_V1.trastuzumab-coGW.SV40) had the highest expression in the brain at 28 days after ICV delivery and the lowest systemic concentration in serum (average 26 ng antibody per mg protein). Western blot analysis showed an equimolar ratio of antibody heavy and light chains, indicating accurate assembly ( Fig.11A and 11B ). Fig.11A and 11B Representative Western blots are shown, demonstrating expression of trastuzumab heavy and light chains in brain lysates (e.g., Figure 2A and 2B ).

[0285] To further improve the current lead engineered vector CB.CI.IL2_V1.Trastuzumab-coGW.SV40, a study was conducted to evaluate the transduction efficiency of CB.CI.IL2_V1.Trastuzumab-coGW.SV40 packaged into AAVhu95 capsids. Briefly, in the AAVhu95 capsid evaluation study, CB.CI.IL2_V1.Trastuzumab-coGW.SV40 and isotype control vectors (i.e., 3bnc117 antibody encoding control vectors) were packaged into AAVhu95 capsids and administered to mice by ICV injection. AAVhu95 transduction efficiency was evaluated by trastuzumab expression using ELISA and by fluorescence imaging. Figure 5A Shown are the expression levels (μg / mL) of trastuzumab compared to capsid control and PBS as measured in serum samples at days -1, 7, 14 and 28 after administration of AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40. Figure 5B Shown are the expression levels (μg / mL) of trastuzumab compared to capsid control and PBS as measured in brain tissue samples at days -1, 7, 14, and 28 after administration of AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40. Figure 6 Vector biodistribution (GC / diploid cell) samples at days -1, 7, 14, and 28 after administration of AAVhu95.CB.CI.IL2_V1.Trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 are shown compared to capsid control and PBS.

[0286] To examine the preclinical activity of rAAV.trastuzumab-coGW (also referred to as AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40 or AAV.trastuzumab), a mouse model as described in Table 3 immediately below, including trastuzumab sensitivity and expected outcomes, was used. Briefly, the xenograft implantation procedure included guide screw implantation and tumor cell transplantation (after >1 week), see also Lal et al., J Neurosurg 92:326-333, 2000.

[0287] Table 3

[0288]

[0289] Preclinical activity was examined in both preventive and therapeutic models (also known as disease-modifying models). Figure 1A A schematic representation of the designed study examining preclinical activity in mice is shown. Briefly, in a prophylactic model (prophylactic model with BT-474 (ER+ / PR+ / HER2+) cell line), guide screws were implanted on days -35 to -28, and Rag1-KO female mice, approximately 7-8 weeks old, were injected ICV with 10 11 GC / mice were treated with AAV, implanted with estrogen pellets on day -1, and intracranially implanted with BT-474 cell lines on day 0. Survival was monitored until human endpoints were reached. Figure 1B Results demonstrating preclinical activity of engineered trastuzumab (antitumor activity against breast cancer brain metastases) are shown as a graph of the probability of mouse survival after tumor implantation. Fig.10 Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 (preventive and prophylactic treatment) is shown. For endpoints, signs of disease progression used included decreased activity, hunched posture, weight loss. The median survival of the designated groups was determined: PBS, n=12, 7 weeks (53 days); isotype control, n=12, 5 weeks (37.5 days); AAV.Trastuzumab, n=12, not reached. These results show that preventive and prophylactic treatment with AAVrh91.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 and AAVhu95.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 prevents the development of HER2+ brain tumors. Pretreatment with AAV.trastuzumab prevented brain xenograft progression, with 100% survival over 10 weeks.

[0290] In addition, the preclinical activity of the BT-474 (ER+ / PR+ / HER2+) mouse model (ie, treatment of breast cancer brain xenografts with AAV.trastuzumab) was examined. Fig.14A A schematic representation of the experimental design of a study examining preclinical activity in mice is shown. Briefly, guide screws were implanted on day -7 (i.e., 7 days before tumor cell implantation), tumor cells were implanted on day 0, and 1e11 (1x10 11AAVhu95.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 was administered ICV at a dose of 1:10 GC / mouse) followed by imaging to monitor tumor burden until humane endpoint. Fig. 14B Shown are quantitative results of tumor burden assessment (bioluminescence assessment) of mouse xenografts (BT-474 (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to PBS and isotype control. Fig. 14C Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 is shown. Briefly, human endpoints included signs of disease progression, such as decreased activity, hunched posture, weight loss. The median survival was determined for the designated groups: PBS, n=8, 4 weeks; isotype control, n=10, 5 weeks; AAV.Trastuzumab, n=14, not reached. These results show that treatment with AAV.Trastuzumab on day +3 inhibited tumor progression.

[0291] In addition, the preclinical activity of the MDA-MB-453 (ER- / PR- / HER2+) (also called MDA-453; highly sensitive to trastuzumab) mouse model was examined. Briefly, in the disease remission model, intracranial implantation of the MDA-453 luciferase+ cell line was performed on day 0, and AAV treatment was performed by ICV injection on day 3 (10 11 GC / mouse), where survival was examined until humane endpoint. Figure 7Quantitative results of tumor bioluminescence assessment of mouse xenografts (MDA-MB-453 (ER- / PR- / HER2+)) after treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype control (isotype control: 47 days; PBS: 44 days; rAAV.Trastuzumab-coGW: >6 weeks). Efficacy of injection of rAAV.Trastuzumab-coGW after establishment of Her2+ cancer in the mouse brain. Complete tumor remissions were observed as measured by tumor growth (chart) and survival (inset). Representative bioluminescent images were taken at weeks 4, 5, and 6 for quantitative analysis of the results of tumor burden assessment by IVIS (bioluminescent assessment) of mouse xenografts (MDA-MB-453 (ER- / PR- / HER2+)) after treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype controls (data not shown). Additionally, representative bioluminescent images were taken for quantitative analysis of the results of tumor bioluminescent assessment of mouse xenografts (MDA-MB-453 (ER- / PR- / HER2+)) after treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype controls. Further, Fig. 9 Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 (disease remission) is shown. The median survival was determined for the indicated groups: PBS, 6 weeks (44 days); isotype control, 6 weeks (47 days); AAV.Trastuzumab, not reached. These results show that tumor-bearing mice treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 achieved complete disease remission with 100% survival over 10 weeks.

[0292] In addition, a dose comparison study was performed using the MDA-MB-453 mouse model to examine long-term transgene expression following a single (or two; repeat dosing on day 59 after tumor cell implantation) ICV administration of AAV.Trastuzumab. Fig.15A Shown is a schematic representation of the experimental design for studies examining long-term transgene expression following single or double (re-dosing) administration of AAV.Trastuzumab. Fig. 15B Shown are trastuzumab expression levels measured by ELISA using serum samples collected on day 98, plotted as μg / mL of trastuzumab. Fig. 15CShown are trastuzumab expression levels measured by ELISA using brain samples (perfused brain homogenates) collected on day 98, plotted as μg / mL of trastuzumab. These results show that trastuzumab is stably expressed for >98 days after a single ICV injection.

[0293] In addition, the preclinical activity of BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts was examined, which provided an evaluation in a more stringent model. Fig. 12A A schematic representation of the experimental design of a study examining preclinical activity in mice is shown. Fig. 12B Shown are quantitative results of tumor bioluminescence assessment of mouse xenografts (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype control. Fig. 12C Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 is shown. These results show that treatment of BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts with AAV.trastuzumab on day +3 delayed tumor progression, with median survival observed of 4 weeks for isotype control, 4 weeks for vehicle control, and 7.5 weeks for AAV.trastuzumab (i.e., approximately two-fold increase in survival).

[0294] In addition, preclinical activity in the MDA-MB-231-HER2 / low tumor model was examined. Fig.13A A schematic representation of the experimental design of a study examining preclinical activity in mice is shown. Fig. 13B Shown is a Kaplan-Meier survival analysis of the survival probability of tumor-bearing mice (MDA-MB-231 HER2 / low tumors) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40. Fig. 13C Shown is the Her2 expression level in MDA-MB-231 cells after surface staining with isotype control antibodies, as measured by flow cytometry. Figure 14 D shows the Her2 expression level in MDA-MB-231 cells after surface staining with Her2 antibodies, as measured by flow cytometry. These results show that the progression of MDA-MB-231HER2 / low tumors was suppressed by AAV.trastuzumab ICV treatment at day +3.

[0295] In conclusion, single-dose ICV administration of AAV.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 resulted in robust anti-tumor responses in the HER2+ breast-to-brain metastasis xenograft mouse model tested.

[0296] AAV-trastuzumab vector treatment driven by CB or UbC promoter leads to the sustained expression of antibody after 28 days. This gene therapy method has the potential of preventing and / or treating HER2+ metastatic brain lesions after a single administration by providing and maintaining the long-term and lasting steady-state level of drugs in the brain after a single treatment of local delivery of trastuzumab, and has the potential of becoming an effective one-time gene therapy method for preventing and / or treating HER2+BCBM. The current treatment strategy for HER2+BCBM uses a combination of different HER2 regulators (anti-HER2 monoclonal antibodies and kinase inhibitors) and chemotherapeutics. In addition, intracranial delivery of AAV-trastuzumab can prevent or significantly delay the appearance of tumor lesions in HER2+ breast cancer patients at high risk of developing BCBM. Alone or in combination with the systemic HER2 kinase inhibitors of the care standard, CNS delivery of AAV-trastuzumab can be used as an alternative method to achieve disease remission or stability, because it can avoid treatment scraping, and cause the long-term and lasting expression of trastuzumab.

