Optimized promoter sequence, intron-free expression construct, and method of use

Abstract

To provide an expression cassette with enhanced gene therapy therapeutic applications through improvements in transgene expression, enhancer and promoter functions that drive transgene expression.SOLUTION: An expression cassette comprises a regulatory element operably linked to a nucleic acid sequence encoding a Factor VIII protein having a B domain deletion (FVIII-BDD) and inverted terminal repeats (ITRs). The expression cassette comprises a sequence that is at least 98% identical to a specific sequence. The ITRs flank the 5' side of the regulatory element. There is no intron between the regulatory element and the nucleic acid sequence.SELECTED DRAWING: Figure 20

Description

[Background technology] 【0001】 〔introduction〕 【0002】 Gene therapy is extremely promising in therapeutic applications involving loss of protein function or activity, for example, due to genetic defects or abnormal protein function or activity, where suppressing the expression of a genetic defect or abnormal protein is desirable. The therapeutic applications of gene therapy can be enhanced by improving transgene expression, the enhancers that drive transgene expression, and the promoter function. This invention addresses this need in particular. [Overview of the project]

[0002] 【0003】 According to the present invention, an expression cassette is provided which includes a nucleic acid sequence encoding factor VIII protein having a B domain deletion (FVIII-BDD).

[0003] 【0004】 In a particular embodiment, the expression cassette contains a sequence that is at least 98% identical to the sequence of SEQ ID NO: 1, or is at least 99% identical to the sequence of SEQ ID NO: 1, contains the sequence of SEQ ID NO: 1, or consists of the sequence of SEQ ID NO: 1.

[0004] 【0005】 In a particular embodiment, the expression cassette includes a regulatory element operably linked to a nucleic acid sequence encoding factor VIII protein having a B domain deletion (FVIII-BDD), wherein there are no introns between the regulatory element and the nucleic acid sequence, and the expression cassette contains a sequence that is at least 91% identical to the sequence of Sequence ID No. 1.

[0005] 【0006】In a particular embodiment, the expression cassette comprises (a) a regulatory element that is at least 90% identical to any of the sequences of SEQ ID NOs: 2 to 67, and (b) a nucleic acid sequence encoding factor VIII protein (FVIII-BDD) having a B-domain deletion, wherein the nucleic acid sequence of (a) is at least 90% identical to the sequence of SEQ ID NO: 77, the regulatory element is operably ligated to the nucleic acid sequence, and there are no introns between the regulatory element and the nucleic acid sequence encoding FVIII-BDD.

[0006] 【0007】 In a particular embodiment, the expression cassette comprises (a) a regulatory element that is at least 90% identical to the sequence of any of SEQ ID NOs: 2 to 67, and (b) a nucleic acid sequence encoding factor VIII protein (FVIII-BDD) having a B-domain deletion, which is at least 90% identical to the sequence of SEQ ID NO: 77, wherein the regulatory element is operably linked to the nucleic acid sequence, and the regulatory element and the nucleic acid sequence encoding FVIII-BDD Between these, there are 0-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 106, or 107 or fewer untranslated nucleic acids.

[0007] 【0008】 In a particular embodiment, the regulatory element in the expression cassette contains a nucleotide sequence that is at least 95% identical to any of SEQ ID NOs: 2 to 67.

[0008] 【0009】 In a particular embodiment, the regulatory element in the expression cassette has the same total number of reduced CpGs as those described in any of the sequences 4-21 or 24-67.

[0009] 【0010】In a particular embodiment, the regulatory element in the expression cassette includes one of the sequences of sequence numbers 2 to 21 or 24 to 67, in which CpG(s) are replaced with CpT, CpA, TpG, or ApG at the same position(s) as described in any of the sequences of sequence numbers 4 to 21 or 24 to 67.

[0010] 【0011】 In a particular embodiment, the nucleic acid sequence in the expression cassette exhibits higher expression compared to expression from an expression cassette having (a) an intron or (b) 10⁸ or more nucleotides of untranslated nucleic acid between the regulatory element and the nucleic acid sequence.

[0011] 【0012】 In a particular embodiment, the encoded FVIII-BDD in the expression cassette exhibits higher biological activity compared to expression from an expression cassette having (a) an intron or (b) 10⁸ or more nucleotides of untranslated nucleic acid between the regulatory element and the nucleic acid sequence.

[0012] 【0013】 In certain embodiments, biological activity is determined by an FVIII assay or a coagulation assay or reduction of bleeding in an FVIII-deficient model.

[0013] 【0014】 In a particular embodiment, the expression cassette is packaged into the AAV vector more efficiently than the packaging of an expression cassette having (a) an intron or (b) 10⁸ or more nucleotides of untranslated nucleic acid between the regulatory element and the nucleic acid sequence.

[0014] 【0015】The present invention provides nucleic acid sequences in which the cytosine-guanine dinucleotide (CpG) of the regulatory element (promoter) is reduced. Exemplary promoters include the TTR (transthyretin gene) promoter and the ApoE / hAAT (human apolipoprotein E gene / human alpha-1 antitrypsin gene) promoter. Similarly, exemplary promoters include the fibrinogen gamma chain gene (FGG) promoter, the albumin promoter, and the serum amyloid A1 gene (SAA1) promoter. Furthermore, an exemplary promoter is the TTR promoter fused to one or more of the hAAT promoter, albumin promoter, and / or SAA1 promoter, resulting in a hybrid promoter or promoter chimera.

[0015] 【0016】 Nucleic acid regulatory elements with reduced CpG include variants that, when introduced into cells, exhibit altered gene expression levels compared to regulatory elements without reduced CpG. In certain embodiments, reduced CpG regulatory elements can provide increased expression of transgenes or heterologous nucleic acids, such as transgenes encoding proteins like blood coagulation factors (e.g., FVIII), in mammals, and can also provide increased efficacy in gene transfer situations by increasing circulating levels of proteins such as blood coagulation factors and achieving hemostasis for beneficial therapeutic outcomes.

[0016] 【0017】 In a particular embodiment, the nucleic acid sequence has at least one fewer CpG than any of the regulatory elements in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0017] 【0018】 In a particular embodiment, the nucleic acid sequence has at least two fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0018] 【0019】 In certain embodiments, the nucleic acid sequence has at least 3 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0019】 【0020】 In certain embodiments, the nucleic acid sequence has at least 4 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0020】 【0021】 In certain embodiments, the nucleic acid sequence has at least 5 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0021】 【0022】 In certain embodiments, the nucleic acid sequence has at least 6 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0022】 【0023】 In certain embodiments, the nucleic acid sequence has at least 7 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0023】 【0024】 In certain embodiments, the nucleic acid sequence has at least 8 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0024】 【0025】 In certain embodiments, the nucleic acid sequence has at least 9 fewer CpGs than a regulatory element that has not had its wild-type CpGs reduced (e.g., any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23). 【0025】 【0026】 In a particular embodiment, the nucleic acid sequence has at least 10 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0026] 【0027】 In a particular embodiment, the nucleic acid sequence has at least 11 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0027] 【0028】 In a particular embodiment, the nucleic acid sequence has at least 12 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0028] 【0029】 In a particular embodiment, the nucleic acid sequence has at least 13 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0029] 【0030】 In a particular embodiment, the nucleic acid sequence has at least 14 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0030] 【0031】 In a particular embodiment, the nucleic acid sequence has at least 15 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0031] 【0032】 In a particular embodiment, the nucleic acid sequence has at least 16 fewer CpGs than the regulatory element in which the wild-type CpG is not reduced (e.g., any of SEQ ID NOs. 2, 3, 22, and 23).

[0032] 【0033】 In a particular embodiment, the nucleic acid sequence has 16 or fewer CpGs, 15 or fewer CpGs, 14 or fewer CpGs, 13 or fewer CpGs, 12 or fewer CpGs, 11 or fewer CpGs, 10 or fewer CpGs, 9 or fewer CpGs, 8 or fewer CpGs, 7 or fewer CpGs, 6 or fewer CpGs, 5 or fewer CpGs, 4 or fewer CpGs, 3 or fewer CpGs, 2 or fewer CpGs, or 1 or fewer CpGs.

[0033] 【0034】 In a particular embodiment, the nucleic acid sequence has at most 15 CpGs, 14 CpGs, 13 CpGs, 12 CpGs, 11 CpGs, 10 CpGs, 9 CpGs, 8 CpGs, 7 CpGs, 6 CpGs, 5 CpGs, 4 CpGs, 3 CpGs, 2 CpGs, or 1 CpG. In a particular embodiment, the nucleic acid sequence does not have any CpGs.

[0034] 【0035】 In certain embodiments, the nucleic acid sequence containing SEQ ID NO: 22 or SEQ ID NO: 23 is modified to have 15 or fewer cytosine-guanine dinucleotides (CpGs), 14 or fewer CpGs, 13 or fewer CpGs, 12 or fewer CpGs, 11 or fewer CpGs, 10 or fewer CpGs, 9 or fewer CpGs, 8 or fewer CpGs, 7 or fewer CpGs, 6 or fewer CpGs, 5 or fewer CpGs, 4 or fewer CpGs, 3 or fewer CpGs, 2 or fewer CpGs, or 1 or 0 CpGs. In certain embodiments, the nucleic acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 23 is modified to have 0 CpGs.

[0035] 【0036】In a particular embodiment, the nucleic acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23 is optionally modified such that all CpG sites except for C in the C / EBP site are replaced with T.

[0036] 【0037】 In a particular embodiment, the nucleic acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C in the C / EBP site are changed to T, except that the first CpG site remains unchanged.

[0037] 【0038】 In a particular embodiment, the nucleic acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C in the C / EBP site are changed to T, while the second CpG site remains unchanged.

[0038] 【0039】 In a particular embodiment, the nucleic acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23 is optionally modified such that all CpG sites except the C in the C / EBP site are changed to T, except that the third CpG site remains unchanged.

[0039] 【0040】 In a particular embodiment, the nucleic acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C in the C / EBP site are changed to T, except that the fourth CpG site remains unchanged.

[0040] 【0041】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all Cs in CpG sites are changed to Ts, except for the C in the C / EBP site, while the fifth CpG site remains unchanged.

[0041] 【0042】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all Cs in CpG sites are changed to Ts, except for the C in the C / EBP site, while the sixth CpG site remains unchanged.

[0042] 【0043】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is modified such that C in any CpG site is changed to T, except that the 7th CpG site remains unchanged.

[0043] 【0044】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all Cs in CpG sites are changed to Ts, except for the C in the C / EBP site, while the 8th CpG site remains unchanged.

[0044] 【0045】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all Cs in CpG sites are changed to Ts, except for C in the C / EBP site, while the 9th CpG site remains unchanged.

[0045] 【0046】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C in the C / EBP site are changed to T, except that the 10th CpG site remains unchanged.

[0046] 【0047】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C in the C / EBP site are changed to T, while the 11th CpG site remains unchanged.

[0047] 【0048】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all Cs in CpG sites are changed to Ts, except for C in the C / EBP site, while the 12th CpG site remains unchanged.

[0048] 【0049】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all Cs in CpG sites are changed to Ts, except for C in the C / EBP site, while the 13th CpG site remains unchanged.

[0049] 【0050】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all C in any CpG site except C in the C / EBP site is changed to T, except that the 14th CpG site remains unchanged.

[0050] 【0051】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C in the C / EBP site are changed to T, except that the 15th CpG site remains unchanged.

[0051] 【0052】 In a particular embodiment, the nucleic acid sequence in either SEQ ID NO: 22 or SEQ ID NO: 23 is optionally modified such that all CpG sites except for the C / EBP site are changed to T, while the 16th CpG site remains unchanged.

[0052] 【0053】 In a particular embodiment, the nucleic acid sequence is modified such that at least the first CpG from the 5' end in any of SEQ ID NOs: 2, 3, 22, and 23 is replaced with something other than a CpG.

[0053] 【0054】In a particular embodiment, the nucleic acid sequence is modified such that at least the second CpG from the 5' end in any of SEQ ID NOs: 2, 3, 22, and 23 is replaced with something other than a CpG.

[0054] 【0055】 In a particular embodiment, the nucleic acid sequence is modified such that at least the third CpG from the 5' end in any of SEQ ID NOs: 2, 3, 22, and 23 is replaced with something other than a CpG.

[0055] 【0056】 In a particular embodiment, the nucleic acid sequence is modified such that at least the fourth CpG from the 5' end in any of SEQ ID NOs: 2, 3, 22, and 23 is replaced with something other than a CpG.

[0056] 【0057】 In a particular embodiment, the nucleic acid sequence is modified such that at least the fifth CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0057] 【0058】 In a particular embodiment, the nucleic acid sequence is modified such that at least the sixth CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0058] 【0059】 In a particular embodiment, the nucleic acid sequence is modified such that at least the seventh CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0059] 【0060】 In a particular embodiment, the nucleic acid sequence is modified such that at least the eighth CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0060] 【0061】In a particular embodiment, the nucleic acid sequence is modified such that at least the 9th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0061] 【0062】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 10th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0062] 【0063】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 11th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0063] 【0064】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 12th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0064] 【0065】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 13th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0065] 【0066】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 14th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0066] 【0067】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 15th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0067] 【0068】 In a particular embodiment, the nucleic acid sequence is modified such that at least the 16th CpG from the 5' end in either SEQ ID NO: 22 or SEQ ID NO: 23 is replaced with something other than a CpG.

[0068] 【0069】 In certain embodiments, one or more cytosines in any of SEQ ID NOs: 2, 3, 22, and 23 are replaced with thymine. In certain embodiments, one or more cytosines in any of SEQ ID NOs: 2, 3, 22, and 23 are replaced with adenine.

[0069] 【0070】 In a particular embodiment, the nucleic acid sequence in any of SEQ ID NOs: 2, 3, 22, and 23 is modified to have a deletion of C in one or more CpGs.

[0070] 【0071】 In a particular embodiment, the nucleic acid sequence in any of SEQ ID NOs: 2, 3, 22, and 23 is modified to have a G deleted in one or more CpGs.

[0071] 【0072】 In a particular embodiment, the nucleic acid sequence in any of SEQ ID NOs. 2, 3, 22, and 23 is modified to have one or more CpGs with C and G deletions.

[0072] 【0073】 Exemplary TTR promoters with reduced CpG are described in SEQ ID NOs: 4 to 13.