[0297] The natural history of tumor progression research

[0298] In this study, the effectiveness of the selected tumor model approach was evaluated. Briefly, in this study, green fluorescent protein-positive (GFP+) breast cancer cells (e.g., BT-474, BT-474 clone 5 (a cell line that is trastuzumab-resistant and invasive in vivo), MDA-MB-453, MDA-MB-231 cells) were sorted and expanded after sorting. Then, 1.5x10 5 The cells were implanted into PBS / matrigel (50:50) (e.g., >1 week after guide screw implantation followed by tumor cell implantation). The advantages of the guide screw method are that it does not require a stereotactic frame, it is streamlined (i.e., more animals per procedure (approximately 6 minutes / mouse)), and the screws can even be implanted several weeks before tumor cell implantation, which allows intratumoral delivery of therapeutic agents (i.e., the screws can be reopened several times). To date, hematoxylin and eosin (H&E) histology has demonstrated a 100% tumor take-away rate (data not shown). In addition, a GFP / luciferase dual reporter breast cancer cell line was generated, which was also used in the studies herein.

[0299] Additionally, a patient-derived xenograft model was developed using a HER2+ breast cancer fragment from (DFCI) and further implanted into 6-8 week old NOD scidγ mice (NSG TM Mouse, Jackson Laboratory) tumor slurry, where the time to progression was between 6-12 months.

[0300] In addition, mouse studies were expanded to include tumor challenge experiments using HER2+ and HER2- xenograft mouse models to confirm anti-tumor efficacy and target specificity, as well as a combination therapy approach including a clinical HER2 kinase inhibitor to maximize the anti-tumor effect. Further studies included evaluation of the AAV-trastuzumab vector packaged into the AAVhu95 capsid. The AAV-trastuzumab vector was then further evaluated for toxicology in rhesus macaques.

[0301] Example 3: Further evaluation of rAAV.trastuzumab in mice

[0302] First, the expression levels of the revised rAAV.Trastuzumab vectors in mice were evaluated. The revised rAAV.Trastuzumab included vectors containing a furin cleavage site in the linker sequence (furin / T2A) between the heavy and light chains of the trastuzumab coding sequence: AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1_Trastuzumab-coGW.SV40.

[0303] Fig.16 and 17 Shown is a comparison of the assessment of trastuzumab expression levels in mice following administration of rAAV.Trastuzumab and amended rAAV.Trastuzumab. Fig.16 The expression level of trastuzumab in serum after administration of AAVhu95M199.CB7.CI.IL2_Trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Trastuzumab-coGW.SV40 into mice is shown. Fig.17Shown is the trastuzumab expression level in the brain (perfused) after administration of AAVhu95M199.CB7.CI.IL2_Trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Trastuzumab-coGW.SV40 in mice.

[0304] Next, a study was conducted to examine the lowest effective dose that confirms the therapeutic effect of rAAV.trastuzumab. Fig.18A A schematic diagram of the experimental design for the study evaluating tumor burden is shown. Briefly, guide screws were implanted seven days before cell implantation (D-7) and MDA-MB-453 cells (2.5x10 5 cells / mouse), and on day 3 after cell implantation, mice were given 1x10 11 GC / mice were administered rAAV.Trastuzumab intracranially (ICV) and imaged at weeks 2, 4, and 6 after cell implantation. Fig.18B Results of tumor burden assessments are shown, plotted as total flux (photons / second) at 2, 4, and 6 weeks post tumor cell implantation in mice administered AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. These results demonstrate the therapeutic efficacy of the rAAV.Trastuzumab vector. Fig.18C The Kaplan-Meier survival curves of mice after administration of AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 are shown.

[0305] Next, in 1x10 10 GC / mouse and 1x10 11Tumor challenge and tumor remission studies were performed in the MDA-MB-453 xenograft mouse model with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 administered to GC / mice. Fig.21A A schematic diagram of the experimental design for the study evaluating tumor priming is shown. Briefly, guide screws were implanted seven days before cell implantation (D-7) and MDA-MB-453 cells (2.5x10 5 cells / mouse), and on day 3 after cell implantation, mice were given 1x10 11 rAAV.Trastuzumab was administered intracranially (ICV) at a dose of 100 GC / mouse and imaging was performed at weeks 2, 4, and 5 after cell implantation. Fig. 21B Tumor growth is shown, which is plotted as 1x10 11 Total flux (photons / second) measured at 2, 4, and 5 weeks after tumor implantation in mice dosed with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at GC / mouse. Fig.22A A schematic diagram of the experimental design for the study evaluating tumor priming is shown. Briefly, guide screws were implanted seven days before cell implantation (D-7) and MDA-MB-453 cells (2.5x10 5 cells / mouse), and on day 3 after cell implantation, mice were given 1x10 10 rAAV.Trastuzumab was administered intracranially (ICV) at a dose of 100 GC / mouse and imaging was performed at weeks 2, 4, and 5 after cell implantation. Fig. 22B Tumor growth is shown, which is plotted as 1x10 10 Total flux (photons / second) measured at 2, 4, and 5 weeks after tumor implantation in mice dosed with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at GC / mouse. Fig.23A The results of tumor burden assessment as examined by imaging are shown, and the results are plotted as 1x10 10 GC / mouse and 1x10 11Total flux (photons / second) in mice administered with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at doses of GC / mouse. Fig. 23B The results of Kaplan-Meier survival analysis are shown, and the results are plotted as 1x10 10 GC / mouse and 1x10 11 The survival probability of mice administered with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at a dose of GC / mouse.

[0306] Additionally, studies evaluating anti-tumor activity were performed in the BT474 clone 5 cell xenograft tumor model. Fig.19A A schematic diagram of the experimental design for the study evaluating antitumor activity is shown. Briefly, guide screws were implanted seven days before cell implantation (D-7), and E2 pellets (1.5×10 5 cells / mouse), and on day 3 after cell implantation, mice were given 1x10 11 GC / mice were administered rAAV.Trastuzumab intracranially (ICV) and imaged at week 2 after cell implantation. Fig.19B Shown are the results of tumor burden assessment, which are plotted as total flux (photons / second) at 2, 4, and 6 weeks after tumor cell implantation in mice administered AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.19C Shown are Kaplan-Meier survival curves of mice (with BT474 xenografts) after administration of AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.

[0307] Next, rAAV vector titration studies were performed and the expression level of trastuzumab was examined. Detection and quantification of trastuzumab were performed by mass spectrometry (LC-MS) and ELISA in perfused mouse brain homogenates. Fig. 20 Results of a vector titration study are shown, plotted as determined by ELISA and mass spectrometry at 1×10 11 GC / mouse and 1×10 10 The expression levels of trastuzumab measured in mice administered rAAV.trastuzumab (AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40) at a dose of 1x10 10 ICV administration of GC / mice resulted in transgene levels similar to those found in NHP brains and was sufficient to induce sustained remissions of tumors in MDA-MB-453 brain xenografts. Low doses of rAAV.trastuzumab inhibited tumor progression in a HER2 cell line-derived xenograft model.

[0308] Additionally, a study was performed to examine the effect of IVIG pretreatment in mice following ICV administration of rAAV.Trastuzumab (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). Briefly, healthy TAG1KO female mice were treated with 1x10 11 GC / mice were administered doses of rAAV.trastuzumab, and blood samples for analysis were collected on day 7, day 14, day 21, and day 3 before day 29 after rAAV administration. Fig.24A Shown are trastuzumab expression levels in serum after administration of rAAV.trastuzumab with and without IVIG pretreatment. Fig. 24B Shown are trastuzumab expression levels in the brain (perfused) at day 30 after administration of rAAV.trastuzumab with and without IVIG pretreatment. These results show that pretreatment with IVIG reduced systemic levels of trastuzumab, but did not reduce levels of trastuzumab in the brain.

[0309] In summary, the above results confirm the engineering of trastuzumab constructs in AAV. 11 At a dose of 1×10 GC / mouse, trastuzumab inhibited tumor progression in all cell line-derived tumor xenograft models tested (BT-474S and R, MDA-MB-453). Additionally, these data show that lower doses (i.e., 1×10 10GC / mice) was still sufficient to induce tumor remission in MDA-MB-453 xenografts (HR- / HER2+).

[0310] Example 4: rAAV Evaluation in Non-Human Primates (NHPs)

[0311] In this study, the efficacy of rAAV.trastuzumab-coGW vectors in non-human primates (NHP; Nab+ female Indian rhesus monkeys) was examined. In short, at -5 days, baseline measurements of clinical pathology samples, neutralizing antibody (NAb) levels were performed, and neurological examinations and nerve conduction velocity (NCV) tests were also performed. At day 0, the AAV vector specified was administered to NHP (injected into the cerebellomedullary cisterna (ICM)), and clinical pathology samples were further collected at this time, serum and cerebrospinal fluid (CSF) samples were collected for analysis, and NAb was evaluated. At the 7th and 21st days, serum and cerebrospinal fluid (CSF) samples were further collected for analysis. At the 14th day, serum and cerebrospinal fluid (CSF) samples were further collected for analysis, and neurological examinations were further performed. At the 36th-37th day after ICM, autopsy was performed, and clinical pathology samples were further collected, and serum and CSF fluid samples were collected, NAb / PBMC were evaluated, and neurological examinations and NCV tests were further performed before autopsy. Throughout the duration of the study, ELISA for trastuzumab and measurement of humoral immune responses (anti-drug antibodies (ADA)) were performed to assess expression and to further guide the duration of the study. Table 3 below shows a summary view of the study design.

[0312] Table 3.

[0313]

[0314]

[0315] Fig.25A Shown are trastuzumab expression levels in CSF as measured by ELISA on day 14 at days 0, 3, 7, 14, 21, and 36-37 following rAAV administration to NHPs of cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)), cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40), and negative controls. Fig.25BShown are the levels of anti-drug antibodies (ADA) as measured in CSF samples collected from NHPs at Day 0, Day 3, Day 7, Day 14, Day 21, and Day 36-37 after administration of rAAV (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). These results show that trastuzumab was detected in the CSF at the terminal study time point and that ADA to trastuzumab were not detected in the CSF.

[0316] Fig.26A Shown are trastuzumab expression levels in serum as measured by ELISA on days 0, 3, 7, 14, 21, and 36-37 after rAAV administration to NHPs of Cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)), Cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). Fig.26B Shown are the levels of anti-drug antibodies as measured in serum samples collected from NHPs at days 0, 3, 7, 14, 21, and 36-37 after administration of rAAV (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. These results show that low systemic trastuzumab was detected in NHP serum by ELISA after ICM treatment with AAV.Trastuzumab.

[0317] Fig. 27 Quantification of trastuzumab protein in NHP brain tissue after ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). Fig.28AQuantification of trastuzumab protein in NHP spinal cord after ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). Fig.28B Quantification of trastuzumab protein in NHP dorsal root ganglia (DRG) after ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40).

[0318] Figures 29A to 29D The average number of reads by 10XGENOMICS (final sequencing depth - 883x10 6 Results of trastuzumab mRNA detection in NHP brain tissue (cerebellum and occipital cortex) using 100 reads / sample (100 reads / sample). No background detection of trastuzumab was observed in untreated monkeys. Fig.29A Shown are the results of trastuzumab mRNA detection in the cerebellum of NHP 18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.29B Shown are the results of trastuzumab mRNA detection in the cerebellum of NHP 20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.29C Shown are the results of trastuzumab mRNA detection in the occipital cortex of NHP 18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Fig.29D Shown are the results of trastuzumab mRNA detection in the occipital cortex of NHP 20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.

[0319] Fig. 30A and 30BThe biodistribution of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) in various tissues of NHPs after ICM administration is shown. Fig. 30A Shown is the DNA biodistribution of AAV.Trastuzumab in various tissues of NHPs after ICM administration. Fig. 30B Shown is the RNA biodistribution of AAV.Trastuzumab in various tissues of NHPs after ICM administration.

[0320] In summary, these results show that treatment with AAV.trastuzumab can both prevent the development of HER2+ CNS tumors, as well as drive tumor regression, in the tumor animal models evaluated to date.

[0321] Example 5: Outcomes of AAV-mediated delivery of trastuzumab to the central nervous system in Her2+ breast cancer brain metastasis and non-human primate xenograft models

[0322] Conversion relevance

[0323] The incidence of breast cancer metastases to the central nervous system (CNS) is increasing due to advances in systemic therapies that better control extracranial disease but potentially enable metastatic cells within the CNS to escape latency. Breast cancer patients with symptomatic brain and / or leptomeningeal metastases have a poor prognosis as current adjuvant therapies fail to prevent CNS recurrence. Repeated intrathecal (IT) delivery of trastuzumab is safe and provides clinical benefit for patients with HER2+ CNS disease. Trastuzumab Approved in protein form as a gold standard treatment for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. To overcome the treatment burden associated with continuous IT drug administration, an adeno-associated viral (AAV) vector was developed to continuously express trastuzumab within the CNS after a single IT injection. Preclinical studies have shown that AAV-trastuzumab induces responses in an orthotopic model of HER2+ brain metastases and is well tolerated in nonhuman primates. These encouraging preclinical results support future clinical studies.

[0324] summary

[0325] Single-dose gene therapy expressing trastuzumab-like anti-Her antibodies within the CNS represents a therapeutic option for disease prevention or treatment.

[0326] An AAV vector (AAV9.UbC.Trastuzumab) expressing an engineered trastuzumab sequence driven by the ubiquitin C (UbC) promoter was developed for IT administration. CNS transgene expression was assessed in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intracisternal (ICM) injection of AAV9.UbC.Trastuzumab, respectively. Real-time PCR, ELISA, western blotting, in situ hybridization, immunohistochemistry, single-cell nuclear RNA sequencing, and liquid chromatography-mass spectrometry were used. HER2+ breast cancer cell line-derived brain xenografts (BT-474 and MDA-MB-453) were used to determine the efficacy of AAV9.UbC.Trastuzumab.

[0327] AAV9.UbC.Trastuzumab (1x10 11 After administration of 10 vector genome copies (GC) / mouse), transgene expression was detected in brain homogenates and serum of Rag1 knockout mice, with levels remaining stable at 28 days post-administration. A single administration of AAV9.UbC.trastuzumab inhibited tumor progression in the xenograft models tested compared to AAV9.UbC.isotype control. In NHPs, AAV9.UbC.trastuzumab (3x10 13 ICM delivery of 100 GC / animal) was well tolerated and resulted in detectable transgene expression in CNS tissues and cerebrospinal fluid after 36-37 days.

[0328] These results suggest that AAV9.UbC.Trastuzumab exhibits robust activity against HER2+ brain orthotopic xenografts. Given the documented clinical safety of AAV9, this gene therapy could represent a viable approach for targeting HER2+ CNS malignancies.

[0329] To meet the need for novel therapies targeting HER2+ CNS metastases, a gene therapy approach was developed that involves engineering a recombinant adeno-associated virus (AAV) vector to constitutively express trastuzumab within the CNS. An effective single-dose gene therapy would provide a sustained local source of trastuzumab, thereby improving patient outcomes.

[0330] Here, it is demonstrated that a single IT administration of AAV9 encoding engineered trastuzumab driven by the human ubiquitin C (UbC) promoter (AAV9.UbC.trastuzumab) results in a robust anti-tumor response in the therapeutic setting for a HER2+ xenograft BCBM model. It is also shown that injection of AAV9.UbC.trastuzumab into the cisterna magna of rhesus monkey NHPs is well tolerated and results in detectable transgenic protein expression at a level expected to exert a therapeutic effect on an orthotopic cell line-derived xenograft BCBM mouse model. In summary, AAV-mediated expression of trastuzumab in the CNS may represent a viable strategy for the treatment and prevention of HER2+ CNS metastases in breast cancer patients.

[0331] Materials and methods

[0332] Vector construction

[0333] The transgenic expression cassette containing the engineered trastuzumab coding sequence was synthesized by GeneArt (ThermoFisher) and engineered as follows: the heavy and light chains were preceded by the human interleukin 2 (IL2) signal peptide (MYRMQLLSCIALSLALVTNS) and separated by a furin cleavage site, followed by a T2A self-cleaving peptide linker and a mutant IL2 signal peptide (MYRMQLLLLIALSLALVTNS). The UbC promoter was used to drive transgenic expression ( Fig.31A ).

[0334] AAV9. Homotype vector controls were engineered to express 3BNC117, a fully human IgG1κ monoclonal antibody against the HIV envelope. All vectors were packaged in AAV9 capsids, produced and purified by the Penn Vector Core as previously described [Lock, M. et al., Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Human Gene Therapy, 2010. 21(10): 1259-71].

[0335] Cell lines and generation of GFP-luciferase+ breast cancer reporter cell lines

[0336] Human breast cancer cell lines MDA-MB-453 (HTB-131) and BT 474 (HTB-20) were obtained from ATCC and maintained according to the supplier's instructions.

[0337] The cell line was transduced with CMV-luciferase-EF1α-copGFP BLIV 2.0 Lentivector lentiviral vector using TransDuxMAX lentiviral transduction reagent according to the supplier's instructions (Systems Biosciences). The transduced cells were then sorted for the top 50% brightest cells expressing green fluorescent protein (GFP).

[0338] Animal procedures

[0339] All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Animals were monitored daily by animal care staff and / or veterinary staff for any signs of distress requiring intervention.

[0340] Vector administration

[0341] Mice: AAV vectors were administered intracerebroventricularly (ICV) to 7- to 8-week-old female B6.129S7-Rag1tm1Mom / J knockout mice (Rag1 KO, Jackson Laboratory strain #002216) by trained laboratory animal technicians or veterinarians (success rate >95%). Blood was collected from the submandibular vein at the scheduled time points ( Fig.31B ).

[0342] Nonhuman primates (NHPs): Adult female Indian rhesus macaques (3-4 years old) were administered 1 mL of AAV vector via intracisterna magna (ICM) as previously described [Katz, N. et al., Standardized Method for Intra-Cisterna Magna Delivery Under Fluoroscopic Guidance in Nonhuman Primates. Human Gene Therapy Methods, 2018. 29(5): pp. 212-219]. Serum, CSF, and tissue samples from Indian rhesus macaques previously treated with AAV9.GFP by ICM were used as negative controls in the transgene expression assay. Blood was collected from a peripheral vein; up to 1 mL of CSF ( Fig.34A Animals were clinically monitored by veterinarians throughout the study, and the main parameters assessed were vital signs, blood and CSF clinical pathology, and comprehensive neurological assessment (mental, posture, proprioception, gait, reflexes, nerve conduction velocity tests).