[0073] 【0074】 Exemplary hybrid promoters with reduced CpG are described in SEQ ID NOs: 14 to 21.

[0074] 【0075】 Exemplary CpG-reduced ApoE / hAAT promoters are described in SEQ ID NOs: 24-67.

[0075] 【0076】In certain embodiments, one or more cytosines in a hybrid promoter, comprising all or part of a TTR promoter fused to all or part of at least one of the hAAT promoter and / or FGG promoter and / or albumin promoter and / or SAA1 promoter, are modified to thymine (C→T) or adenine (G→A). The TTR promoter may be fused to any one or a combination of the aforementioned promoters in any 5'→3' orientation.

[0076] 【0077】 In certain embodiments, the hybrid promoter has a 5'→3' oriented TTR / hAAT or hAAT / TTR. In certain embodiments, the hybrid promoter has a 5'→3' oriented TTR / FGG or FGG / TTR. In certain embodiments, the hybrid promoter has a 5'→3' oriented TTR / hAAT / albumin or hAAT / TTR / albumin or albumin / TTR / hAAT or TTR / albumin / hAAT, hAAT / albumin / TTR or albumin / hAAT / TTR, etc.

[0077] 【0078】 In a particular embodiment, the hybrid promoter has a 5'→3' oriented TTR / FGG / albumin or hAAT / TTR / FGG or FGG / TTR / hAAT or TTR / FGG / hAAT, etc.

[0078] 【0079】 In a particular embodiment, the hybrid promoter has a TTR promoter fused to all or some of the four promoters: the hAAT promoter, the FGG promoter, the albumin promoter, and the SAA1 promoter. The TTR promoter may be fused to all or some of the aforementioned promoters in any 5'→3' orientation and in any promoter order.

[0079] 【0080】 In a particular embodiment, the nucleic acid or polynucleotide of the present invention, such as a nucleic acid sequence with reduced CpG, is operably linked to a transgene.

[0080] 【0081】 In certain embodiments, the nucleic acids or polynucleotides of the present invention, such as the modified SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 22, or SEQ ID NOs. 23 described herein, are operably linked to a transgene.

[0081] 【0082】 In certain embodiments, the nucleic acids or polynucleotides of the present invention, such as the modified SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 23 described herein, confer transcription on an operablely linked transgene that is within approximately 5% to 100% of the transcription conferred by the unmodified SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 23, or within approximately 50% of the transcription conferred by the unmodified SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 23, or within approximately 25% to 50% of the transcription conferred by the unmodified SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 2.

[0082] 【0083】 In certain embodiments, the modified nucleic acids or polynucleotides described herein, such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 23, are located at the 5' position of the transgene.

[0083] 【0084】 In a particular embodiment, the introduced gene encodes a blood coagulation or coagulation protein.

[0084] 【0085】 In certain embodiments, the transgene encodes factor IX (FIX), factor VIII (FVIII), factor VII (FVII), or protein C.

[0085] 【0086】 In a particular embodiment, the transgene encodes factor VIII, which has a sequence that is at least 95% identical to the sequence of sequence number 68.

[0086] 【0087】In certain embodiments, the transgene is transcribed into an inhibitory RNA. In certain embodiments, the inhibitory RNA includes antisense RNA, microRNA (miRNA), or small interfering RNA (siRNA).

[0087] 【0088】 In a particular embodiment, the transgene encodes a therapeutic protein that is expressed in liver cells and secreted into the systemic circulation.

[0088] 【0089】 In certain embodiments, therapeutic proteins treat or prevent neurodegenerative diseases or central nervous system (CNS) diseases.

[0089] 【0090】 In a particular embodiment, the therapeutic protein is a protective ApoE isoform.

[0090] 【0091】 In a particular embodiment, the therapeutic protein is the ApoE ε2 isoform.

[0091] 【0092】 In certain embodiments, therapeutic proteins treat or prevent autoimmune or allergic diseases.

[0092] 【0093】 In a particular embodiment, the therapeutic protein is a fusion protein comprising an undesirable antigen and a leader sequence that drives the secretion of the therapeutic protein from cells.

[0093] 【0094】 In certain embodiments, the undesirable antigen is the extracellular domain or fragment thereof of myelin oligodendrocyte glycoprotein (MOG).

[0094] 【0095】In a particular embodiment, the expression cassette comprises a nucleic acid sequence or polynucleotide of the present invention, such as the modified SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 23, operably linked to the transgene, wherein the nucleic acid sequence or polynucleotide is located upstream of the 5' end of the transgene, and optionally comprises zero untranslated nucleic acid sequences located between the nucleic acid sequence or polynucleotide and the 5' end of the transgene. There are up to 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 106, or 107 or fewer subnets.

[0095] 【0096】 In a particular embodiment, the expression cassette includes a first nucleotide sequence having 95% or more sequence identity with any of the sequences of SEQ ID NOs: 4 to 21 or SEQ ID NOs: 24 to 67, and the first nucleotide located upstream of the 5' end of the second nucleotide sequence has 95% or more sequence identity with the sequence of SEQ ID NOs: 77, and optionally, a first nucleotide sequence located between the first nucleotide sequence and the 5' end of the second nucleotide sequence The untranslated nucleic acid sequence contains 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 106, or 107 or fewer nucleic acids.

[0096] 【0097】 In a particular embodiment, the expression cassette includes the sequence of SEQ ID NO: 1 or a polynucleotide having at least 98% sequence identity with respect to the sequence of SEQ ID NO: 1.

[0097] 【0098】In a particular embodiment, the expression cassette comprises a polynucleotide having at least 99% sequence identity with respect to the sequence of SEQ ID NO: 1.

[0098] 【0099】 In a particular embodiment, the expression cassette essentially consists of Sequence ID No. 1.

[0099] 【0100】 In a particular embodiment, the transgene or second nucleotide sequence comprises a nucleic acid sequence encoding factor VIII (FVIII) (FVIII-BDD) having a B-domain deletion, wherein the nucleic acid sequence encodes an FVII-BDD protein having FVIII blood coagulation activity and having at least 90% sequence identity with the sequence of SEQ ID NO: 68.

[0100] 【0101】 In a particular embodiment, the transgene or second nucleotide sequence comprises a nucleic acid sequence encoding factor VIII (FVIII) (FVIII-BDD) having a B-domain deletion, the nucleic acid sequence having 90% or more sequence identity with the sequence of Sequence ID No. 77, and encoding a protein having FVIII blood coagulation activity.

[0101] 【0102】 In certain embodiments, the untranslated nucleic acid sequence is either not an intron or does not contain an intron.

[0102] 【0103】 In a particular embodiment, the first nucleotide sequence includes a nucleic acid sequence that is at least 95% identical to either SEQ ID NOs: 4 to 21 or SEQ ID NOs: 24 to 67.

[0103] 【0104】 In a particular embodiment, the first nucleotide sequence comprises a nucleic acid sequence that is at least 95% identical to any of the sequences of SEQ ID NOs: 4 to 21 or SEQ ID NOs: 24 to 67, and has the same total number of reduced CpGs as described in any of the sequences of SEQ ID NOs: 4 to 21 or SEQ ID NOs: 24 to 67.

[0104] 【0105】In a particular embodiment, the first nucleotide sequence includes a nucleic acid sequence in which CpG(s) are substituted with CpT, CpA, TpG, or ApG at the same position(s) described in any of the sequences of Sequence IDs 4-21 or 24-67, and which is at least 95% identical to any of Sequence IDs 4-21 or 24-67.

[0105] 【0106】 In a particular embodiment, the second nucleotide sequence exhibits higher expression compared to a polynucleotide having 10⁸ or more nucleotides between the first nucleotide sequence and the 5' end of the second nucleotide sequence.

[0106] 【0107】 In certain embodiments, the second nucleotide exhibits higher biological activity compared to expression from a polynucleotide having 10⁸ or more nucleotides between the first nucleotide sequence and the 5' end of the second nucleotide sequence.

[0107] 【0108】 In certain embodiments, biological activity is determined by an FVIII assay or a coagulation assay or reduction of bleeding in an FVIII-deficient model.

[0108] 【0109】 In a particular embodiment, the second nucleotide sequence is packaged into the AAV vector more efficiently than packaging a polynucleotide having 10⁸ or more nucleotides between the first nucleotide sequence and the 5' end of the second nucleotide sequence.

[0109] 【0110】 In certain embodiments, the adeno-associated virus (AAV) vector comprises a nucleic acid sequence, or polynucleotide or expression cassette, as described herein.

[0110] 【0111】In a particular embodiment, the AAV vector comprises a) an AAV capsid, and b) one or more AAV terminal inversion sequences (ITRs), which are AAV ITRs (or more) adjacent to the 5' or 3' end of a nucleic acid sequence, polynucleotide, and / or transgene.

[0111] 【0112】 In a particular embodiment, the AAV vector further includes an intron located within an adjacent 5' or 3' ITR.

[0112] 【0113】 In a particular embodiment, an intron or one or more ITRs are modified to have a reduced CpG.

[0113] 【0114】 In a particular embodiment, the AAV capsid serotype is a modified or variant AAV VP1, VP2 and / or VP3 capsid having 90% or more sequence identity with the AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, AAV-2i8, SEQ ID NO: 91 or SEQ ID NO: 92 VP1, VP2 and / or VP3 sequences, or AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8, SEQ ID NO: 91 or SEQ ID NO: 92 The capsid contains a capsid having 95% or more sequence identity to the VP1, VP2, and / or VP3 sequences, or a capsid having 100% sequence identity to the AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8, SEQ ID NO: 91, or SEQ ID NO: 92 VP1, VP2, and / or VP3 sequences.

[0114] 【0115】In a particular embodiment, the ITR includes one or more ITRs of any AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74 AAV serotypes, or combinations thereof.

[0115] 【0116】 In a particular embodiment, the AAV vector further comprises an ITR, a poly-A signal, and / or an intron sequence.

[0116] 【0117】 In a particular embodiment, the AAV vector described herein is any pharmaceutical composition.

[0117] 【0118】 In certain embodiments, the pharmaceutical composition includes a biocompatible carrier or excipient.

[0118] 【0119】 In a particular embodiment, the pharmaceutical composition further comprises an empty AAV capsid.

[0119] 【0120】 In certain embodiments, the composition or pharmaceutical composition includes ratios of empty AAV capsid to AAV vector within or between approximately 100:1 to 50:1, approximately 50:1 to 25:1, approximately 25:1 to 10:1, approximately 10:1 to 1:1, approximately 1:10 to 1:10, approximately 1:10 to 1:25, approximately 1:25 to 1:50, or approximately 1:50 to 1:100.

[0120] 【0121】 In certain embodiments, the ratio of empty AAV capsid to AAV vector in the composition or pharmaceutical composition is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0121] 【0122】 In certain embodiments, the compositions or pharmaceutical compositions described herein further comprise a surfactant.

[0122] 【0123】 In a particular embodiment, a method is provided for treating a human being in need of gene therapy.

[0123] 【0124】 In certain embodiments, humans require blood coagulation or coagulation factors.

[0124] 【0125】 In a particular embodiment, a method for treating a human being comprises the steps of (a) preparing an expression cassette, a polynucleotide, an AAV vector, or a pharmaceutical composition as described herein, and (b) administering a certain amount of the expression cassette, polynucleotide, AAV vector, or pharmaceutical composition to a human being.

[0125] 【0126】 In certain embodiments, a human being has hemophilia A or hemophilia B.

[0126] 【0127】 In certain embodiments, the AAV vector is administered to a human being intravenously, intraarterially, intraluminally, intramuscularly, or by catheter.

[0127] 【0128】 In certain embodiments, blood coagulation or coagulation factors are expressed at increased levels after administration.

[0128] 【0129】 In certain embodiments, blood coagulation or coagulation factors are expressed at levels exceeding 1% of those found in humans who do not require blood coagulation or coagulation factors.

[0129] 【0130】 In certain embodiments, blood coagulation or coagulation factors are expressed at approximately 1% to 40% of the levels found in humans who do not require blood coagulation or coagulation factors.

[0130] 【0131】 In certain embodiments, blood coagulation or coagulation factors are expressed at approximately 5% to 30% of the levels found in humans who do not require blood coagulation or coagulation factors.

[0131] 【0132】In a particular embodiment, the AAV vector is approximately 1 × 10¹⁶ per kilogram of human body weight. 8 ~Approx. 1×10 14 It is administered within the range of vector genome (vg / kg). [Brief explanation of the drawing]

[0132] [Figure 1] This figure shows the levels of human factor VX (hFIX) measured by an activity assay in mouse plasma 24 hours after hydrodynamic delivery of CpG-reduced ApoE / hAAT regulatory element-hFIX coding constructs labeled "CpG1" to "CpG22". Sequence IDs 24 to 67 correspond to regulatory elements CpG1-ApoE / hAAT to CpG22-ApoE / hAAT, respectively, with and without restriction enzyme sites, as further described in Example 13. The hFIX level is expressed as a multiple of the reference plasmid containing the non-CpG-reduced ApoE / hAAT (Sequence ID 23). [Figure 2] This figure shows the levels of human factor VIII (hFVIII) antigen in mouse plasma, measured by ELISA assay, 24 hours after hydrodynamic delivery of CpG-reduced TTRm promoter hFVIII coding constructs labeled "CpG1" to "CpG5". Sequence IDs 4 to 21 correspond to promoters CpG1-TTRm to CpG5-TTRm, respectively, with and without adjacent restriction enzyme sites, and are further described in Example 13. Levels are expressed as a percentage of normal human plasma FVIII, where 100% = 150 ng / mL. "TTRm" refers to the hFVIII coding construct containing the TTRm promoter (Sequence ID 3) without CpG reduction. [Figure 3]This figure shows the hFVIII antigen levels in mouse plasma, measured by ELISA assay, 24 hours after hydrodynamic delivery of CpG-reduced hybrid promoter hFVIII coding constructs labeled "Hybrid 6" to "Hybrid 9". Sequence IDs 14 to 21 correspond to CpG-reduced promoter hybrids 6 to 9, respectively. Levels are expressed as a percentage of normal human plasma FVIII, where 100% = 150 ng / mL. "TTRm" refers to the hFVIII coding construct containing the non-CpG-reduced TTRm promoter (Sequence ID 3). [Figure 4] This figure shows the hFVIII antigen levels in mouse plasma, measured by ELISA assay, 2, 4, and 8 weeks after delivery of hybrid 6, hybrid 7, hybrid 8, and hybrid 9 promoter-hFVIII constructs with unreduced (TTRm) and reduced CpG at a dose of 6.4e11 vector genome (vg) / kg. Levels are expressed as a percentage of normal human plasma FVIII, where 100% = 150 ng / mL. [Figure 5] This figure shows hFVIII antigen levels measured by ELISA assay in mouse plasma 8 weeks after delivery of hybrid 7 and hybrid 9 promoter-hFVIII constructs, where AAV capsid-formed CpG was not reduced (TTRm-hFVIII) at doses of 2.56e11, 6.4e11, and 1.6e12 vg / kg. Levels are expressed as a percentage of normal human plasma FVIII, where 100% = 150 ng / mL. [Figure 6]This figure shows the expression of hFVIII mRNA in the liver, brain, testes, spleen, and kidneys of mice 8 weeks after intravenous administration of hybrid 7 and hybrid 9 promoter-hFVIII constructs at a dose of 6.4e11 vg / kg, where AAV-formed CpG was not reduced (TTRm-hFVIII). hFVIII RNA was not observed in any tissue other than the liver, demonstrating the liver specificity of the promoter. The results for the liver, brain, testes, spleen, and kidneys are shown from left to right, for TTRm-hFVIII, hybrid 7-hFVIII, and hybrid 9-hFVIII, respectively. [Figure 7] This figure shows a schematic comparison between an expression cassette with an intron, referred to as "AAV-WINT" (TTRm-intron-hFVIII-BDD), and an expression cassette without an intron, referred to as "AAV-INTL" (TTRm-hFVIII-BDD without intron, SEQ ID NO: 1). AAV-WINT has a synthetic intron (SEQ ID NO: 93) located between the TTRm promoter and the transgene encoding B-domain deletion human factor VIII (hFVIII-BDD), which is not present in AAV-INTL. The codon-optimized nucleic acid sequences in these cassettes encoding hFVIII-BDD are described in SEQ ID NO: 77. [Figure 8] This figure shows the hFVIII levels detected by ELISA performed on mouse plasma samples for a 6.4e9 vg / mouse dose in Study #1. The results are the mean values ​​for animals (n=5) in each treatment group. Error bars represent the standard deviation. [Figure 9] This figure shows the hFVIII levels detected by ELISA performed on mouse plasma samples for a 1.6e10 vg / mouse dose in Study #1. The results are the mean values ​​for animals in each treatment group (TTRm hFVIII, n=4; TTRm hFVIII without introns, n=5). Error bars represent the standard deviation. [Figure 10]This figure shows the hFVIII levels detected by ELISA performed on mouse plasma samples from Study #2. The results are the mean values ​​for animals (n=10) in each treatment group. Error bars represent the standard deviation. [Figure 11] This figure shows the hFVIII levels detected by ELISA performed on non-human primate (NHP) plasma samples from Study #1. Individual monkey results are shown for low-dose (2e12 vg / kg) groups 1 (AAV-WINT, lines and squares, n=2) and 2 (AAV-INTL, SEQ ID NO: 1, lines and triangles, n=3). One animal in the low-dose AAV-WINT group was removed due to positive neutralizing antibodies against AAV observed in a sample 8 days prior to administration. [Figure 12] This figure shows the hFVIII levels detected by ELISA performed on NHP plasma samples from Study #1. Individual monkey results are shown for high-dose (6e12 vg / kg) groups 3 (AAV-WINT, lines and squares, n=2) and 4 (AAV-INTL, SEQ ID NO: 1, lines and triangles, n=3). One animal in the high-dose AAV-WINT group, P0101, was removed due to the absence of FVIII expression during treatment. [Figure 13] This figure shows the hFVIII levels detected by ELISA performed on plasma samples from NHP in Study #2. Individual monkey results for AAV-WINT (lines and squares, n=5) and AAV-INTL (Sequence ID 1, lines and triangles, n=5) at 2e12 vg / kg doses are shown. [Figure 14] This figure shows the results of cell-based vector efficacy assays at three different MOIs. The cell supernatant was evaluated for hFVIII activity with Chromogenix Coatest SP4, and the results are the mean values ​​of two biological replicates assayed in double replicates. Error bars represent the standard deviation. "AAV-WINT" and "AAV-INTL" are the original undiluted stock vials of the virus, while "AAV-WINT Dosage" and "AAV-INTL Dosage" show the material diluted for injection. [Figure 15]This figure shows the results of Figure 14, replotted as a multiple change relative to AAV-WINT for each MOI. [Figure 16] This figure shows the evaluation of vector potency in in vitro cell-based vector potency assays at three different MOIs. Cell supernatants were evaluated for hFVIII activity with Chromogenix Coatest SP4, and the mean values ​​are from two biological replicates assayed in double replicates. Error bars represent the standard deviation. AAV-WINT and AAV-INTL (SEQ ID NO: 1) are original undiluted stock vials of virus from two different lots. [Figure 17] This figure shows a comparison of expression cassettes for in vitro hFVIII levels assayed with Chromogenix Coatest SP4 from the supernatant of Huh7 cells transfected with independent plasmid DNA preparations (preps) of mTTR-intron-hFVIII-BDD and mTTR-hFVIII-BDD (SEQ ID NO: 1). Individual data points are shown as black circles, and each bar represents the mean value (n=2) of two biological replicates assayed with double repeats. Error bars represent the standard deviation. [Figure 18] This figure shows the daily FVIII activity levels in four human subjects (Participant 1 (circle), Participant 2 (square), Participant 3 (triangle), and Participant 4 (diamond)) who were injected with a 5 × 10¹¹ vg / kg AAV-INTL hFVIII expression cassette (SEQ ID NO: 1) capsid-formed in the LK03 AAV vector (SEQ ID NO: 91), referred to herein as LK03-INTL hFVIII-BDD. [Figure 19] This figure shows the weekly mean FVIII activity levels in four human subjects (Participant 1 (circle), Participant 2 (square), Participant 3 (triangle), and Participant 4 (diamond)) who were injected with 5 × 10¹¹ vg / kg of LK03-INTL hFVIII-BDD. [Figure 20]This figure shows the 4-week block mean of FVIII activity levels in four human subjects (Participant 1 (circle), Participant 2 (square), Participant 3 (triangle), and Participant 4 (diamond)) who were injected with 5 × 10¹¹ vg / kg of LK03-INTL hFVIII-BDD. [Modes for carrying out the invention] 【0133】 [Detailed explanation] 【0153】 Intron-free expression cassette nucleic acid sequences for the expression of factor VIII (FVIII) and FVIII having a deleted B domain (FVIII-BDD) (e.g., human FVIII (hFVIII) and human FVIII-BDD (hFVIII-BDD)) are disclosed herein. Researchers have reported that the inclusion of introns in expression cassettes, including those in AAV delivery vectors, may contribute to increased transgene expression (Huang et al., 1990, Nucl. Acid Res., 18:937-947; Choi et al., 2014, Mol. Brain, 7:17; Powell et al., 2015, Discov. Med., 19:49-57; Lu et al., 2017, Hum. Gene Ther., 28:125-134). Surprisingly, as disclosed herein, partial or complete removal of introns resulted in increased AAV vector potency and transgene (in this case, factor VIII) expression levels in cell cultures, mice, and non-human primates. “Intron-free” expression cassette designs represent an improvement to vectors for the treatment of blood coagulation disorders such as hemophilia A, potentially providing efficacy at lower vector doses, reducing manufacturing barriers such as cost and time, and offering benefits to patient safety and outcomes. 【0134】 【0154】Also disclosed are nucleic acid sequences with reduced CpG compared to a reference wild-type mammal (e.g., human) sequence and / or nucleic acid sequences having less than 100% sequence identity to a reference wild-type mammal (e.g., human) sequence. The CpG-reduced nucleic acid sequences include one or more of the following promoters: TTR promoter, ApoE / hAAT promoter, FGG promoter, albumin promoter, and SAA1 promoter. The CpG-reduced nucleic acid sequences include fusions and hybrids of the TTR promoter and at least one or more of the following promoters: ApoE / hAAT promoter, FGG promoter, albumin promoter, and SAA1 promoter. 【0135】 【0155】 The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Examples of polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Examples of polynucleotides include naturally occurring polynucleotides, synthetic polynucleotides, and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides may be single-stranded, double-stranded or triple-stranded, linear or cyclic, and may have any length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction. 【0136】 【0156】As used herein, “modify” or “variant” and their grammatical variations mean that a nucleic acid, polynucleotide, or subsequence thereof deviates from a reference sequence. Therefore, modified and variant sequences may have substantially the same, higher, or lower expression, activity, or function as the reference sequence, but at least retain some partial activity or function of the reference sequence. Specific examples of modified or variant sequences include the CpG-reduced TTR promoter, ApoE / hAAT promoter, FGG promoter, albumin promoter, and SAA1 promoter. 【0137】 【0157】 A "nucleic acid" or "polynucleotide" variant refers to a genetically modified sequence compared to the wild type. A nucleic acid or polynucleotide variant may refer to a sequence in which codons are altered but which retains at least partial sequence identity with respect to a reference sequence, such as the wild-type sequence. A nucleic acid or polynucleotide encoding a protein can be genetically modified without altering the encoded protein sequence. Alternatively, the sequence can be genetically modified to encode a variant protein. For example, some codons in such a nucleic acid variant may be altered without altering the amino acids of the protein it encodes (e.g., CpG reduction). 【0138】 【0158】 Expression vectors with promoters containing reduced CpG may show improved expression compared to promoters with unreduced CpG content. When comparing expression, promoters with reduced CpG are compared to wild-type or unreduced promoters. 【0139】 【0159】 The terms “variant” or “modified” do not need to appear in any reference herein to CpG-reduced nucleic acid sequences. Similarly, terms such as “CpG-reduced nucleic acid” may omit “variant” or “modified,” but references to “CpG-reduced nucleic acid” encompass variants at the genetic level. 【0140】 【0160】 A specific example of a variant is a nucleic acid with reduced CpG. CpG reduction can be achieved by changing a C or G nucleotide to a different nucleotide, for example, changing C to T or G to A. CpG reduction can also be achieved by deleting a C nucleotide, or a G nucleotide, or both C and G nucleotides. 【0141】 【0161】 "Variant or modified" FVIII refers to FVIII or FVIII-BDD that is genetically modified compared to unmodified wild-type FVIII or FVIII-BDD (SEQ ID NO: 68). Such variants may also be referred to as "nucleic acid variants encoding factor VIII (FVIII)." 【0142】 【0162】 "Variant Factor VIII (FVIII)" can also refer to a modified FVIII protein in which the modified protein has amino acid changes compared to wild-type FVIII. When comparing activity and / or stability, if the encoded variant FVIII protein retains the B-domain, it is appropriate to compare it to wild-type FVIII, and if the encoded variant FVIII protein has a B-domain deletion, it is similarly compared to wild-type FVIII which also has a B-domain deletion. 【0143】 【0163】 Variant FVIII is part of the B-domain. Therefore, FVIII-BDD contains a portion of the B-domain. Typically, a large portion of the B-domain is deleted in FVIII-BDD. 【0144】 【0164】Variant FVIII may contain the "SQ" sequence described as SFSQNPPVLKRHQR (SEQ ID NO: 69). Typically, such variant FVIII having SQ (FVIII / SQ) has BDD, for example, with at least all or part of BD deleted. Variant FVIII such as FVIII-BDD may have all or part of the "SQ" sequence, i.e., all or part of SEQ ID NO: 69. Therefore, for example, variant FVIII-BDD having the SQ sequence (SFSQNPPVLKRHQR, SEQ ID NO: 69) may have the amino acid sequence SFSQNPPVLKRHQR (Sequence ID 69) It may have all or only a part of the following. For example, FVIII-BDD includes SFSQNPPVLKRHQR (Sequence ID 69) SFSQNPPVLKRHQR may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid residues. Therefore, SFSQNPPVLKRHQR has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 internal deletions and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino or carboxy terminal deletions. (Sequence ID 69) This is contained in the variant FVIII protein described herein. 【0145】 【0165】 "Polypeptides," "proteins," and "peptides" encoded by "nucleic acid" or "polynucleotide" sequences include full-length natural sequences as well as functional partial sequences, modified forms, or sequence variants, insofar as the partial sequences, modified forms, or variants retain some functionality of the naturally occurring full-length protein. For example, a nucleic acid encoding an FVIII protein (e.g., a nucleic acid with reduced CpG) may have the B-domain deletion described herein and retain coagulation function. In the methods and uses of the present invention, such polypeptides, proteins, and peptides encoded by nucleic acid sequences may be, but do not need to be, identical to endogenous proteins that are deficient, underexpressed, or missing in the treated mammal. 【0146】 【0166】 For example, and without limitation, modifications include one or more nucleotide or amino acid substitutions (e.g., 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 500 to 750, 750 to 850 or more nucleotides or residues). An example of nucleic acid modification, as described herein, is CpG reduction. 【0147】 【0167】 Examples of amino acid modifications include conservative amino acid substitutions or deletions (e.g., partial sequences or fragments) of a reference sequence, such as FVIII, which has a B domain deletion. In certain embodiments, the modified or variant sequence retains at least some of the function or activity of the unmodified sequence. 【0148】 【0168】 This specification clearly encompasses all mammalian and non-mammalian forms of nucleic acids, whether known or unknown, including other mammalian forms of promoters with reduced CpG values. 【0149】 【0169】The term “vector” refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV), or other vehicle that can be manipulated by the insertion or incorporation of nucleic acids. Vectors can be used for genetic manipulation (i.e., “vector cloning”), for introducing / transferring polynucleotides into cells and / or organs, and for transcribing or translating the inserted polynucleotides in cells. An “expression vector” is a vector containing a gene or nucleic acid sequence that has essential regulatory regions required for expression in a host cell. Vector nucleic acid sequences generally contain at least one origin of replication for propagation in cells, and optionally further elements such as heterologous nucleic acid sequences, expression regulatory elements (e.g., promoters, enhancers), introns, terminal inversion sequences (ITRs), optionally selectable markers, polyadenylation signals, etc. 【0150】 【0170】 As disclosed herein, intron-deficient vectors exhibited superior characteristics compared to the same vectors having synthetic introns. Accordingly, the present invention relates to an expression cassette comprising a transgene operably linked to a regulatory element, wherein the regulatory element (e.g., a CpG-reduced promoter as described herein) is positioned upstream of the 5' end of the transgene, and between the regulatory element and the 5' end of the transgene are 0-5, 5-10, 10-15, 15-20, and 20- An expression cassette is provided containing 25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 106, or 107 or fewer nucleotides. 【0151】 【0171】The present invention also relates to an expression cassette comprising a first nucleotide sequence having 95% or more sequence identity with any of the sequences of SEQ ID NOs: 2 to 67, wherein the first nucleotide sequence is positioned upstream of the 5' end of a second nucleotide sequence having 95% or more sequence identity with the sequence of SEQ ID NO: 77, and between the 5' ends of the first nucleotide sequence and the second nucleotide sequence are 0 to 5 or 5 to 10 untranslated nucleic acid sequences. An expression cassette is provided containing 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 106, or 107 or fewer nucleic acids. 【0152】 【0172】 The present invention further relates to an expression cassette comprising a first nucleotide sequence having 95% or more sequence identity with any of the sequences of SEQ ID NOs: 2 to 67, wherein the first nucleotide sequence is located upstream of the 5' end of a second nucleotide sequence having 95% or more sequence identity (e.g., 95%, 96%, 97%, 98%, or 99% or more sequence identity) with any of the sequences of SEQ ID NOs: 71 to 88, and the 5' ends of the first nucleotide sequence and the second nucleotide sequence An expression cassette is provided in which, between the untranslated nucleic acid sequence, there are 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 106, or 107 or fewer nucleotides.