[0343] Autopsy

[0344] Mice: For vector expression studies, animals were deeply anesthetized and systemically perfused with saline through the left ventricle at the planned study endpoint. Brain, heart, and liver tissues were snap frozen for analysis.

[0345] NHP: On days 36-37 after vehicle administration, animals were deeply anesthetized and euthanized by intravenous (iv) overdose of pentobarbital followed by cerebral perfusion by injection of 600 mL of saline into the jugular vein. Various tissues were collected for histopathological analysis, including adrenal gland, aorta, bone marrow, femur, bone, sternum, brain (right hemisphere), brain (caudate nucleus), brain (cerebellum), brain (frontal cortex), brain (hippocampus), brain (medulla), brain (occipital cortex), brain (parietal cortex), brain (temporal cortex), brain (thalamus), brain (olfactory bulb), chest tattoo, deep cervical lymph nodes, cervical dorsal root ganglia, thoracic dorsal root ganglia, lumbar dorsal root ganglia, sacral dorsal root ganglia, esophagus, eyes, gallbladder, heart, large intestine, colon, small intestine (duodenum), small intestine (jejunum), kidney (left), kidney (right), liver (left lobe), liver (right lobe), liver (middle lobe), liver (caudate lobe), lung (left), lung (right), lymph nodes: axillary, lymph nodes: cervical, lymph nodes: inguinal, lymph nodes: mesenteric. Muscle (quadriceps), Muscle (gastrocnemius), Nerve (optic nerve (cranial nerve II)), Nerve (sciatic), Nerve (sural), Nerve (median, proximal), Nerve (median, distal), Nerve (median, epon), Nerve (tibia), Ovaries, Pancreas, Salivary glands, Submandibular, Injection site (skin and surrounding tissue), Injection site (spinal cord), Spinal cord (cervical), Spinal cord (thoracic), Spinal cord (lumbar), Spleen, Thymus, Thyroid gland with parathyroid glands, Trachea, Trigeminal nerve + ganglion, Bladder, Macroscopic lesions.

[0346] Tumor Models

[0347] Breast cancer cell lines (250,000 cells / mouse) were resuspended in 1:1 DPBS / Matrigel and implanted into 7- to 8-week-old female Rag1 KO mice via guide screws as previously described [Lal, S. et al., An implantable guide-screw system for brain tumor studies in small animals. Journal of Neurosurgery, 2000. 92(2): p. 326-33]. Three to four days after transplantation, mice were randomly assigned to treatment groups using an online randomization tool (random.org) and treated with AAV vectors by ICV injection. Tumor progression was monitored using the IVIS Spectrum in vivo imaging system (PerkinElmer). Animals were euthanized at humane endpoints (signs of cachexia, lethargy, hunched posture).

[0348] The tumor status of estrogen receptor (ER), progesterone receptor (PR), and HER2 receptors was confirmed by immunohistochemistry of formalin-fixed brain sections.

[0349] Histopathology

[0350] A veterinary pathologist certified by the American College of Veterinary Pathologist performed histopathological evaluations of NHP tissues using formalin-fixed, paraffin-embedded tissues stained with hematoxylin and eosin.

[0351] Immunohistochemistry (IHC)

[0352] Immunohistochemistry was performed on sections of formalin-fixed paraffin-embedded tissues on a Leica Bond Rx automatic stainer using the Bond polymer detection system (Leica Biosystems, DS9800) and DAB as the chromogen according to standard IHC protocols. A 30-minute incubation time was applied to the primary antibodies using the specified conditions to detect estrogen receptor (ER), progesterone receptor (PR) and HER2. After staining, the slides were dehydrated with ethanol and xylene and coverslips were placed.

[0353] Biodistribution of vector DNA and RNA

[0354] DNA and RNA were extracted from snap-frozen tissue samples using the QIAmp mini extraction kit (Qiagen). Vector GC levels were determined by qPCR using vector-specific primers and probes.

[0355] Single-cell nuclear RNA sequencing

[0356] Nuclei isolation and snRNA-seq

[0357] To isolate nuclei from frozen tissue samples, a modified version of a previously published nuclear isolation procedure was used [Zhu, Y. et al., Spatiotemporal transcriptomic divergence across human and macaque brain development. Science, 2018. 362(6420); Kati J. Ernst, KO, Josephine Bageritz, Jan-Philipp Mallm, Andrea Wittmann, Kendra K. Maaβ, Svenja Leible, Michael Boutros, Stefan M. Pfister, Marc Zuckermann, David TW Jones, Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues. bioRxiv, 2020]. For these separations, all buffers and samples were maintained on ice throughout the procedure to maintain nuclear integrity. Buffers were typically made as described below and cooled in advance, and then supplemented immediately before use to make "complete" buffers at final concentrations of 1 mM DTT, 0.8 U / μl RNase inhibitor (Protect RNase inhibitor, Roche) and 1X protease inhibitor (Complete Mini EDTA-free, Roche). To isolate nuclei, about 25 mg of frozen tissue was minced with a scalpel and transferred to a pre-cooled 2 ml Dounce homogenizer with 1 ml of cold complete lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl pH 8.0 and 0.1% Triton X-100). The tissue was homogenized by 10 strokes each with pestle A followed by pestle B, and then filtered through pre-wetted 100 μM and 30 μM filters, collecting the filtered sample into a sterile tube. The homogenizer and filter were washed with an additional 3 volumes of complete lysis buffer and collected with the filtered sample.Then, the sample of two volumes is laid on the top of the cold complete separation buffer (1.8M sucrose, 3mM magnesium acetate and 10mM Tris-HCl pH 8.0) of one volume, and rotated 45 minutes with 21,000xg at 4 ℃.Then, carefully remove supernatant, and add 100 μ l complete resuspension buffer (250mM sucrose, 25mM KCl, 5mMMgCl2, 20mM Tris-HCl pH 7.2) in pipe without mixing.Make sample incubated on ice 10-15 minute, and then make nucleus resuspended completely by gently pipetting up and down 20-30 times, and count with automatic cell counter (Countess 3, Thermo Fisher Scientific).For submitting single nuclear transcriptomics sample, according to the sample loading guide of manufacturer, concentration is adjusted to about 1x10. 6 nuclei / ml (range 0.7-1.2x10 6 Nuclei / ml). To identify individual nuclear transcriptomes, 10x Genomics Chromium Controller and 3' Gene Expression Assay (version 3) were used to achieve approximately 10,000 partitioned nuclei per sample according to the manufacturer's protocol. Nuclear partitioning, reverse transcription and cleanup, cDNA amplification, and library construction and cleanup were all performed as described in the manufacturer's protocol, and the library was sequenced on an Illumina Next Seq 2000. The final sequencing depth of the average read was 883x106 reads / sample.

[0358] snRNA-seq analysis

[0359] After sequencing, the demultiplexed Fastq files were aligned to a custom reference genome consisting of a rhesus macaque reference (Mmul 10) and a fully annotated plasmid sequence for generating rAAV vectors through the Cell Ranger counting pipeline (10x Genomics). The count matrix generated by Cell Ranger was then further analyzed in R using the package Seurat (version 4) and as described (Hao, Y. et al. Cell 184, 3573-3587e3529 (2021)). Each individual sample dataset was normalized using the sctransform function, and principal component analysis (PCA), uniform manifold approximation and projection (UMAP), and cell nucleus clustering were performed on all individual datasets using standard functions in Seurat.

[0360] Antibody

[0361] Manufacturer information for all antibodies is listed in the table immediately below.

[0362]

[0363]

[0364] In situ hybridization (ISH)

[0365] ISH was performed on sections of formalin-fixed paraffin-embedded tissue. The sections were manually processed using the RNAscope 2.5HD detection kit (Advanced Cell Diagnostics, ACD) according to the manufacturer's instructions. The probe consisting of the ZZ probe pair was manufactured by ACD and was designed not to detect endogenous sequences in NHP tissues. Fast Red was used as a chromogen to show the sites of binding probes. The sections were mounted in Fluoromount with DAPI to counterstain the nuclei and imaged with rhodamine and DAPI filter sets.

[0366] Extraction of proteins for ELISA and western blot assays

[0367] Snap frozen brain tissue was homogenized in lysis buffer (20 mM Tris-HCl pH: 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1X EDTA-free protease inhibitor tablet) with 5 mm stainless steel beads using a bead mill homogenizer. The lysate was clarified by centrifugation and the protein concentration was determined by BCA assay.

[0368] Enzyme-linked immunosorbent assay (ELISA)

[0369] Samples and reagents were diluted in DPBS or blocking buffer (5% BSA, 0.05% Tween-20, DPBS) for capture for detection. CSF was diluted 1:5 in blocking buffer. Brain homogenate was diluted to 2 mg total protein / mL in blocking buffer. ELISA assays were performed as previously described [Maple, L. et al., Development and validation of ELISA for herceptin detection in human serum. J Immunol Methods, 2004. 295(1-2): 169-82].

[0370] Trastuzumab

[0371] Mouse: First, 2 μg / mL human ErbB2 / Her2 Fc chimera protein was coated into a high binding 96-well plate. Trastuzumab was detected using an HRP-conjugated anti-human kappa light chain antibody.

[0372] NHP: 1 μg / mL NHP pre-adsorbed mouse anti-human IgG antibody was used for capture. Mouse pre-adsorbed HRP-conjugated goat anti-human IgG Fc antibody was used for detection.

[0373] 0.5 μg / mL anti-HER2-Tra-hlgG1 antibody was used to generate a standard curve.