[0153] 【0173】The present invention further provides an expression cassette having a non-coding nucleic acid positioned between a regulatory element and an introduced gene, wherein the non-coding nucleic acid is not an intron. Such an expression cassette may be referred to as an intron-free cassette.

[0154] 【0174】 An intron is a sequence containing a donor site and a splice acceptor site, which allows cellular mechanisms to cleave untranslated nucleotide sequences by splicing during the process of RNA maturation into mRNA. As used herein, "intron-free" refers to an untranslated nucleic acid sequence that lacks a donor site and a splice acceptor site, but does not mean that the untranslated nucleic acid sequence lacks other sites such as restriction enzyme recognition / cleavage sites, Kozak sequences, or transcription factor recognition / binding sites. In other words, "intron-free" does not mean that the nucleic acid sequence is completely lacking any untranslated nucleic acid sequence(s).

[0155] 【0175】 AAV vectors are derived from adeno-associated viruses. AAV vectors are useful as gene therapy vectors because they can penetrate cells and introduce nucleic acids / genetic material, which can then be stably maintained within the cells. Since AAV is not associated with pathogenic disease in humans, AAV vectors can deliver heterologous nucleic acid sequences (i.e., encoding therapeutic proteins and inhibitory RNAs) to human patients without causing substantial AAV disease or illness.

[0156] 【0176】The term "recombinant" as a modifier for vectors such as recombinant AAV (rAAV) vectors, and as a modifier for sequences such as recombinant polynucleotides and polypeptides, means that the composition has been manipulated (i.e., modified) in a manner that would not normally occur. A specific example of a recombinant AAV vector is one in which nucleic acids (heterogeneous sequences) not commonly present in the wild-type AAV genome have been inserted into the viral genome. Another example of a recombinant AAV vector is one in which nucleic acids (e.g., genes) encoding a therapeutic protein or polynucleotide sequence have been cloned into a vector, with or without the gene having the commonly associated 5', 3', and / or intron regions within the AAV genome. The term "recombinant" is not always used in relation to AAV vectors and polynucleotide sequences, and despite any such omissions, recombinant forms including AAV vectors and polynucleotides are clearly encompassed.

[0157] 【0177】 An "rAAV vector" is obtained from the wild-type AAV genome by using molecular methods to remove all or part of the wild-type AAV genome and replace it with non-natural (heterogeneous) nucleic acids, such as nucleic acids encoding therapeutic proteins or polynucleotide sequences. Typically, with respect to rAAV vectors, one or both terminal inversion (ITR) sequences of the AAV genome are retained. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with non-natural sequences related to the AAV genome nucleic acid, such as heterogeneous nucleic acids encoding therapeutic proteins or polynucleotide sequences. Therefore, the incorporation of non-natural (heterogeneous) sequences defines AAV as a "recombinant" AAV vector, which may be referred to as an "rAAV vector."

[0158] 【0178】Recombinant AAV vector sequences may be packaged, which are referred to herein as “particles” for subsequent cell infection (transduction) ex vivo, in vitro, or in vivo. When a recombinant vector sequence is capsid-formed or packaged into an AAV particle, the particle may also be referred to as “rAAV,” “rAAV particle,” or “rAAV virion.” Such rAAV, rAAV particle, and rAAV virion contain proteins that capsid-form or package the vector genome. A specific example, in the case of AAV, is the capsid protein.

[0159] 【0179】 The term "vector genome," or more conveniently abbreviated as "vg," refers to the portion of a recombinant plasmid sequence that is ultimately packaged or capsid-formed to create rAAV particles. When a recombinant plasmid is used to construct or produce a recombinant AAV vector, the AAV vector genome does not include the portion of the plasmid that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," and while it is crucial for plasmid cloning and amplification—processes required for propagation and recombinant AAV vector production—it is not itself packaged or capsid-formed into rAAV particles. Therefore, the "vector genome" refers to the nucleic acid that is packaged or capsid-formed by rAAV.

[0160] 【0180】"AAV helper function" refers to AAV-derived coding sequences (proteins) that can be expressed to sequentially provide trans-functioning AAV gene products and AAV vectors with respect to proliferative AAV replication and packaging. Therefore, AAV helper function includes both rep and cap, which are the primary AAV open reading frames (ORFs). Rep expression products have been shown to possess many functions, including, among others, recognition, binding, and nicking of AAV DNA replication origins; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. Cap expression products (capsids) provide essential packaging functions. AAV helper function is used to complement trans-functioning AAV that is lacking in the AAV vector genome.

[0161] 【0181】 An "AAV helper construct" generally refers to a nucleic acid sequence containing a nucleotide sequence that provides missing AAV functionality from an AAV vector, which should be used, for example, to produce a transduction AAV vector for the delivery of a target nucleic acid sequence to a subject in gene therapy. AAV helper constructs are generally used to provide transient expression of AAV rev and / or cap genes to complement missing AAV functionality necessary for AAV vector replication and capsid formation. Helper constructs generally lack AAV ITRs, cannot replicate, and cannot package themselves. AAV helper constructs can exist in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Numerous AAV helper constructs are described, such as the plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products (see, e.g., Samulski et al. (1989) J. Virol. 63: pp. 3822-3828, and McCarty et al. (1991) J. Virol. 65: pp. 2936-2945). Numerous other vectors encoding Rep and / or Cap expression products are described (see, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237).

[0162] 【0182】The term "accessory function" refers to non-AAV-derived viral / cellular functions on which AAV can replicate. This term encompasses proteins and RNAs required for AAV replication, including regions involved in AAV gene transcription, step-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and activation of AAV capsid packaging. Virus-based accessory functions may originate from any known helper viruses, such as adenoviruses, herpesviruses (herpes simplex virus type 1), and vaccinia viruses.

[0163] 【0183】An "accessory function vector" generally refers to a nucleic acid molecule containing a polynucleotide sequence that provides accessory function. Such a sequence resides on the accessory function vector and can be lanceted into a suitable host cell. Accessory function vectors can support rAVV virion production in host cells. Accessory function vectors can exist in the form of plasmids, phages, transposons, or cosmids. Furthermore, complete complement of the adenovirus gene is not required for accessory function. For example, adenovirus mutants that are unable to perform DNA replication and late gene synthesis have been reported to be able to replicate with AAV (Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al., (1971) Virology 45:317). Similarly, mutants in the E2B and E3 regions have been shown to support AAV replication, suggesting that the E2B and E3 regions are unlikely to be involved in providing accessory function (Carter et al., (1983) Virology 126:505). Adenoviruses lacking the E1 region or missing the E4 region are unable to support AAV replication. Therefore, the E1A and E4 regions appear to be directly or indirectly necessary for AAV replication (Laughlin et al., (1982) J. Virol. 41: 868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78: 1925; Carter et al., (1983) Virology 126: 505).Other characterized adenovirus variants include E1B (Laughlin et al. (1982), see above; Janik et al., (1981), see above; Ostrove et al., (1980) Virology 104:502), E2A (Handa et al., (1975) J.Gen.Virol.29:239; Strauss et al., (1976) J.Virol.17:140; Myers et al., (1980) J.Virol.35:665; Jay et al., (1981) Proc.Natl.Acad.Sci.USA 78:2927; Myers et al., (1981) J.Biol.Chem.256:567), E2B (Carter, Adeno-Associated Virus Helper Functions, I CRC Handbook of Examples include Parvoviruses (P. Tijssen, ed., 1990), E3 (Carter et al., (1983), see above), and E4 (Carter et al., (1983), see above; Carter et al., (1995)). Studies of accessory functions provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, with E1B55k potentially required for AAV virion production, while E1B19k is not (Samulski et al. (1988) J. Virol. 62: pp. 206-210). Furthermore, International Publication No. 97 / 17458 and Matshushita et al., (1998) Gene Therapy 5: pp. 938-945 describe accessory function vectors encoding various adenovirus genes. Exemplary accessory functional vectors include the adenovirus VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72kD coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the intact E1B 55k coding region. Such accessory functional vectors are described, for example, in International Publication No. 01 / 83797.

[0164] 【0184】As used herein, the term “serotype” refers to a characteristic of an AAV that has a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies against a particular AAV compared to another AAV. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0165] 【0185】 Traditionally, a serotype means that the virus of interest has been tested against serums specific to all existing characterized serotypes for neutralizing activity. As more naturally occurring virus isolates are discovered and / or capsid variants are produced, there may or may not be serological differences between them and any of the existing serotypes. Therefore, if a new virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. Often, according to the traditional definition of serotype, serological testing for neutralizing activity to determine whether a variant virus with a capsid sequence modification is a different serotype has not yet been performed for variant viruses with capsid sequence modifications. Therefore, for convenience and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g., AAV) and serologically non-distinguishable viruses that may exist within a given subgroup or variant of a serotype.

[0166] 【0186】rAAV vectors can encompass any viral strain or serotype. For example, but not limited to, an rAAV vector genome or particle (capsid such as VP1, VP2, and / or VP3) may be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10, or AAV-2i8. Such vectors may be based on the same strain or serotype (or subgroup or variant), or they may be different from each other. For example, but not limited to, the genome or particle (capsid) of an rAAV plasmid or vector based on a single serotype genome may be identical to one or more of the capsid proteins that package the vector. Furthermore, the genome of the rAAV plasmid or vector may be based on an AAV serotype genome different from one or more of the capsid proteins that package the vector genome, in which case at least one of the three capsid proteins may be, for example, a different AAV serotype, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, LK03 (SEQ ID NO: 91), SPK (SEQ ID NO: 92), or a variant thereof. More specifically, the rAAV2 vector genome may contain the AAV2 ITR, but is a capsid derived from different serotypes, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, LK03 (SEQ ID NO: 91), SPK (SEQ ID NO: 92), or their variants. Therefore, AAV vectors contain gene / protein sequences identical to those characteristic of specific serotypes and mixed serotypes, also known as pseudotypes.

[0167] 【0187】In a particular embodiment, the rAAV vector contains, or consists of, at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) the same capsid sequences as one or more AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, LK03 (SEQ ID NO: 91), SPK (SEQ ID NO: 92) capsid proteins (VP1, VP2, and / or VP3 sequences). In a particular embodiment, the rAAV vector contains or consists of sequences that are identical to at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) one or more AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, or -rh10 ITR(or) ITR(or).

[0168] 【0188】 In certain embodiments, the rAAV vectors include, for example, AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, and AAV-2i8, and their variants (e.g., ITR and capsid variants such as amino acid insertions, additions, substitutions, and deletions), as described in International Publication No. 2013 / 158879 (International Application PCT / US2013 / 037170), International Publication No. 2015 / 013313 (International Application PCT / US2014 / 047670), and U.S. Patent No. 2013 / 0059732 (U.S. Patent Application No. 13 / 594,773, disclosing LK01, LK02, LK03 (SEQ ID NO: 91), etc.).

[0169] 【0189】rAAVs such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, LK03 (SEQ ID NO: 91), SPK (SEQ ID NO: 92), and variants, hybrids, and chimeric sequences can be constructed using recombination techniques known to those skilled in the art to contain one or more functional AAV ITR sequences and one or more heterologous polynucleotide sequences (transgenes) adjacent to them. Such AAV vectors typically retain at least one(s) functional adjacent ITR sequences as required for rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome contains sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0170] 【0190】 As used herein, the terms “true AAV vector” or “true rAAV vector” refer to an AAV vector containing heterologous nucleic acid capable of infecting target cells. This term excludes empty vectors (those not containing heterologous nucleic acid) and AAV vectors lacking a complete insertion (e.g., heterologous nucleic acid fragment) or AAV vectors containing host cell nucleic acid.

[0171] 【0191】 The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0172] 【0192】 Nucleic acids include genomic cDNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA).

[0173] 【0193】Nucleic acids include naturally occurring polynucleotides, synthetic polynucleotides, and intentionally modified or altered polynucleotides. Nucleic acids may be single-stranded, double-stranded, or triple-stranded, linear or cyclic, and may have any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction.

[0174] 【0194】 A “heterogeneous” nucleic acid sequence refers to a nucleotide inserted into an AAV plasmid or vector for the purpose of vector-mediated introduction / delivery of a polynucleotide into a cell. Heterogeneous nucleic acid sequences are different from, i.e., unnatural with respect to, AAV nucleic acids. Once introduced / delivered into a cell, a heterogeneous nucleic acid sequence contained within a vector may be expressed (e.g., transcribed and translated where appropriate). Alternatively, a heterogeneous polynucleotide contained within a vector and introduced / delivered into a cell may not be expressed. The term “heterogeneous” is not necessarily used herein in relation to nucleic acid sequences and polynucleotides, and references to nucleic acid sequences and polynucleotides are intended to encompass heterogeneous nucleic acid sequences and polynucleotides, even in the absence of the modifier “heterogeneous,” and despite omission.

[0175] 【0195】 "Transgene" is used herein for convenience to refer to a nucleic acid that is intended to be introduced into or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a therapeutic protein or a heterologous nucleic acid encoding a polynucleotide sequence. Transgenes and heterologous nucleic acids / polynucleotide sequences are used interchangeably herein.

[0176] 【0196】In cells containing a transgene, the transgene is introduced / transferred by plasmid or AAV vector, or by "transduction" or "transfection" of the cell. The terms "transduction" and "transfection" refer to the introduction of molecules such as nucleic acids into host cells (e.g., HEK293) or cells or organs of an organism. The transgene may or may not be integrated into the genomic nucleic acid of the recipient cell.

[0177] 【0197】 "Nucleic acids," "polynucleotides," "heteronucleotides," "transgenes," and "CpG-reduced nucleic acid sequences" include full-length sequences and functional subsequences, as long as the subsequences retain some degree of functionality of the full-length sequence. Nucleic acids, polynucleotides, heteronucleotides, transgenes, and CpG-reduced nucleic acid sequences.

[0178] 【0198】 Polynucleotides, proteins, and peptides encoded by nucleic acid sequences, such as heterogeneous nucleic acids, include full-length sequences like naturally occurring proteins, as well as functional partial sequences, modified forms, or sequence variants, as long as they retain some degree of functionality of the full-length protein. Such polypeptides, proteins, and peptides encoded by nucleic acid sequences may, but do not need to be, identical to endogenous proteins that are deficient, underexpressed, or missing in the treated mammal.

[0179] 【0199】"Host cell" means, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or are used as AAV vector plasmids, AAV helper constructs, accessory functional vectors, or other transfer DNA. This term encompasses the offspring of the original cell being transfected. Therefore, "host cell" generally refers to a cell transfected with an exogenous DNA sequence. It is understood that offspring of a single parental cell may not necessarily be completely identical to the original parent in morphology or genomic or total DNA complement due to spontaneous, accidental, or planned mutations. An example host cell is human embryonic kidney (HEK) cells such as HEK293.

[0180] 【0200】 A "transduced cell" is a cell into which a transgene has been introduced. Therefore, a "transduced" cell refers to a genetic change in a cell after the uptake of an exogenous molecule, such as nucleic acid (e.g., a transgene). Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or its offspring. Cells may be propagated (cultured), the introduced protein may be expressed, the nucleic acid may be transcribed, or vectors such as rAAV may be produced by the cells. With regard to the use and methods of gene therapy, transduced cells may include organs or tissues, and may be present in the target accordingly.

[0181] 【0201】 As used herein, the terms “stable” or “stable integration” in relation to cells mean that a selectable marker or nucleic acid sequence, such as a heterologous nucleic acid sequence, or a plasmid or vector, is inserted into a chromosome (e.g., by homologous recombination, non-homologous end rejoining, transfection, etc.) or maintained outside the chromosome in a recipient cell or host organism, and remains on the chromosome or is maintained outside the chromosome for a period of time.

[0182] 【0202】A "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro under appropriate culture conditions. A cell line may, but does not have to be, a clonal population derived from a single progenitor cell. Spontaneous or induced changes may occur in a cell line during storage or transfer of such a clonal population, and during long-term subculturing in tissue culture. Therefore, the progeny cells obtained from a cell line may not be exactly identical to the ancestral cells or culture. An exemplary cell line applicable to the purification method of the present invention is HEK293.

[0183] 【0203】 A “regulatory expression element” refers to a nucleic acid sequence(s) that affects the expression of a operably linked nucleic acid. Examples of regulatory elements include promoters and enhancers, as described herein. An rAAV vector may contain one or more “regulatory expression elements.” Typically, such elements are included to promote proper heterologous polynucleotide transcription and, where appropriate, translation (e.g., promoters, enhancers, splicing signals for introns, accurate reading frames for genes to enable in-frame translation of mRNA, and stop codons). Such elements typically act in the cis and are referred to as “cis-acting” elements, but may also act in the trans.

[0184] 【0204】Expression regulation can be achieved at levels such as transcription, translation, splicing, and message stability. Typically, regulatory elements that modulate transcription are juxtaposed near the 5' end of the nucleic acid being transcribed (i.e., "upstream"). Regulatory elements can also be located at the 3' end of the sequence being transcribed (i.e., "downstream") or within the transcript (e.g., in an intron). Regulatory elements (e.g., CpG-reduced TTR, ApoE / hAAT, FGG, albumin, and SAA1 promoters and their fusions / hybrids) can be located adjacent to the sequence being transcribed, slightly away from it (e.g., 1-10, 10-25, 25-50, 50-100, 100-500 or more nucleotides from a polynucleotide), or even considerably away from the 5' or 3' end. Nevertheless, due to the length limitations of rAAV vectors, regulatory sequences are usually located within 1-1000 nucleotides of the nucleic acid being transcribed.

[0185] 【0205】 Functionally, the expression of operably linked nucleic acids is controllable, at least to some extent, by elements (e.g., promoters, enhancers, etc.) such that the elements regulate the transcription of the nucleic acid and, where appropriate, the translation of the transcript. A specific example of an expression regulatory element is a promoter, which is usually located at 5' of the sequence being transcribed. Promoters typically increase the expression from operably linked nucleic acids compared to the amount expressed in the absence of the promoter (when it is present).

[0186] 【0206】 Examples of promoters include the TTR and ApoE / hAAT promoters, including CpG-reduced versions and hybrid forms of the TTR promoter disclosed herein. Further examples of promoters include the ApoE / hAAT, FGG, albumin, and SAA1 promoters, including their CpG-reduced versions and hybrid forms.

[0187] 【0207】As used herein, "enhancer" may refer to a sequence positioned adjacent to a nucleic acid sequence, such as a heterologous nucleic acid sequence. Enhancer elements are typically positioned upstream (5') of a promoter element, but can also function and be positioned downstream (3') of the sequence, or even within the sequence. Therefore, enhancer elements can be positioned upstream or downstream of a heterologous nucleic acid encoding a selectable marker and / or a therapeutic protein or polynucleotide sequence, for example, within 100, 200, or 300 or more base pairs. Enhancer elements typically increase the expression of the operablely linked nucleic acid beyond the expression confeded by the promoter element.

[0188] 【0208】 The term "operably linked" means that regulatory sequences necessary for the expression of a nucleic acid sequence are positioned appropriately relative to the sequence to achieve nucleic acid sequence expression. This same definition may, in some cases, apply to the arrangement of nucleic acid sequences and transcriptional regulatory elements (e.g., promoters, enhancers, and termination elements) in expression vectors, such as rAAV vectors.

[0189] 【0209】 In examples of expression regulatory elements operably linked to nucleic acids, the relationship is such that the regulatory element modulates the expression of the nucleic acid. More specifically, for example, two operably linked DNA sequences mean that the two DNA sequences are arranged (cis or trans) in such a relationship that at least one of the DNA sequences can exert a regulatory effect on the other sequence.

[0190] 【0210】 Therefore, further elements of the vector include, but are not limited to, expression regulatory (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylated (polyA) sequences) adjacent to sequences such as one or more copies of an AAV ITR sequence (e.g., heterologous sequences), or introns.

[0191] 【0211】 Further elements include, for example, filler or stuffer polynucleotide sequences to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept DNA insertions having a size range of approximately 4kb to 5.2kb, or slightly larger. Therefore, for shorter sequences, stuffers or fillers are included to adjust the length, either near or at the normal size of the viral genome sequence that is acceptable for vector packaging into rAAV particles. In certain embodiments, the filler / stuffer nucleic acid sequence is an untranslated (non-protein-coding) segment of the nucleic acid. For nucleic acid sequences less than 4.7kb, the filler or stuffer polynucleotide sequence, when combined with the sequence (e.g., inserted into a vector), has a total length of approximately 3.0kb to 5.5kb, or approximately 4.0kb to 5.0kb, or approximately 4.3kb to 4.8kb.

[0192] 【0212】 If wild-type heterologous nucleic acids or transgenes are too large to be packaged within AAV vector particles, the heterologous nucleic acids may be provided in a modified, fragmented, or cleaved form for packaging in and delivery by the AAV vector, so that functional proteins or nucleic acid products, such as therapeutic proteins or nucleic acid products, are ultimately provided.

[0193] 【0213】 In a particular embodiment, heterologous nucleic acids encoding a protein (e.g., a therapeutic protein) are provided in a modified or cleaved form, or the heterologous nucleic acids are provided in a multiple construct delivered by separate multiplex AAV vectors.

[0194] 【0214】 In a particular embodiment, the coding heterologous polynucleotide is provided as a cleavage variant that maintains the functionality of the coding protein (e.g., a therapeutic protein), including the removal of a portion necessary for its function, so that the size of the coding heterologous polynucleotide is reduced for packaging in an AAV vector.

[0195] 【0215】 In a particular embodiment, heterogeneous nucleic acids are provided in a split AAV vector, each providing a nucleic acid that encodes a different portion of a protein (e.g., a therapeutic protein), and thus delivers multiple portions of the protein (e.g., a therapeutic protein) that associate and function in cells.

[0196] 【0216】 In certain embodiments, heterogeneous nucleic acids are provided by a dual AAV vector using duplication, trans-splicing, or hybrid trans-splicing dual vector technology. In certain embodiments, two duplicated AAV vectors are used that combine in cells to produce a fully expressed cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0197] 【0217】 The terms "identity" and "homology," and their grammatical variations, mean that two or more reference objects are the same when they are "aligned" sequences. For example, if two polypeptide sequences are identical, they have the same amino acid sequence, at least within a reference region or portion. If two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within a reference region or portion. Identity can extend across a defined region (region or domain) of a sequence. The "region" or "domain" of identity refers to a part of two or more identical reference objects. Therefore, if two protein or nucleic acid sequences are identical across one or more sequence regions or domains, they share identity within that region. An "aligned" sequence refers to a polynucleotide or protein (amino acid) sequence that, compared to a reference sequence, often contains corrections regarding missing or additional bases or amino acids (gaps).

[0198] 【0218】Identity can extend over the entire length or part of the sequence. In certain embodiments, the length of sequences sharing percent identity is 2, 3, 4, or 5 or more adjacent nucleic acids or amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In certain embodiments, the length of sequences sharing identity is 21 or more adjacent nucleic acids or amino acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. In a particular embodiment, the length of a sequence sharing identity is 41 or more adjacent nucleic acids or amino acids, for example, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc. In a particular embodiment, the length of a sequence sharing identity is 50 or more adjacent nucleic acids or amino acids, for example, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1,000, etc.

[0199] 【0219】As described herein, nucleic acid variants, such as CpG-reduced promoters including hybrid forms, are different from the wild type but may exhibit sequence identity with respect to the wild type promoter. In CpG-reduced promoters including hybrid forms, the CpG-reduced promoter is typically at least about 70% identical, more typically at least about 75% identical, and even more typically about 80% to 90% identical to the wild type promoter at the nucleotide sequence level. For example, a CpG-reduced promoter may have 70% to 99% identity with respect to the wild type promoter. Therefore, a CpG-reduced promoter may have 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 99%, and 75% to 99% identity with respect to the wild type promoter.

[0200] 【0220】 At the amino acid sequence level, variants such as variant FVIII or hFVIII-BDD protein are at least about 70% identical, more typically at least about 75% identical, or about 80% identical, and even more typically about 85% identical, or about 90% identical, to the reference sequence. In certain embodiments, variants such as variant FVIII or hFVIII-BDD protein may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity with respect to the reference sequence, for example, wild-type FVIII protein with or without a B domain.

[0201] 【0221】The term “homogenetic” or “homonymy” means that two or more reference objects share at least partially identity over a given region or part. The “region, region, or domain” of homology or identity means that parts of two or more reference objects share homology or are identical. Thus, if two sequences are identical over one or more sequence regions, the two sequences share identity over those regions. “Substantial homology” means that a molecule is structurally or functionally conserved such that it has, or is expected to have, at least partially structure or function (e.g., biological function or activity) of a reference molecule with which it shares homology, or of a related / corresponding region or part of a reference molecule.

[0202] 【0222】The degree of identity (homology) or "percent identity" between two sequences can be verified using computer programs and / or mathematical algorithms. For the purposes of this invention, the comparison of nucleic acid sequences is performed using the GCG Wisconsin Package version 9.1, available from Genetics Computer Group, Madison, Wisconsin. For convenience, the default parameters specified by the program (gap creation penalty = 12, gap extension penalty = 4) are for use herein to compare sequence identity. Alternatively, the level of identity and similarity between nucleic acid sequences and amino acid sequences may be determined using the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the World Wide Web at ncbi.nlm.nih.gov / blast / ; Altschul et al., 1990, J Mol Biol 215: pp. 403-410) with a gapped alignment using the default parameters. For polynucleotide sequence comparison, the BLASTP algorithm is typically used in combination with scoring matrices such as PAM100, PAM250, BLOSUM62, or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J.Mol.Biol.147:195 (1981)). Programs for quantifying protein structure similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0203] 【0223】A “therapeutic protein” is a peptide or protein that, in a particular embodiment, can alleviate or reduce symptoms caused by an insufficient amount of protein, the absence or defect of protein in a cell or subject. A “therapeutic” protein encoded by a transgene can confer benefits to a subject, for example, to correct a genetic defect or to correct a gene deficiency (loss of expression or function).

[0204] 【0224】 For example, without limitation, heterogeneous nucleic acids encoding gene products useful by the present invention (e.g., therapeutic proteins) include: hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, any blood coagulation factor: factor VII, factor VIII, factor IX, factor X, factor XI, factor V, factor XII, factor II, von Willebrand factor deficiency, combined FV / FVIII deficiency, thalassemia, vitamin K epoxydoreductase C1 deficiency, gamma carboxylase deficiency. Examples of conditions that may be used in the treatment of diseases or disorders include, but are not limited to, congestion or blood coagulation (bleeding) disorders such as: anemia; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulation disorders, and disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, warfarin, and small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, and storage pool deficiency. In certain embodiments, the subject has a blood coagulation disorder. In certain embodiments, the subjects include hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiencies of any coagulation factor: factor VII, factor VIII, factor IX, factor X, factor XI, factor V, factor XII, factor II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxydoreductase C1 deficiency, or gamma carboxylase deficiency.