[0374] Anti-drug antibodies (ADA)

[0375] 1 μg / mL anti-HER2-Tra-hlgG1 antibody was used for capture. 1 μg / mL anti-trastuzumab antibody was used to generate a standard curve. 2 μg / mL HRP-conjugated ErbB2 / Her2 antibody was used for detection.

[0376] Western blotting

[0377] Brain lysates (25 μg / lane total protein) were heat denatured in reducing LDS sample buffer, separated by SDS-PAGE, and transferred to PVDF membranes. The electroblotted membranes were blocked in 5% skim milk. Goat anti-human IgG FC antibody was used to probe the trastuzumab heavy chain, or goat anti-human kappa light chain antibody was used to probe the trastuzumab light chain. HRP-conjugated donkey anti-goat IgG antibody was used to detect the heavy and light chains. The blots were developed using a chemiluminescent substrate.

[0378] Liquid chromatography mass spectrometry (LC-MS)

[0379] Detection of trastuzumab in mouse and NHP brain samples by LC-MS was also performed by LC-MS.

[0380] LC-MS analysis

[0381] Mouse brain lysate

[0382] The characteristic peptides for LCMS analysis and multiple reaction monitoring (MRM) transitions were selected from previous literature [Russo, R. et al., Ultra-performance liquid chromatography / multiple reaction monitoring mass spectrometry quantification of trastuzumabin human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions. Rapid Communications in Mass Spectrometry, 2017. 31(14): p. 1184-1192]. Tissue samples were homogenized in lysis buffer (50 mM ammonium bicarbonate, 6 M urea, 0.15% sodium deoxycholate) at a ratio of 7 μL buffer to about 1 mg tissue. The homogenate was clarified and the protein concentration of the clarified lysate was determined by BCA. Trastuzumab calibration curve was created in the clarified lysate of control mice, and its range was 68-7277ng / mL. Twenty μL calibration standards, animal samples, clarified lysate blanks and clarified lysate double blanks were plated in duplicate to 1mL 96-well plates. 50:50 methanol:acetone was added to each well, the plate was vortexed, and heating was not used to dry in Eppendorf Vaccufuge. The sample was reconstituted in 6M urea, reduced by shaking at 500rpm for 1 hour at 37°C in 8mM (2-carboxyethyl) phosphine, and then alkylated in 30mM iodoacetamide at room temperature in the dark for 30 minutes. Standards, samples and blanks were spiked with 10nM heavy labeled endopeptide standard solution (DTYIHWVR; SEQ ID NO:56) of 20 μL. Samples were diluted 1:3 with 0.1mg / mL trypsin prepared in 50mM ammonium bicarbonate. The samples were digested overnight at 37°C with shaking at 500 rpm. The digestion was quenched to a final concentration of 0.7% formic acid. Solid phase extraction was performed using Waters Oasis HLB sorbent. The eluted peptides were dried in an Eppendorf Vaccufuge without heating and reconstituted in 5:95 acetonitrile:water and 0.1% formic acid.LCMS analysis was performed using Agilent 1260 Infinity II HPLC and Agilent TQ 6495 mass spectrometers. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The stationary phase was Waters Premier HSS T3 1.8 μm 2.1x100 mm, using a gradient of 15% mobile phase B to 21% mobile phase B for 8 minutes. Data were analyzed and quantified using Skyline by the ratio of the renormalization method and 1 / x*x regression weighting [MacLean, B. et al., Skyline: An open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 2010.26 (7): pp. 966-968]. The calculated concentration was normalized relative to BCA protein content.

[0383] NHP brain lysate

[0384] Trastuzumab pull-down: Human HER2 coupled magnetic beads were reorganized and washed according to the supplier's advice (Acro Biosystems MBS-K006). One hundred μL of beads of each sample were pelletized in a microcentrifuge tube using a magnetic separator, and the supernatant was discarded. As described in the LC-MS analysis of mouse brain lysate sections, protein lysate samples from NHP brain homogenates were prepared. The sample was diluted to 2 mg / mL in PBS-T, and 1 ml of diluted lysate was added to a tube with pelletized beads. The sample was incubated overnight at 4 ° C on a tube rotator. After incubation, beads were pelletized using a magnetic separator, and supernatant was discarded. PBS-T was used to wash beads by vortexing the tube for 20 seconds, beads were pelletized on a magnetic separator, and supernatant was discarded, and a total of 4 times were performed.

[0385] Protein-bound beads were prepared according to the SP3 protocol [Hughes, CS et al., Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nature Protocols, 2019.14(1):68-85]. A heavy labeled internal standard peptide (DTYIHWVR; SEQ ID NO:56) was added to the solution for residence time reference and normalization. Each sample was divided into two vials for targeted and untargeted LCMS analysis.

[0386] As discussed in the LC-MS analysis of mouse brain lysate sections, vial one was analyzed using a targeted MRM acquisition method. Using a 10 ng / mL positive control as a reference, the data were analyzed and quantified using Skyline by ratio renormalization and linearity by zero single point calibration regression [MacLean, B. et al., Skyline: An open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 2010. 26(7): pp. 966-968].

[0387] Vial two was analyzed using a Thermo UltiMate 3000RSLNano LC system and a Thermo Q-Exactive HFOrbitrap mass spectrometer. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The stationary phase was Easy-Spray PepMap Neo 2μm C18 75μm X 150mm, and the gradient used was 4% mobile phase B to 50% mobile phase B over 65 minutes, followed by a high organic wash and re-equilibration. Data acquisition was performed using a top 10 data dependent acquisition method. MS1 resolution was set to 60,000 with an AGC target set to 1e6 with a maximum IT of 60 milliseconds, a scan of 300-1500 m / z, and an isolation window of 2 m / z. MS2 resolution was set to 30,000 with an AGC target of 1e6, a maximum IT of 120 ms, a scan of 50-2000 m / z, and an NCE of 27. The minimum intensity threshold was set to 8.3e3. Charge state exclusions were set to unassigned, 1, and >7, and dynamic exclusions were set to 5 seconds. The data were analyzed using Skyline with peptide settings set to a maximum of 1 trypsin missed cleavage site and fixed carbamidomethyl cysteine. Skyline transition settings were set to charge states of 2-4, ion charge of 1, and ion types of y, b, and p. Skyline product ions were set from ion 1 to last ion. Peptide map coverage was manually reviewed for accuracy. Relative quantification of heavy and light chains was calculated using the average MS1 peak area of ​​the 3 most abundant peptides in each chain.

[0388] Statistical analysis

[0389] Unpaired two-tailed Student t-test was used in GraphPad Prism 9.5.1 to analyze the difference of trastuzumab expression level between treatment group and control group. T-test with logarithmic transformation value was used to calculate the comparison of the number of viral genome copies between different treatment groups. GraphPad Prism 9.5.1 was used to draw the estimated Kaplan-Meier survival probability of tumor stimulation experiment in mice, and the log-rank test was used to compare the differences between groups.

[0390] result

[0391] ICV delivery of AAV9.UbC.Trastuzumab vector results in transgene expression in Rag1 KO mice

[0392] To achieve stable trastuzumab expression in vivo, healthy young adult Rag1 KO female mice were injected ICV with (1x10 11GC / mice)AAV9.UbC.Trastuzumab, and quantification of trastuzumab levels in systemic circulation and brain parenchyma ( Fig.31A and 31B ). AAV9.UbC.Trastuzumab vector (1x10 11 GC / mice) resulted in robust systemic and local transgene expression after 4 weeks (signal increased 100-1000-fold over background, Fig.31C and 31D ). Biodistribution studies in brain and liver tissues confirmed that the vector delivered ( Fig.31E ) and transgenic expression ( Fig.31F ) was successful in AAV-treated mice (AAV genome copies per microgram of DNA: signal increased 600-3,000-fold over background; transgene RNA transcripts: signal increased 5,000-42,000-fold over background). Western blot analysis confirmed the expression of human IgG antibody heavy and light chains in whole brain lysates of mice that received AAV-trastuzumab by ICV injection ( Figure 31G The identity of the expressed transgene was confirmed to be trastuzumab by LC-MS, where multiple reaction monitoring conversion was performed by quantification of a unique peptide (DTYIHWVR; SEQ ID NO: 56) present in brain homogenates of ICV-treated mice receiving AAV9.UbC.trastuzumab (signal ≥588-fold above background compared to PBS control; Fig.31H ).

[0393] Figures 31A-31H It was shown that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Fig.31A A schematic diagram of the AAV vector genome is shown. Fig.31B Figure 2 shows ICV delivery of trastuzumab-encoding AAV9 vector (1×10 11 Schematic diagram of the assessment of transgene expression in vivo after GC / mice). Fig.31C The results show that the AAV9.UbC.Trastuzumab vector (1×10 11 Longitudinal quantification of trastuzumab by ELISA in the sera of Rag1 KO mice treated ICV with either GC / mouse; n=10) or PBS (n=10). Fig.31D The results show that the AAV9.UbC.Trastuzumab vector (1×10 11 Longitudinal quantification of trastuzumab by ELISA in perfused brain tissue of Rag1 KO mice treated ICV with either GC / mouse; n=10) or PBS (n=10). Fig.34Eshows DNA biodistribution, and Fig.31F Shown are RNA biodistribution analyses by qPCR of brain and liver tissues of Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab or PBS. Figure 31G Western blot analysis of trastuzumab heavy and light chains in brain lysates of Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab (n=4) or PBS (n=2) is shown. Purified trastuzumab (10 ng / lane) and β-actin were used as positive and loading controls, respectively. Fig.31H Shown are brain homogenates of Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab (n=4) or PBS (n=2) to confirm the identity of trastuzumab by LC-MS. Data are shown as individual data points and mean±SEM.