[0205] 【0225】In certain embodiments, the subjects include, for example, lung diseases (e.g., cystic fibrosis), bleeding disorders (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood disorders (e.g., anemia), neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS)), neuropathy (e.g., epilepsy), lysosomal storage disorders (e.g., aspartylglucosamineuria, Batten's disease, late-onset childhood neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease). Glycogen storage disease type I, II and III, glycogen storage disease type II (Pompe disease), glycogen storage disease type III (GSDIII, Kohli's disease); ganglioside monosialic acid 2 (GM2)-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis type I and II), mucolipidosis type II (I-cell disease), mucolipidosis type III (pseudo-Harler disease) and mucolipidosis type IV, polysaccharide storage disease (Harler disease and its variants, Hunter disease, Sanfilipo type A, Sanfilipo type B, Sanfilipo type B, Sanfilipo type B) (Filippo type C, Sanfilippo type D, Morquio type A and Morquio type B, Maratho-Lamy and Sly disease), Niemann-Pick disease types A / B, Niemann-Pick type C1 and Niemann-Pick disease type C2, and Schindler disease types I and II), inflammatory disorders (e.g., hereditary angioedema (HAE)), copper or iron storage disorders (e.g., Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, cancer, type 1 or type 2 diabetes, adenosine deamylase deficiency, metabolic diseases or disorders (e.g., glycogen storage disease, methylmalonic acidemia, ornithine transcalculus) Having a disease or disorder including bamylase deficiency, hypophosphatasia, very long chain acyl-CoA dehydrogenase deficiency (VLCAD), galactosemia), autoimmune diseases (e.g., multiple sclerosis, type 1 diabetes mellitus, celiac disease, neuromyelitis optica (NMO), immunothrombocytopenia (ITP, idiopathic thrombocytopenic purpura), Addison's disease, myasthenia gravis), diseases of parenchymal organs (e.g., brain, liver, kidneys, heart), or infectious viral diseases (e.g., hepatitis B and C, human immunodeficiency virus (HIV), etc.), bacterial diseases, or fungal diseases.

[0206] 【0226】 In certain embodiments, the subject has a disease that affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemopalsy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0207] 【0227】 Apolipoprotein E (ApoE) is a major cholesterol carrier involved in lipid transport and brain injury repair. Human ApoE isoforms have been suggested to differentially affect amyloid-beta (Aβ) clearance or synthesis in vivo. The epsilon 4 (ε4) allele of ApoE has been associated with an increased risk of Alzheimer's disease (AD), while the presence of the ApoE ε2 allele appears to reduce AD ​​risk and is considered a protective ApoE isoform. As used herein, the term “protective ApoE isoform” refers to an ApoE isoform that reduces one or more symptoms or signs of Alzheimer's disease (e.g., physical, physiological, biochemical, histological, behavioral). Protective ApoE isoforms also refer to ApoE isoforms that can reduce the risk of Alzheimer's disease by at least 5%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0208] 【0228】 In certain embodiments, the present invention provides a method for delivering a protective ApoE isoform (e.g., ApoE ε2) to the CNS of a subject (e.g., a mammal) by delivery or administration to a target non-CNS cell, organ, or tissue (e.g., not to cerebrospinal fluid (CSF) or the brain).

[0209] 【0229】 In a particular embodiment, rAAV particles comprising an AAV capsid protein and a vector containing a nucleic acid encoding a protective ApoE isoform (e.g., ApoE ε2) are inserted between pairs of AAV terminal inversion sequences (ITRs) in a manner effective for transducing non-CNS cells (e.g., liver cells) in a subject (e.g., mammals), so that non-CNS cells (e.g., liver cells) secrete a protective ApoE isoform into the systemic circulation (vascular system and blood vessels) of the subject. The circulating protective ApoE isoform crosses the blood-brain barrier and enters the CNS (e.g., cerebrospinal fluid (CSF) or brain, e.g., brain parenchyma).

[0210] 【0230】 In certain embodiments, the present invention provides a vector, expression cassette, or nucleic acid encoding a protective ApoE isoform (e.g., ApoE ε2) expressed in the liver or in hepatocytes.

[0211] 【0231】In certain embodiments, heterologous nucleic acids include insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), progesterone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), and transforming growth factor α (TGF). α) Encodes a protein selected from the group consisting of platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenesis protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neuruturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgesock, and tyrosine hydroxylase.

[0212] 【0232】 In a particular embodiment, the heterologous nucleic acid sequence encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-36), monocyte chemotactic proteins, leukemia inhibitors, granulocyte-macrophage colony-stimulating factors, Fas ligands, tumor necrosis factor α and tumor necrosis factor β, interferon α, interferon β and interferon γ, stem cell factors, flk-2 / flt3 ligands, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I MHC molecules and class II MHC molecules.

[0213] 【0233】In certain embodiments, heterogeneous nucleic acids include CFTR (Cystic Fibrosis Transmembrane Regulatory Factor Protein), blood coagulation (coagulation) factors (Factor VIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor VIIa, Protein C, etc.), acquisition of functional blood coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α-antitrypsin, and adenosine deaminase (ADA). ), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage disorders (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or insulin-like growth factor 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and neurotrophic factor-4, brain-derived neurotrophic factor, glial cell-derived growth factor, transforming Growth factor α and transforming growth factor β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hitch). Per lindow (VHL), adenomatous polyposis (APC), peptides with immunomodulatory properties, immunotolerogenic or immunogenic peptides or proteins such as Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (colloidemia), LCA5 (LCA-lebercilin), ornithine keto acid aminotransferase (rotational atrophy), retinosoxin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C),Encodings include X-linked retinitis pigmentosa GTPase (XLRP), MERTK (RP: AR form of retinitis pigmentosa), DFNB1 (connexin 26, hearing loss), ACHM2, ACHM3 and ACHM4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, captesin A, GM2-AP, Niemann-Pick C1 (NPC1), VPC2, sphindolipid-activating protein, one or more zinc finger nucleases for genome editing, and one or more donor sequences used as repair templates for genome editing.

[0214] 【0234】 In certain embodiments, the protein encoded by a heterologous nucleic acid comprises a gene-editing nuclease. In certain embodiments, the gene-editing nuclease comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). In certain embodiments, the gene-editing nuclease comprises functional type II CRISPR-Cas9.

[0215] 【0235】Other heteronucleotides that may be used in conjunction with the present invention, and optionally expressed in the liver or liver cells (e.g., hepatocytes) to provide beneficial effects, may encode gene products (e.g., therapeutic proteins), such as: GAA (acid alpha-glucosidase) for Pompe disease; ATP7B (copper transport ATPase 2) for Wilson's disease; alpha-galactosidase (GLA) for Fabry disease; ASS1 (arginosuccinate synthase) for citrullinemia type 1; beta-glucocerebrosidase for Gaucher disease type 1; beta-hexosaminidase A for Tay-Sachs disease; SERPING1 (C1 protease inhibitor, C1 esterase inhibitor (C1E1)) for hereditary angioedema (HAE); glucose-6-phosphatase for glycogen storage disease type 1 (GSDI); and glycogen storage disease type 3 (GSD). III. Glycogen-debranching enzyme (GDE) for the treatment of Kohli's disease; Niemann-Pick C1 protein (NPC intracellular cholesterol transporter 1, NPC1) for the treatment of Niemann-Pick disease; erythropoietin (EPO) for the treatment of anemia; interferon-alpha, interferon-beta, and interferon-gamma for the treatment of various immune disorders, viral infections, and cancer; interleukins (IL) containing any one of IL-1 to IL-36 and their corresponding receptors for the treatment of various inflammatory diseases or immunodeficiencies; chemokines containing chemokine (CXC motif) ligand 5 (CXCL5) for the treatment of immune disorders; Granulocyte colony-stimulating factor (G-CSF) for the treatment of immune disorders such as Roon's disease; granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment of various human inflammatory diseases; macrophage colony-stimulating factor (M-CSF) for the treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for the treatment of epithelial tissue damage; chemokines such as monocyte chemotactic protein-1 (MCP-1) for the treatment of recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for the treatment of various immune disorders; alpha-1 antitrypsin for the treatment of emphysema or chronic obstructive pulmonary disease (COPD); alpha-L-iduronidase for the treatment of mucopolysaccharidosis I (MSP I); ornithine transcarbamylase (OTC) for the treatment of OTC deficiency;Phenylalanine hydroxylase (PAH) or phenylalanine ammonia lyase (PAL) for the treatment of phenylketonuria (PKU); lipoprotein lipase for the treatment of lipoprotein lipase deficiency; apolipoprotein for the treatment of apolipoprotein (Apo) AI deficiency; low-density lipoprotein receptor (LDL-R) for the treatment of familial hypercholesterolemia (FH); albumin for the treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrase; porphobi Examples include, but are not limited to, linogen deaminase; cystathionine beta-synthase for the treatment of homocystinuria; branched-chain keto acid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl-CoA carboxylase; methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassettes, subfamily A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; and dystrophin.

[0216] 【0236】 In certain embodiments, the subject has an autoimmune disease or disorder (e.g., multiple sclerosis, anti-MAG peripheral neuropathy, type 1 diabetes, Braves' disease, rheumatoid arthritis, proteoglycan-induced arthritis (PGIA), or myasthenia gravis), an allergy, or an allergic disease.

[0217] 【0237】Mature myelin oligodendrocyte glycoproteins (MOGs) are associated with the lipid bilayer. MOGs are characterized by an IgV-like extracellular domain, a single-bypass transmembrane protein, a membrane-binding domain, and a cytoplasmic end. The extracellular IgV-like domain is referred to herein as mini-MOG (mMOG). MOGs are primarily found in the oligodendrocyte membrane and contribute somewhat to the final composition of myelin. Autoimmune responses to MOGs are associated with the development and pathogenesis of multiple sclerosis.

[0218] 【0238】 In a particular embodiment, the therapeutic protein is a fusion protein comprising an undesirable antigen and a leader sequence for cell secretion.

[0219] 【0239】 In a particular embodiment, the therapeutic protein is a fusion protein comprising the extracellular domain of MOG, or a fragment thereof, and a leader sequence for cell secretion.

[0220] 【0240】 In a particular embodiment, the expression cassette includes a regulatory element operably linked to a nucleic acid encoding a fusion protein containing an undesirable antigen and a leader sequence for cell secretion.

[0221] 【0241】 In certain embodiments, the undesirable antigen includes a self-antigen, an autoantigen, or a protein or peptide having structural similarity or sequence identity to a self-antigen or autoantigen. In certain embodiments, the protein or peptide having structural similarity or sequence identity to a self-antigen or autoantigen is a microbial protein or peptide. In certain embodiments, the undesirable antigen includes an allergen.

[0222] 【0242】 In certain embodiments, the allergen includes plant, insect, or animal allergens. In certain embodiments, the undesirable antigen includes myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), protelipid protein (PLP), or partial sequences thereof.

[0223] 【0243】 In certain embodiments, MOG lacks all or part of its transmembrane domain. In certain embodiments, MOG contains or consists of amino acids 1-117 of mature MOG. In certain embodiments, a MOG subsequence is a subsequence of its extracellular domain or a subsequence of its transmembrane domain. In certain embodiments, MOG contains or consists of amino acids 35-55, 118-132, 181-195, or 186-200 of mature MOG. In certain embodiments, MOG contains or consists of amino acids 1-20, 11-30, 21-40, 31-50, etc., of mature MOG.

[0224] 【0244】 In certain embodiments, the present invention provides a method for suppressing, reducing, or inhibiting a cell-mediated or antibody-mediated immune response to an undesirable antigen in a mammal. In certain embodiments, the method comprises the steps of preparing an expression cassette, particle, or pharmaceutical composition or LNP composition described herein, and administering an amount of the expression cassette, particle, or pharmaceutical composition or LNP composition sufficient to suppress, reduce, or inhibit a cell-mediated or antibody-mediated immune response to an undesirable antigen to a mammal (the fusion protein is expressed in the mammal).

[0225] 【0245】 In certain embodiments, the present invention provides a method for inducing tolerance to an undesirable antigen in a mammal. In certain embodiments, the method comprises the steps of preparing an expression cassette, particles, or pharmaceutical composition or LNP composition as described herein, and administering an expression cassette, particles, or pharmaceutical composition or LNP composition in an amount sufficient to induce tolerance to the undesirable antigen to a mammal (the fusion protein is expressed in the mammal).

[0226] 【0246】 In certain embodiments, the present invention provides a method for treating a subject (e.g., a human) that requires a fusion protein. In certain embodiments, the method comprises the steps of preparing an expression cassette, particles, or pharmaceutical composition or LNP composition as described herein, and administering a certain amount of the expression cassette, particles, or pharmaceutical or LNP composition to a subject (e.g., a human) (in which the fusion protein is expressed).

[0227] 【0247】 In certain embodiments, the subject (e.g., a human) has an autoimmune disease or disorder. In certain embodiments, the subject (e.g., a human) has an allergy or an allergic disease or disorder.

[0228] 【0248】 In certain embodiments, the subject (e.g., human) has multiple sclerosis, anti-MAG peripheral neuropathy, type 1 diabetes, Graves' disease, rheumatoid arthritis, proteoglycan-induced arthritis (PGIA), or myasthenia gravis.

[0229] 【0249】 In this specification, “undesirable antigen” is a self-antigen or autoantigen that can induce, provide, enhance and / or stimulate immune tolerance to the antigen itself or a protein containing all or part of the antigen, and / or suppress, inhibit, reduce and / or decrease the immune response induced to the antigen itself or a protein containing all or part of the antigen. Undesirable antigens also include allergens or allergenic antigens that, when used herein, can induce, provide, enhance and / or stimulate immune tolerance to allergens, as well as allergens and allergenic antigens that suppress, inhibit, reduce and / or decrease the immune response induced to allergens or objects containing allergens.

[0230] 【0250】The undesirable antigens described herein also include allogeneic antigens, transplant antigens, or minor histocompatibility antigens that may cause rejection of cells, tissues, or organs after transplantation into a subject. Subjects typically recognize transplanted cells, tissues, or organs as foreign and express an immune response against them. Therefore, the methods of the present invention relate to preventing or reducing rejection of cells, tissues, or organs after transplantation into a subject.

[0231] 【0251】 While we do not wish to be constrained by any particular theory or mechanism, it is thought that undesirable antigens function by binding to or activating T regulatory cells (Tregs), thereby preventing, suppressing, inhibiting, reducing, decreasing, or otherwise downregulating the immune response. This binding to or activation of Tregs may then lead to immune tolerance to self-antigens or autoantigens.

[0232] 【0252】 As used herein, a “leader” sequence is an amino acid sequence that, when ligated to a protein, provides or promotes the secretion of the ligated protein from the cell on which it is expressed. A leader sequence may also be referred to as a secretion sequence as used herein. While such leader and secretion sequences are intended to provide or promote cellular secretion, they may not necessarily promote secretion if they are ligated to a protein that has a signal sequence that can prevent the secretion of the protein.

[0233] 【0253】 In certain embodiments, the undesirable antigen includes an autoimmune disease protein or a subsequence thereof. The autoimmune disease protein encompasses any antigen (e.g., a protein, a subsequence thereof, or a peptide) that contributes to the onset and / or progression of an autoimmune disease. Since the sequence or structure of proteins from other organisms may resemble self-antigens or autoantigens, such autoimmune disease proteins may originate from other organisms, such as microorganisms.

[0234] 【0254】In certain embodiments, the autoimmune disease proteins include myelin oligodendrocyte glycoprotein (MOG, e.g., for multiple sclerosis), myelin basic protein (MBP, e.g., for multiple sclerosis), proteolipidoprotein (PLP, for multiple sclerosis), myelin-associated glycoprotein (MAG, for anti-MAG peripheral neuropathy), insulin (e.g., for type 1 diabetes), islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP, e.g., for type 1 diabetes), and preprosinus. Phosphorus (e.g., for type 1 diabetes), glutamate decarboxylase (GAD, e.g., for type 1 diabetes), tyrosine phosphatase-like autoantigen (e.g., for type 1 diabetes), insulinoma antigen-2 (e.g., for type 1 diabetes), islet cell antigen (e.g., for type 1 diabetes), thyroid-stimulating hormone (TSH) receptor (e.g., for Graves' disease), thyrotropin receptor (e.g., for Graves' disease), chondroitin sulfate proteoglycan (e.g., for rheumatoid arthritis), CD4+ T cell epitope (e.g., GRVRVNSAY) (Sequence ID 98) ) (for example, for proteoglycan-induced arthritis (PGIA) or rheumatoid arthritis), or acetylcholine receptors (for example, for myasthenia gravis).

[0235] 【0255】 In certain embodiments, the autoimmune disease protein is a mammalian myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), proteolipidoprotein (PLP), or a subsequence thereof. In some embodiments, the autoimmune disease protein is a human protein, such as human myelin basic protein (MBP), human proteolipidoprotein (PLP), human myelin oligodendrocyte glycoprotein (MOG), or a subsequence thereof.

[0236] 【0256】Other heterogeneous nucleic acids encoding useful gene products according to the present invention include, but are not limited to, luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein, cyan fluorescent protein, enhanced GFP, enhanced YFP, photoactivatable GFP, Discosoma species fluorescent protein (dsRed), mFruits, mCherry, TagRFP, eqFP611, photoswitchable fluorescent proteins (e.g., Dronpa and EosFP), chloramphenicol acetyltransferase, Halo-tag fusion protein, alkaline phosphatase, horseradish peroxidase, and beta-galactosidase as reporters or detectable markers.

[0237] 【0257】 In certain embodiments, heterologous nucleic acids include inhibitory DNA or encode inhibitory RNA (RNAi). Examples of inhibitory RNA include, but are not limited to, small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, and antisense RNA.

[0238] 【0258】 In certain embodiments, heterologous nucleic acids encode inhibitory nucleic acids. In certain embodiments, inhibitory nucleic acids are selected from the group consisting of siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA. In certain embodiments, inhibitory nucleic acids include the huntingtin (HTT) gene, which is associated with dentatorubral-pallidoluysian atrophy (atropine 1, ATN1); the androgen receptor on the X chromosome in spinal bulbar atrophy; and human ataxin-1, ataxin-2, ataxin-3, and ataxin-7, Ca in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17). v2.1 P / Q voltage-gated calcium channel (CACNA1A), TATA-binding protein, ataxin 8 reverse strand (ATXN8OS), serine / threonine protein phosphatase 2A 55kDa regulatory subunit B beta isoform; FMR1 (Fragile X intellectual disability protein 1) in Fragile X syndrome; FMR1 (Fragile X intellectual disability protein 1) in Fragile X-associated tremor / ataxia syndrome; FMR1 (Fragile X intellectual disability protein 2) or AF4 / FMR2 family member 2 in Fragile XE intellectual disability; myotonin-protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; superoxide dimystase 1 (SOD) in amyotrophic lateral sclerosis; genes involved in the development of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV in HIV infection Tat, a transcription gene for human immunodeficiency virus transactivator; HIV TAR, human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; Roussarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or organ dysfunction or renal impairment after kidney transplantation; protein kinase N3 (PKN3) in progressive recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 (PSMB 8), metastatic melanoma; proteasome subunit beta-10 (PSMB 10) MECL1, also known as metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors; apoptosis inhibitor in chronic myeloid leukemia; B-cell CLL / lymphoma (BCL-2); ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors;Diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection; beta-catenin in familial adenomatous polyposis; adrenergic receptor, glaucoma; RTP801 / Redd1, also known as DNA damage-induced transcript 4 protein in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal angiogenesis; caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6A in congenital onychoplasty N17K mutant protein; influenza A virus genome / genome sequence in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola filovirus genome / gene sequence in Ebola infection; hepatitis B and C virus genome / gene sequence in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequence in HSV infection; coxsackievirus B3 genome / gene sequence in coxsackievirus B3 infection; silencing of pathogenic alleles of genes like tosin A (TOR1A) in primary dystonia (allele-specific silencing); specific pan-class I and HLA alleles in grafts; and binding to genes or gene transcripts associated with polynucleotide repeat diseases selected from the mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP).

[0239] 【0259】Nucleic acid molecules, vectors such as cloning vectors and expression vectors (e.g., vector genomes), and plasmids can be prepared using recombinant DNA techniques. The availability of nucleotide sequence information allows for the preparation of nucleic acid molecules by various means. For example, heterologous nucleic acids, including vectors or plasmids, can be prepared using various standard cloning, recombinant DNA techniques, and chemical synthesis techniques by cell expression or in vitro transplantation. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, etc. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences, (2) antibody screening to detect polypeptides with shared structural characteristics, for example, using expression libraries, (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest, (4) computer search of sequence databases of relevant sequences, and (5) differential screening of subtracted nucleic acid libraries.

[0240] 【0260】 The nucleic acids of the present invention can be maintained as DNA in any convenient cloning vector. In certain embodiments, the clones are maintained in plasmid cloning / expression vectors such as pBlurscript or pBluecript II (Stratagene, La Jolla, CA) grown in suitable Escherichia coli (E. coli) host cells. Alternatively, the nucleic acids are maintained in vectors suitable for expression in mammalian cells.

[0241] 【0261】Methods known to those skilled in the art for generating rAAV virions include, for example, transfection using an AAV vector and AAV helper sequence, or transfection using a recombinant AAV vector, an AAV helper vector, and an accessory functional vector, in conjunction with co-infection with one or more AAV helper viruses (e.g., adenovirus, herpesvirus, or vacciniavirus). Methods for generating rAAV virions are described, for example, but are not limited to, U.S. Patents 6,001,650 and 6,004,797. Following recombinant rAAV vector production (i.e., vector generation in a vector culture system), rAA virions can be obtained from host cells and cell culture supernatants and may be purified as described herein.

[0242] 【0262】 Methods for determining the infectivity titer of rAAV vectors containing transgenes are known in the art (e.g., Zhen et al., (2004) Hum. Gene Ther. (2004) 15:709). Methods for assaying AAV vector particles with empty capsids and packaged genomes are known (e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; see Sommer et al., Molec. Ther. (2003) 7:122-128).

[0243] 【0263】 To determine the degraded / denatured capsid, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis, which involves using any gel (e.g., a gradient gel) capable of separating the three capsid proteins, followed by running the gel until the sample is separated, and then blotting the gel onto a nylon or nitrocellulose membrane. Next, an anti-AAV capsid antibody is used as the primary antibody to bind to the denatured capsid protein (see, e.g., Wobus et al., J. Virol. (2000) 74: pp. 9281-9293). The secondary antibody, which binds to the primary antibody, contains means for detecting the primary antibody. The binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsid.

[0244] 【0264】 rAAV vectors and other compositions, active ingredients, drugs, and biologics (proteins) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo.

[0245] 【0265】 When used as a modifier for a composition, the term "isolated" means that the composition is either prepared by human hands or completely or at least partially isolated from their naturally occurring in vivo environment. Generally, isolated compositions substantially lack one or more of the materials they normally associate with, such as one or more proteins, nucleic acids, lipids, carbohydrates, or cell membranes.

[0246] 【0266】 With respect to proteins, the terms “isolated protein” or “isolated and purified protein” are used herein as sometimes applicable. This term primarily refers to proteins produced by the expression of nucleic acid molecules. Alternatively, it refers to proteins that are sufficiently isolated from other naturally associated proteins to exist in a “substantially pure” form.

[0247] 【0267】 The term "isolated" does not exclude the compositions described herein or combinations produced by human hands, such as rAAV and / or pharmaceutical formulations. The term "isolated" also does not exclude alternative physical forms such as hybrids / chimeras, multimers / oligomers, modified (e.g., phosphorylated, glycosylated, lipid-added) forms or derivatized forms, or forms expressed in host cells produced by human hands.

[0248] 【0268】The term "substantially pure" refers to a preparation containing at least 50% to 60% by weight of the compound of interest (e.g., nucleic acids, oligonucleotides, proteins, etc.). The preparation may contain at least 75% by weight, or about 90% to 99% by weight, of the composition of interest. Purity is measured by a method appropriate to the compound of interest (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0249] 【0269】 When referring to a specific nucleotide or amino acid sequence, the phrase "essentially derived from" means a sequence that possesses the characteristics of the given sequence. For example, when used in relation to nucleic acids or amino acid sequences, this phrase includes the sequence itself, as well as molecular modifications that do not affect the sequence's fundamental and novel features.

[0250] 【0270】 In certain embodiments, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutically active substance that does not induce an adverse immune response in the recipient of the composition and can be administered without excessive toxicity.

[0251] 【0271】 As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation of a gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, the material can be administered to a subject without causing substantially undesirable biological effects. Such pharmaceutical compositions can therefore be used when administering nucleic acids, vectors, viral particles, or proteins to a subject.

[0252] 【0272】Examples of pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, physiological saline, glycerol, sugar, and ethanol. Furthermore, pharmaceutically acceptable salts, such as mineral salts including hydrochloride, hydrobromide, phosphate, and sulfate, and salts of organic acids such as acetate, propionate, malonate, and benzoate, may also be included. In addition, auxiliary substances such as wetting agents or emulsifiers and pH buffering agents may be present in such a vehicle.

[0253] 【0273】 The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. The salt tends to be more soluble in aqueous or protic solvents than the corresponding free base form. In other cases, the preparation may be a lyophilized powder that is combined with a buffer before use and may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 0.2% sucrose, and 2% to 7% mannitol.

[0254] 【0274】 Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions, and suspension media, coatings, isotonic and absorption enhancers, or retarders, that are suitable for pharmaceutically effective administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may also contain anti-precipitation agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplemental active compounds (e.g., preservatives, antimicrobial agents, antiviral agents, and antifungal agents) may also be incorporated into the composition.

[0255] 【0275】 Pharmaceutical compositions may be formulated to be compatible with specific routes of administration or delivery described herein or known to those skilled in the art. Accordingly, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration by various routes.

[0256] 【0276】Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions of the active compound, and these preparations are typically sterile and may be isotonic with the blood of the recipient. Examples include, but are not limited to, water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal oil, vegetable oil, or synthetic oil. Aqueous injection suspensions may contain, but are not limited to, substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0257] 【0277】 Furthermore, the suspension of the active compound may be prepared as a suitable oil injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain a suitable stabilizer or an active agent that enhances the solubility of the compound to enable the preparation of a highly concentrated solution. 【0278】 Cosolvents or auxiliary agents may be added to the formulation, for example, cosolvents containing hydroxyl groups or other polar groups, such as alcohols like isopropyl alcohol; glycols like propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers; glycerol; polyethylene alcohol and polyoxyethylene Examples of auxiliary agents include, but are not limited to, fatty acid esters. Examples of auxiliary agents include, but are not limited to, surfactants such as soy lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0258] 【0279】 After a medical composition is prepared, it may be placed in a suitable container and labeled with respect to its therapeutic properties. Such labeling may include the amount, frequency, and method of administration.

[0259] 【0280】 The compositions, methods, and suitable pharmaceutical compositions and delivery systems of the present invention are known in the art (for example, Remington: The Science and Practice of Pharmacy(2003) 20th edition, Mack Publishing, Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th edition, Mack Publishing, Easton, PA; The Merck Index (1996) 12th edition, Merck Publishing Group, White House, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing, Lancaster, Pa; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th edition, Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al. Drug Delivery Systems (1980), edited by R. L. Juliano, Oxford, NY, pp. 253-315.

[0260] 【0281】 In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the present invention are delivered or administered by AAV vector particles. In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the present invention may be delivered or administered by other types of viral particles, including retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, Epstein-Barr viruses, vaccinia viruses, and human cytomegalovirus particles.

[0261] 【0282】 In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the present invention are delivered or administered using nonviral delivery systems. Nonviral delivery systems include, for example, chemical methods such as liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles, and physical methods such as gene guns, electroporation, particle guns, ultrasound, and magnetofection.