[0394] Preventing off-target transduction does not affect trastuzumab expression in the brain of Rag1 KO mice

[0395] Cardiotoxicity is a significant but largely reversible complication of systemic trastuzumab therapy observed in a subset of patients with metastatic HER2+ breast cancer. The incidence of trastuzumab-related cardiotoxicity varies widely depending on risk factors such as pre-existing cardiac dysfunction; the likelihood of developing any type of cardiotoxicity peaks at 11 months after the start of treatment and decreases over time [Bouwer, NI, et al., Cardiotoxicity during long-term trastuzumab use in patients with HER2-positive metastatic breast cancer: who needs cardiac monitoring? Breast Cancer Res Treat, 2021.186(3):851-862]. Since AAV vectors can leak from the CSF into the general circulation to transduce off-target organs after IT delivery, we evaluated whether intravenous plasma-derived pooled human immunoglobulins (IVIg; a source of anti-AAV9 neutralizing antibodies) could reduce extracranial organ transduction after ICV AAV vector delivery in mice [Boutin, S. et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Human Gene Therapy, 2010. 21(6): 704-12]. ICV injection of AAV9.UbC.Trastuzumab (1x10 11 Mice that received IVIg 2 hours before GC / mice) showed a 40-fold decrease in trastuzumab concentration in serum ( Fig.32A ), while levels in the brain remained unaffected ( Fig.32B Vector biodistribution analysis confirmed these findings ( Fig.32C), showing a reduction in systemic but not brain levels of trastuzumab, due to the effective blockade of only liver and heart transduction after IVIg pretreatment. In humans, pre-existing immunity to recombinant AAV vectors after natural exposure to wild-type AAV can greatly reduce tissue transduction. However, since CSF contains only a small fraction of NAbs compared to blood [Horiuchi, M. et al., Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors. Mol Ther Methods Clin Dev, 2022. 27: 272-280; Verdera, HC, K. Kuranda and F. Mingozzi, AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther, 2020. 28(3): 723-746], pre-existing immunity to AAV may not prevent this gene therapy approach for most potential patients.

[0396] Figures 32A to 32C The results show that the CNS transduction efficiency of AAV9.UbC.trastuzumab is not affected by pretreatment with intravenous immunoglobulin (IVIg) containing broadly neutralizing antibodies against AAV9. Adult female Rag1 KO mice were given ICV AAV9.UbC.trastuzumab (1×10 11 GC / mice; n=5) were pretreated with IVIg or control serum from C57BL6 / J donor mice 2 hours before. Mice treated with PBS ICV (n=3) were used as controls. Fig.32A shows the levels of trastuzumab in serum over time, and Fig.32B Shown are trastuzumab levels in perfused brain tissue (day 30) of ICV-treated mice measured by ELISA. Fig.32C Shown is DNA biodistribution analysis of AAV9.UbC.Trastuzumab vectors in brain, liver, and heart 30 days after vector administration by qPCR. Data are shown as individual data points and mean ± SEM. ***p≤0.0001; ns, not significant.

[0397] Treatment with AAV9.UbC.Trastuzumab inhibits tumor progression in an orthotopic xenograft mouse model of HER2+ breast cancer brain metastasis

[0398] Next, we attempted to evaluate the anti-tumor efficacy of AAV9.UbC.trastuzumab ICV administration in an immunocompromised mouse model that retains NK cell-mediated ADCC activity. Orthotopic xenograft Rag1 KO mouse models were generated by transplanting GFP / luciferase reporter gene HER2+ cell lines (BT-474 or MDA-MB-453) into the right prefrontal lobe using a guide screw system [Lal, S. et al., Implantable guide screw system for small animal brain tumor research. Journal of Neurosurgery, 2000.92 (2): Pages 326-33]. The hormone receptor and HER2 status of each cell line was confirmed by IHC staining of tumor-bearing brain sections of Rag1 KO mice (data not shown). Three days after tumor cell implantation, animals were randomly assigned to treatment groups and received AAV9.UbC.trastuzumab or AAV9.UbC.isotype control vectors (1x10 11 GC / mice); tumor growth was monitored by bioluminescence imaging (data not shown).

[0399] AAV9.UbC.trastuzumab administration delayed tumor progression in BT-474 tumor-bearing mice compared to mice treated with AAV9.UbC.isotype control ( Fig.33A ), and extend OS( Fig.33B ) (3.8 weeks vs. 9.9 weeks, respectively). Six of 12 mice treated with AAV9.UbC.trastuzumab remained asymptomatic for >12 weeks and were considered to be in complete remission. ICV treatment of Rag1 KO mice bearing MDA-MB-453 xenograft tumors with AAV9.UbC.trastuzumab achieved a 100% response rate ( Fig.33C ), and all animals remained in remission >12 weeks after treatment; the OS of mice treated ICV with AAV9.UbC.isotype control vector was 5.5 weeks ( Fig.33D Analysis of brain homogenates from Rag1 KO mice orthotopically implanted with MDA-MB-453 104 days after ICV administration of AAV9.UbC.Trastuzumab showed sustained trastuzumab expression (range 23.7-49.5 ng trastuzumab / mg total protein; Fig.33E ). Taken together, these results provide principle evidence supporting direct CNS delivery of AAV9.UbC.trastuzumab vectors to target HER2+ BCBM.

[0400] Figures 33A to 33EThe results show that a single ICV administration of AAV9.UbC.trastuzumab resulted in tumor regression in a xenograft mouse model of HER2+ breast to brain metastases. Adult female Rag1 KO mice were injected ICV with 1×10 11 GC / animals AAV9.UbC.Trastuzumab or AAV9.Isotype control. Fig.33A Quantification of total photon flux by bioluminescent in vivo imaging system (IVIS) is shown, and Fig.33B Shown are Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from the BT-474 cell line and treated ICV with AAV vectors (AAV9.Isotype control: n=9; or AAV9.UbC.Trastuzumab: n=12). Fig.33C Shown are representative bioluminescent IVIS images (week 4, left panel), total photon flux signal quantification (right panel), and Fig.33D Shown are Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from the MDA-MB-453 cell line after ICV treatment (AAV9.Isotype control: n=8; or AAV9.UbC.Trastuzumab: n=7). Fig.33E Shows Fig.33C and Fig.33D Long-term trastuzumab expression quantified by ELISA in perfused brain tissue at day 104 after ICV administration of AAV9.UbC.trastuzumab in a subset of mice shown (n=5). ***p<0.0001

[0401] Successful expression of therapeutic antibody proteins in the CSF of NHPs treated with AAV9.UbC.Trastuzumab

[0402] NHPs are more comparable to humans in size and have stronger immunological similarities, making them the gold standard model species for evaluating AAV safety and CNS vector distribution [Gopinath, C. et al., Contemporary Animal Models for Human Gene Therapy Applications. Contemporary Gene Therapy, 2015.15(6): p. 531-40]. In order to achieve extensive vector distribution in the CNS, ICM injections (3x10 13GC / animal, n=2 / group) AAV9-UbC. Trastuzumab was delivered into the CSF of adult female Indian rhesus macaques with pre-existing AAV9 neutralizing antibodies as previously described [Hinderer, C. et al., Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna. Mol Therapeutics Clinical Development, 2014.1: p. 14051]. Body weight and comprehensive serum chemistry remained within normal parameters compared to baseline measurements. Throughout the study, no physical defects or clinical toxicity signs were observed, and animals were euthanized at their planned endpoints on days 36-37. No gross lesions or histopathological abnormalities were observed after autopsy.

[0403] We then aimed to assess whether ICM injection of AAV9.UbC.Trastuzumab vectors resulted in detectable transgene expression in the serum and CSF of treated animals as a longitudinal surrogate marker of CNS gene transfer. Samples were collected at baseline and weekly intervals until planned euthanasia ( Fig.34A Serum and CSF samples collected from NHPs previously treated with AAV9.GFP ICM were used as negative controls. Quantification of trastuzumab levels in 2 / 2 NHPs that received AAV9.UbC.trastuzumab by ICM injection demonstrated that the drug was expressed in the CSF ( Fig.34B ) rather than in serum ( Fig.34C ) Evidence is accumulating that the development of anti-drug antibodies (ADA) against trastuzumab may aid in CSF drug clearance ( Fig.34D and 34E ), the ADA was in the CSF of 1 / 2 NHP treated with AAV9.UbC.Trastuzumab at a later time point ( Fig.34D ) and serum ( Fig.34E In summary, these results confirm that AA9.UbC.Trastuzumab successfully expressed trastuzumab in the CSF of NHPs after ICM administration.

[0404] Figures 34A to 34E Shown is the detection of trastuzumab in the CSF of female NHPs following ICM administration of an AAV9 vector encoding trastuzumab. Fig.34A Experimental ICM delivery of 3 × 10 13Schematic design of AAV9.UbC.Trastuzumab (n=2) vectors for GC / animal to inform blood and CSF collection schedule for survival and planned end of study time point. 3x10 of AAV9 vector expressing GFP were injected into 13 GC ICM was injected into NHP#3 and used as a negative control. Fig.34D ) and serum ( Fig.34E ) and anti-drug antibodies against trastuzumab were quantified by ELISA, and the CSF ( Fig.34B ) and serum ( Fig.34C Detection of trastuzumab (a humanized IgG1 antibody) in patients with

[0405] ICM delivery of AAV vectors encoding trastuzumab resulted in widespread transduction and transgene expression in CNS tissues. Fig.35A ) showed that compared with patients receiving ICM AAV9.UbC.Trastuzumab (3x10 13 Vector GC was 100-10,000-fold higher in the brain and spinal cord compared to the heart and liver of NHPs (100-10,000 GC / animal). The presence of transgene transcripts in the CNS, heart, and liver were assessed following transduction. Consistent with the DNA biodistribution data, transgene mRNA expression was 100-1,000-fold higher in the brain and spinal cord regions compared to the heart and liver of these animals ( Fig.35B In paraffin-embedded sections of the cerebellum and occipital cortex, in situ hybridization with probes specific for the engineered trastuzumab version (3x10 13 Single-cell nuclear RNA sequencing further confirmed the presence of transgene transcripts in the cerebellum and occipital cortex of NHPs receiving ICM AAV9.UbC.trastuzumab, although different levels of vector transduction were observed in these regions (0.6 to 1% of total trastuzumab-positive cells in the cerebellum, compared to 0.02-0.08% of total trastuzumab-positive cells in the occipital cortex; Figures 36A to 36D ). These data suggest that extracranial transduction by AAV leaked from the CSF was largely prevented in these animals because they had pre-existing levels of anti-AAV9 neutralizing antibodies prior to ICM treatment.

[0406] Fig.35A , 35B and 36A to 36D show the use of 3×10 13Whole CNS gene delivery and transgene transcription after ICM treatment of GC / animals with AAV9.UbC.Trastuzumab. Fig.35A Shown are absolute quantification of AAV vector GC in NHPs at days 36-37 after ICM administration of AAV9.UbC.Trastuzumab (n=2) vectors. Fig.35B Shown are transcripts in selected tissues in NHPs by qPCR at days 36-37 after ICM administration of AAV9.UbC.Trastuzumab (n=2) vectors.

[0407] Figures 36A to 36D Shown are the results of single-cell nuclear RNA sequencing showing the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex of animals injected ICM with AAV9.UbC.trastuzumab vector.

[0408] Next, the identity of AAV-encoded trastuzumab in NHP CNS tissue was analyzed using LC-MS targeted and non-targeted proteomics methods (based on detection of unique trastuzumab-derived peptides and peptide mapping, respectively). In order to reduce matrix effects and improve assay sensitivity, NHP brain homogenates were prepared under non-denaturing conditions, and trastuzumab was pulled down using HER2-coated magnetic beads. Ten nanograms of purified research-grade trastuzumab or rituximab (anti-human CD20) antibodies were added to brain homogenates consisting of 1 mL of a solution containing 1 mg of total protein from an untreated NHP control to provide positive or negative input controls for pull-down assays, respectively. After incubation and recovery of the HER2-coated magnetic beads, the antibodies bound to the beads were released by proteolysis, and the peptide fragments were analyzed by LC-MS. For the targeted proteomics approach, the amount of trastuzumab recovered from the positive input control was used for normalization; as expected, binding of the antibody to HER2 was specific, as essentially no rituximab was recovered in the pull-down. The presence of trastuzumab in brain homogenates of NHPs receiving ICMAAV9.UbC.trastuzumab was confirmed by detection of unique trastuzumab-derived peptides and by peptide mapping of trastuzumab heavy and light chains ( Fig.37A ).

[0409] ELISA was used to further determine the abundance of human IgG protein (as a surrogate for trastuzumab) in multiple brain regions and spinal cord of NHPs that received ICM AAV9.UbC.Trastuzumab. CNS tissue samples collected from NHPs previously treated with AAV9.GFP ICM were used as negative controls. Fig.37C), human IgG levels in both the cerebellum and medulla of NHPs treated with AAV9.UbC.trastuzumab appear to be consistently high (2-4ng trastuzumab / mg total protein). The cerebellum is a hotspot for HER2+BCBM, and persistent expression of trastuzumab in this region can effectively target the tumor bed and slow disease progression [Kyeong, S. et al., Subtypes of breast cancer show different spatial distributions of brain metastases. PloS One, 2017.12(11): p. e0188542]. Compared with the brain of these animals, trastuzumab levels in the spinal cord appear to be slightly higher (3-10ng / mg total protein; Fig.37E ). ICM vector delivery reportedly resulted in higher vector transduction of the spinal cord compared to lumbar puncture, further validating the choice of this route of administration, implying that leptomeningeal metastasis along the spinal cord could also be a viable target using this approach [Hinderer, C. et al., Extensive gene transfer in the central nervous system of cynomolgus monkeys following delivery of AAV9 into the cisterna magna. Mol Ther Appl Clin Dev, 2014.1: p. 14051].

[0410] Tumor remission in mice treated with low-dose AAV9.UbC.trastuzumab ICV achieved CNS transgene expression levels similar to NHPs

[0411] Since the levels of trastuzumab expression achieved in the NHP brain parenchyma were lower than those observed in the tumor challenge experiments in mice presented above, it was investigated whether treatment of tumor-bearing mice with doses of AAV9.UbC.trastuzumab that reproduced the levels of transgene expression in the CNS of NHPs would maintain adequate antitumor activity. 11 GC / mouse up to 1x10 10 GC / mice resulted in trastuzumab accumulation in the brain parenchyma of healthy female Rag1 KO mice that was similar to that observed in the cerebellum and medulla of ICM-treated NHPs (1.6-3.5 ng / mg total protein; Fig.37B ). Compared with animals treated with AAV9.UbC.isotype control, animals treated with 1x10 10ICV treatment of Rag1 KO mice bearing orthotopic brain xenografts derived from the MDA-MB-453 cell line with AAV9.UbC.Trastuzumab in GC / mice resulted in a 100% response rate, with all mice exhibiting complete remission more than 12 weeks after transplantation (OS 7.6 weeks; Fig.37D and 37F ). These results demonstrate that local expression of AAV-encoded trastuzumab in the CNS of tumor-bearing mice provides a robust anti-tumor response even at lower therapeutic drug concentration levels observed in ICM-treated NHPs. In conclusion, ICM delivery of AAV9.UbC.trastuzumab in NHPs is well tolerated and results in CNS transgene expression at levels that demonstrate a therapeutic effect.

[0412] Figures 37A to 37F It is shown that ICM delivers 3×10 13 GC / animal AAV9.UbC.Trastuzumab resulted in expression of transgenic protein in NHP CNS tissue at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. The experimental protocol is shown in Fig.34A middle. Fig.37A Results of LC-MS analysis for detection of a unique AAV-encoded trastuzumab-derived peptide (DTYIHWVR; SEQ ID NO: 56) in NHP brain homogenates are shown. Trastuzumab levels are presented as fold change relative to the input reference positive control (10 ng trastuzumab). Fig.37B Female Rag1 KO mice were shown to have received ICV administration of 1 × 10 10 Trastuzumab expression in perfused brain tissue 28 days after AAV9.isotype control (n=8) or AAV9.UbC.trastuzumab (n=8) in GC / animals as measured by ELISA. Fig.37C Trastuzumab protein expression in perfused brain regions was shown by determining tissue levels of human IgG by ELISA. Fig.37D Figure 3 shows the intracranial tumor cells implanted with reporter gene MDA MB 453 breast cancer cells, using 1×10 10 Quantification of total photon flux by IVIS imaging of adult female Rag1 KO mice ICV treated with AAV9.isotype control (n=8) or AAV9.UbC.trastuzumab (n=8) GC / animal. Fig.37E Shown is the expression of trastuzumab protein in the spinal cord, quantified by determining tissue levels of human IgG by ELISA. Fig.37F Figure 3 shows the intracranial tumor cells implanted with reporter gene MDAMB 453 breast cancer cells, with 1×10 10Kaplan-Meier survival curves of adult female Rag1 KO mice ICV treated with AAV9.isotype control (n=8) or AAV9.UbC.trastuzumab (n=8) in GC / animal. ***p<0.0001

[0413] discuss

[0414] The mechanisms that contribute to the transition from tumor cell quiescence to the onset of symptomatic CNS metastases are unclear, but they typically manifest in the brain parenchyma and leptomeninges approximately 2-3 years after the initial breast cancer diagnosis (80% versus 11-20% of cases, respectively) [Pestalozzi, BC, et al., Identifying breast cancer patients at risk for Central Nervous System (CNS) metastases in trials of the International Breast Cancer Study Group (IBCSG). Annals of Oncology, 2006. 17(6): 935-44; Scott, BJ and S. Kesari, Leptomeningeal metastases in breast cancer. Am J Cancer Res, 2013. 3(2): 117-26]. The overall prognosis and survival of patients diagnosed with BCBMs depend on independent factors, including tumor molecular subtype, Karnofsky performance status, and the size and number of tumors present; for example, having >3 BCBM lesions at diagnosis is associated with a worse prognos...

Claims

1. A recombinant adeno-associated virus (rAAV), comprising an adeno-associated virus (AAV) capsid containing a vector genome, wherein the vector genome comprises: (a) AAV – 5' inverted terminal repeat (ITR), (b) an expression cassette, the expression cassette comprising a coding sequence for an anti-Her2 antibody, the anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising: (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody heavy chain, (ii) a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence that is at least 95% identical to SEQ ID NO: 3, wherein the nucleic acid sequence encodes an anti-Her2 antibody heavy chain, (iii) a furin cleavage site, (iv) T2A element connector, (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody light chain, (vi) a nucleic acid sequence comprising SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: 5, wherein the nucleic acid sequence encodes an anti-Her2 antibody light chain, and a regulatory control sequence operably linked to the sequence in the expression cassette, wherein the regulatory sequence comprises a ubiquitin C (UbC) promoter, an optional enhancer, an intron, and a polyadenylation (polyA) sequence, and (c) AAV–3’ITR.

2. The rAAV of claim 1, wherein the nucleic acid sequence encoding the leader peptide of (i) comprises SEQ ID NO:

7.

3. The rAAV according to claim 1 or 2, wherein the nucleic acid sequence encoding the leader peptide of (v) comprises SEQ ID NO:

9.

4. The rAAV according to any one of claims 1 to 3, wherein the anti-Her2 antibody encoding sequence comprises the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 95% identical to SEQ ID NO:

29.

5. The rAAV according to any one of claims 1 to 4, wherein the UbC promoter has a nucleic acid sequence of SEQ ID NO:

24.

6. The rAAV according to any one of claims 1 to 5, wherein the polyA sequence is an SV40 polyA sequence having a nucleic acid sequence of SEQ ID NO:

23.

7. The rAAV according to any one of claims 1 to 6, wherein the intron is a chimeric intron having a nucleic acid sequence of SEQ ID NO:

22.

8. The rAAV of any one of claims 1 to 7, wherein the AAV ITRs are from AAV2.

9. The rAAV of any one of claims 1 to 8, wherein the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 26 or a nucleic acid sequence that is at least 99% identical to SEQ ID NO:

26.

10. The rAAV according to any one of claims 1 to 9, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO: 25 or a sequence at least 99% identical to SEQ ID NO:

25.

11. A recombinant adeno-associated virus (rAAV), comprising an adeno-associated virus (AAV) capsid containing a vector genome, wherein the vector genome comprises: (a) AAV – 5' inverted terminal repeat (ITR), (b) an expression cassette, the expression cassette comprising a coding sequence for an anti-Her2 antibody, the anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising: (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody heavy chain, (ii) a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence that is at least 95% identical to SEQ ID NO: 3, wherein the nucleic acid sequence encodes an anti-Her2 antibody heavy chain, (iii) a furin cleavage site, (iv) T2A element connector, (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody light chain, (vi) a nucleic acid sequence comprising SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: 5, wherein the nucleic acid sequence encodes an anti-Her2 antibody light chain, and a regulatory control sequence operably linked to the sequence in the expression cassette, wherein the regulatory sequence comprises one or more of: a promoter and a polyA sequence, the promoter being a chicken β-actin promoter or a CB7 hybrid promoter comprising a cytomegalovirus immediate early (CMVIE) enhancer, a chicken β-actin promoter, and a chimeric intron comprising a chicken β-actin intron, the polyA sequence being an SV40 polyA sequence, and (c) AAV–3’ITR.

12. The rAAV according to claim 11, wherein the promoter is the CB7 hybrid promoter having a nucleic acid sequence of SEQ ID NO:

21.

13. The rAAV of claim 11 or 12, wherein the expression cassette comprises the nucleic acid sequence of SEQ ID NO:28, a nucleic acid sequence at least 99% identical to SEQ ID NO:28, a nucleic acid sequence of SEQ ID NO:2, or a nucleic acid sequence at least 99% identical to SEQ ID NO:

2.

14. The rAAV of claim 11 or any one of claims 11 to 13, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO:27, a sequence at least 99% identical to SEQ ID NO:27, SEQ ID NO:50, a sequence at least 99% identical to SEQ ID NO:50, SEQ ID NO:1, or a sequence at least 99% identical to SEQ ID NO:

1.

15. The rAAV of any one of claims 1 to 14, wherein the AAV capsid is a clade F AAV capsid.

16. The rAAV of any one of claims 1 to 15, wherein the AAV capsid is an AAVhu68 capsid.

17. The rAAV of any one of claims 1 to 14, wherein the AAV capsid is an AAVhu95 capsid, an AAVhu96 capsid, an AAV9 capsid, or an AAVrh91 capsid.

18. The rAAV of any one of claims 1 to 17, for use in treating one or more of HER2-positive cancer, HER2-positive breast cancer with brain metastases, or HER2-positive trastuzumab-resistant cancer.

19. The rAAV of any one of claims 1 to 18, wherein the rAAV is formulated for systemic, central nervous system, and / or intratumoral delivery.

20. A composition comprising a stock solution of rAAV according to any one of claims 1 to 19 and an aqueous suspension medium.

21. The composition of claim 20, wherein the suspension is formulated for intrathecal delivery, optionally wherein the intrathecal delivery is an intracerebroventricular (ICV) injection or an intracisternal (ICM) injection.

22. A pharmaceutical composition comprising the rAAV according to any one of claims 1 to 19 and an aqueous formulation buffer.

23. The pharmaceutical composition of claim 22, formulated for intrathecal delivery.

24. The pharmaceutical composition of claim 22, formulated for intracerebroventricular (ICV) injection or intracisternal (ICM) injection.

25. A recombinant nucleic acid molecule comprising: (a) AAV – 5' inverted terminal repeat (ITR), (b) an expression cassette, the expression cassette comprising: at least one open reading frame (ORF), the ORF comprising an anti-Her2 antibody heavy chain and an anti-Her2 antibody light chain, and a nucleic acid sequence operably linked thereto for regulating the expression of the anti-Her2 heavy chain and the anti-Her2 light chain, and (c) AAV–3’ITR, Wherein the expression cassette comprises: (i) a promoter, wherein the promoter is: (A) Ubiquitin C (UbC) promoter, or (B) a CB7 hybrid promoter comprising a CMVIE enhancer, a chicken β-actin promoter, and a chimeric intron comprising a chicken β-actin splice donor and a rabbit β-globin splice acceptor, the chicken β-actin splice donor comprising a chicken β-actin intron, and / or (ii) an intron, said intron being a chimeric intron, and (iii) at least one ORF, the at least one ORF comprising: a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 3, wherein the nucleic acid sequence encodes an anti-Her2 heavy chain, (v) a furin cleavage site, (vi) T2A connector, (vii) a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 light chain, (viii) a nucleic acid sequence comprising SEQ ID NO: 5, wherein the nucleic acid sequence encodes an anti-Her2 light chain, (ix) the SV40 polyadenylation (polyA) sequence, and Wherein the expression cassette further comprises a spacer sequence.

26. The recombinant nucleic acid molecule of claim 25, wherein the expression cassette comprises the nucleic acid sequence of SEQ ID NO:

26.

27. The recombinant nucleic acid molecule according to claim 25, comprising the nucleic acid sequence of SEQ ID NO:

25.

28. A packaging host cell comprising the recombinant nucleic acid molecule according to any one of claims 25 to 27.

29. The packaging host cell of claim 28, further comprising an AAV rep coding sequence operably linked to a sequence that expresses a rep protein in the packaging host cell, an AAV capsid coding sequence operably linked to a sequence that expresses an AAV capsid protein in the packaging host cell, and helper virus functions necessary to allow packaging of the expression cassette and the AAV ITR into the AAV capsid.

30. The packaging host cell of claim 28 or 29, wherein the AAV capsid is AAVhu68.

31. The packaging host cell of claim 28 or 29, wherein the AAV capsid is AAV9, AAVrh91, AAVhu95 or AAVhu96.

32. An rAAV production system that can be used to produce the rAAV according to any one of claims 1 to 19, wherein the production system comprises a cell culture, and the cell culture comprises the packaging host cell according to any one of claims 28 to 31.

33. The rAAV production system of claim 32, wherein the AAV capsid is AAVhu68.

34. The rAAV production system of claim 32, wherein the AAV capsid is AAV9, AAVrh91, AAVhu95 or AAVhu96.

35. The rAAV production system according to any one of claims 32 to 34, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO:

25.

36. The rAAV production system according to any one of claims 32 to 34, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO: 27 or SEQ ID NO:

1.

37. A method for treating HER2-positive brain metastatic cancer, the method comprising administering to a subject a suspension of the rAAV according to any one of claims 1 to 19 in a formulation buffer.

38. A method for treating brain metastatic breast cancer, the method comprising administering to a subject a suspension of the rAAV according to any one of claims 1 to 19 in a formulation buffer.

39. The method of claim 37 or 38, wherein the suspension is administered intrathecally.

40. The method according to any one of claims 37 to 39, wherein the suspension is administered in the absence of a chemical or physical disruptor of the blood-brain barrier.

41. The method of claim 37 or 38, wherein the suspension is administered by direct injection into the tumor bed or by an Ommaya device.

42. A recombinant AAV (rAAV) according to claims 1 to 19 or a composition according to any one of claims 20 to 22, for use in the preparation of a medicament for treating HER2-positive brain metastatic cancer, optionally wherein the HER2-positive brain metastatic cancer is brain metastatic breast cancer.

43. An anti-tumor regimen comprising administering the rAAV of any one of claims 1 to 19, the composition of any one of claims 20 to 22, or the pharmaceutical composition of any one of claims 23 to 24 in combination with a biopharmaceutical, a small molecule, an anti-tumor agent, radiation and / or a chemotherapeutic agent.

44. A method for treating trastuzumab-resistant HER2-positive cancer, the method comprising administering to a subject a suspension of the rAAV of claim 1 in a formulation buffer.

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