[0262] 【0283】In certain embodiments, the nucleic acids, polynucleotides and expression cassettes of the invention are delivered as naked DNA, minicircles, transposons, or closed-ended linear double-stranded DNA. 【0263】 【0284】 In certain embodiments, the nucleic acids, polynucleotides and expression cassettes of the invention are further encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles and delivered or administered in AAV vector particles or other particles. 【0264】 【0285】 "Lipid nanoparticles" or "LNPs" are useful for the delivery of AAV and refer to lipid-based vesicles having nanoscale dimensions, i.e., dimensions of about 10 nm to about 1000 nm, or about 50 nm to about 500 nm, or about 75 nm to about 127 nm. Without wishing to be bound by theory, LNPs are thought to provide partial or complete shielding of nucleic acids, polynucleotides, expression cassettes, or AAV vectors from the immune system. This shielding allows for the delivery of nucleic acids, polynucleotides, expression cassettes, or AAV vectors while avoiding the induction of a substantial immune response thereto in vivo. The shielding may also allow for repeated administration in vivo (e.g., in a subject such as a human) without the induction of a substantial immune response to the nucleic acid, polynucleotide, expression cassette or AAV vector. 【0265】 【0286】The pI (isoelectric point) of AAV is in the range of approximately 6 to 6.5. Therefore, the surface of AAV has a slightly negative charge. Thus, the inclusion of cationic lipids such as aminolipids may be beneficial for LNPs. Exemplary aminolipids are U.S. Patents 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,6 This is described in Patent No. 51, and in U.S. Patent Application Publications No. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125.

[0266] 【0287】 The terms “cationic lipid” and “amino lipid” are used interchangeably in this specification and encompass lipids and salts thereof having one, two, or three or more fatty acid or fatty alkyl chains and pH titrable amino groups (e.g., alkylamino or dialkylamino groups). Cationic lipids are typically protonated (i.e., positively charged) at pH below their pKa and substantially neutral at pH above their pKa. Cationic lipids may also be titrable cationic lipids. In certain embodiments, cationic lipids include a protonable tertiary amine (e.g., pH titrable) group; a C18 alkyl chain (where each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds); and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0267] 【0288】As cationic lipids, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (also known as DLin-K-C2-DMA, DLin-C2K-DMA, XTC2 and C2K), Examples include, but are not limited to, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (Din-M-C2-DMA, also known as MC2), 4-(dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (DLin-M-C3-DMA, also known as MC3), their salts, and mixtures thereof. Other cationic lipids include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0268] 【0289】Further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (Dlin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), and 1,2-dilinoleyloxy-3-morpholino Propane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMAP), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-RMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (Dlin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOPA), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleyloxy)prop N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleioyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRI E) 2,3-Dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), dioctadecylamideglycylspermine (DOG3), 3-dimethylamino-2-(cholesta-5-en-3-beta-oxybutane-4-yl)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-en-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl Examples include, but are not limited to, til-1-(cis,cis-9',1,2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (D2S), or mixtures thereof.

[0269] 【0290】Numerous commercially available preparations of cationic lipids, such as LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO / BRL), may be used.

[0270] 【0291】 In a particular embodiment, cationic lipids are present in an amount of about 10% by weight of LNPs to about 85% by weight of lipid nanoparticles, or about 50% by weight of LNPs to about 75% by weight of LNPs.

[0271] 【0292】 Sterols can impart fluidity to LNPs. As used herein, “sterol” refers to any naturally occurring sterol of plant (phytosterol) or animal (animal sterol) origin, as well as synthetic sterols that do not exist naturally, all characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Sterols can be any sterol commonly used in the field of liposomes, lipid vesicles, or lipid particle preparations, most typically cholesterol. Examples of phytosterols include campestrol, sitosterol, and stigmasterol. Sterols also encompass sterol-modified lipids, such as those described in U.S. Patent Application Publication 2011 / 0177156. In certain embodiments, sterols may be present in amounts ranging from about 5% by weight of the LNP to about 50% by weight of the lipid nanoparticles, or from about 10% by weight to about 25% by weight of the LNP.

[0272] 【0293】LNPs contain neutral lipids. Neutral lipids may include any lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided, in particular, by considerations of particle size and essential stability. In certain embodiments, the neutral lipid components may be lipids having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0273] 【0294】 Lipids having acyl chain groups of various chain lengths and degrees of saturation are available or can be isolated or synthesized by known techniques. In certain embodiments, lipids containing saturated fatty acids having carbon chain lengths in the range of C14 to C22 may be used. In certain embodiments, lipids having monounsaturated or diunsaturated fatty acids having carbon chain lengths in the range of C14 to C22 may be used. Furthermore, lipids having mixtures of saturated and unsaturated fatty acids may be used. Exemplary neutral lipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), or any related phosphatidylcholine. Neutral lipids may also consist of phospholipids having other head groups such as sphingomyelin, dihydrosphingomyelin, or serine and inositol.

[0274] 【0295】 In certain embodiments, neutral lipids may be present in amounts ranging from about 0.1% by weight of lipid nanoparticles to about 75% by weight of LNPs, or from about 5% by weight of LNPs to about 15% by weight of LNPs.

[0275] 【0296】LNP-encapsulated nucleic acids, expression cassettes, and AAV vectors can be incorporated into pharmaceutical compositions, such as pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions are particularly useful for the administration and delivery of LNP-encapsulated nucleic acids, expression cassettes, and AAV vectors to subjects in vivo or ex vivo.

[0276] 【0297】 The LNP preparation may be combined with further components, including, but not limited to, polyethylene glycol (PEG) and sterols.

[0277] 【0298】 The term "PEG" refers to polyethylene glycol, a linear, water-soluble polymer of ethylene PEG repeating units having two terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG2000 has an average molecular weight of approximately 2,000 daltons, and PEG5000 has an average molecular weight of approximately 5,000 daltons. PEGs are commercially available from Sigma Chemical and other companies, and include, but are not limited to, the following functional PEGs: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM).

[0278] 【0299】In certain embodiments, PEG may be polyethylene glycol having an average molecular weight of about 550 daltons to about 10,000 daltons, and may optionally be substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, PEG may be substituted with methyl groups at the position of the terminal hydroxyl group. In certain embodiments, PEG may have an average molecular weight of about 750 daltons to about 5,000 daltons, or about 1,000 daltons to about 5,000 daltons, or about 1,500 daltons to about 3,000 daltons, or about 2,000 daltons or about 750 daltons. PEG may optionally be substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, the terminal hydroxyl group may be substituted with a methoxy or methyl group.

[0279] 【0300】 Examples of PEG-modified lipids include, but are not limited to, PEG-dialkyloxypropyl conjugates (PEG-DAAs) described in U.S. Patents 8,936,942 and 7,803,397. Useful PEG-modified lipids (or lipid-polyoxyethylene conjugates) have various "fixed" lipid moieties, and the PEG moiety can be immobilized on the surface of lipid vesicles. Suitable examples of PEG-modified lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates described in U.S. Patent 5,820,873 (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. In certain embodiments, the PEG-modified lipids may be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, PEG may be present in an amount of about 0.5% by weight to about 20% by weight of LNP, or about 5% by weight to about 15% by weight of LNP.

[0280] 【0301】Furthermore, the LNP may be PEG-modified and sterol-modified. The LNP may be combined with further components to form the same LNP or separate LNPs. In other words, the same LNP may be PEG-modified and sterol-modified, or the first LNP may be PEG-modified and the second LNP may be sterol-modified. Optionally, the first modified LNP and the second modified LNP may be combined.

[0281] 【0302】 In one embodiment, before encapsulation, the LNP may have a size in the range of about 10 nm to 500 nm, or about 50 nm to about 200 nm, or about 75 nm to about 125 nm. In one particular embodiment, the nucleic acid, expression vector, or AAV vector encapsulated with the LNP may have a size in the range of about 10 nm to 500 nm.

[0282] 【0303】 "Effective amount" or "sufficient amount" means an amount, in single or multiple doses, that provides a detectable response, expected outcome, or desired outcome, or any benefit to any measurable or detectable amount, for any duration (long or short) in a subject of any measurable or detectable degree, either alone or in combination with one or more other compositions (such as therapeutic agents or immunosuppressants, including prednisone), treatments, protocols, or therapeutic regimens or agents.

[0283] 【0304】 Dosages may vary and depend on the type, progression, severity, frequency, duration, or likelihood of the disease being treated, the desired clinical endpoint, previous or concurrent treatments, the subject's overall health status, age, sex, race, or immunological eligibility, and other factors understood by those skilled in the art. Dosage, frequency, duration, or number of doses may be relatively increased or decreased as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy and the subject's condition. Factors that may affect the dose and timing required to provide a sufficient amount to deliver therapeutic or prophylactic benefits will be understood by those skilled in the art.

[0284] 【0305】 The dose for achieving a therapeutic effect, for example, the dose of vector genome per kilogram of body weight (vg / kg), is variable based on several factors including, but not limited to, the route of administration, the level of heterologous polynucleotide expression required to achieve the therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein. One of ordinary skill in the art can determine the rAAV / vector genome dose range for treating a patient with a specific disease or disorder based on the factors described above, as well as other factors. 【0285】 【0306】 Generally, the dose is at least 1×10 8 , for example, 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1×10 14 vector genome (vg / kg) or more. In mice, doses in the range of 1×10 10 ~1×10 11 vg / kg, and in dogs, doses in the range of 1×10 12 ~1×10 13 vg / kg of AAV have been effective. The dose can be less, for example, a dose less than 6×10 12 vector genome (vg / kg) per kilogram. More specifically, a dose of about 1×10 11 vg / kg to about 5×10 12 vg / kg, or about 5×10 11 vg / kg to about 2×10 12 vg / kg, or about 5×10 11 vg / kg to about 1×10 12 vg / kg. 【0286】 【0307】For Pompe disease, the effective dose is the amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal in the body tissues of the subject, thereby reducing lysosomal alterations, or, for example, improving muscle tone and / or muscle strength and / or respiratory function in the subject. The effective dose can be determined, for example, by observing the dynamics of GAA uptake by plasma-derived myoblasts. Myoblast GAA uptake rates (K uptake) of approximately 141 nM to 147 nM may appear to be effective (see, e.g., Maga et al., J. Biol. Chem. 2012). In animal models, plasma GAA activity levels higher than approximately 1,000 nmol / hr / mL, e.g., approximately 1,000 nmol / hr / mL to approximately 2,000 nmol / hr / mL, have been observed to be therapeutically effective.

[0287] 【0308】 For example, using hemophilia B, it is generally believed that, in order to achieve therapeutic efficacy, a blood coagulation factor concentration higher than 1% of the normal level is required to change a severe disease phenotype to a moderate one. The severe phenotype is characterized by joint damage and cataract-threatening bleeding. To change a moderate disease phenotype to a mild disease phenotype, a blood coagulation factor concentration higher than 5% of the normal level is considered necessary.

[0288] 【0309】 The diagnosis and classification of disease severity for hemophilia A and hemophilia B are based on the results of factor VIII and factor IX activity assays, respectively. The two main assays used to assess factor activity are a one-step assay (OSA) based on activated partial thromboplastin time (aPTT) and a two-step chromophore assay (CSA) using a factor Xa-based enzymatic chromophore substrate reaction. Such assays are well known in the art and are further described in Adcock et al., 2018, Int. J. Lab. Hem., 40: pp. 621-629.

[0289] 【0310】FVIII levels in normal humans are approximately 150 ng / mL (plasma) to 200 ng / mL (plasma), but may be lower (e.g., in the range of approximately 100 ng / mL to 150 ng / mL) or higher (e.g., in the range of approximately 200 ng / mL to 300 ng / mL), and may still be considered normal due to functional coagulation, such as that determined by a one-step aPTT coagulation assay. Therefore, therapeutic effects can be achieved by the expression of FVIII or hFVIII-BDD such that the total amount of FVIII in the subject / human exceeds 1% of the FVIII present in normal subjects / humans, e.g., 1% of 100 ng / mL to 300 ng / mL.

[0290] 【0311】 rAAV vectors can typically be at the lower end of the dose range, such that there is no substantial immune response to FVIII or AAV vectors. More specifically, the maximum dose is 6 × 10¹² cells vg / kg, but doses below that, for example, around 5 × 10¹² cells, are not effective. 11 pieces~approx. 5×10 12 Each VG / kg, or more specifically, approximately 5 × 10 11 vg / kg or approximately 1 × 10⁻⁶ 12 Individual vg / kg.

[0291] 【0312】 In certain embodiments, the rAAV vector dose is at a level that delivers a safe and effective amount of FVIII to provide therapeutic benefit to subjects with hemophilia A who have inhibitory antibodies against FVIII (hemophilia A with inhibitors).

[0292] 【0313】 An “effective dose” or “sufficient dose” for treatment (for example, to induce remission or to provide therapeutic benefit or improvement) is usually effective to provide a measurable response to one, more or all adverse symptoms, consequences or complications of the disease, or one or more adverse symptoms, disorders, diseases, conditions, or complications caused by or associated with the disease, but a satisfactory outcome is to reduce, lessen, inhibit, suppress, limit or control the progression or worsening of the disease.

[0293] 【0314】 An effective or sufficient amount may be provided in a single dose, but does not have to be; multiple doses may be required, and it may be administered alone or in combination with another composition (e.g., an active agent), treatment, protocol, or treatment regimen, but does not have to be. For example, the amount may be increased proportionally, as indicated by the needs of the subject, the type, condition, and severity of the disease being treated, or the side effects of the treatment (if any). Furthermore, an effective or sufficient amount may not be effective or sufficient if it is provided in single or multiple doses without the use of a second composition (e.g., another drug or active agent), treatment, protocol, or treatment regimen, because in addition to such dose, further doses, amounts, or durations, or further compositions (e.g., drugs or active agents), treatments, protocols, or treatment regimens may be included in such a way as to be deemed effective or sufficient for a given subject. The amount deemed effective also includes amounts that result in the use of another treatment, treatment regimen or protocol, for example, a reduction in the administration of recombinant coagulation factor proteins (e.g., FVIII) for the treatment of coagulation disorders (e.g., hemophilia A, or hemophilia A with inhibitory antibodies against FVIII, also known as hemophilia A with inhibitors).

[0294] 【0315】 Accordingly, the methods and uses of the present invention encompass, in particular, methods and uses that result in a reduction of the need for or use of other compounds, active substances, drugs, treatment regimens, treatment protocols, processes, or therapies. For example, with respect to blood coagulation disorders, the methods or uses of the present invention have therapeutic benefits when, in a given subject, there is a less frequent, reduced dose, or elimination of recombinant coagulation factor proteins to compensate for a deficiency or lack (abnormal or mutated) endogenous coagulation factor in the subject. Accordingly, the present invention provides methods and uses that reduce the need for or use of other treatments or therapies.

[0295] 【0316】An effective or sufficient dose does not need to be effective in all subjects being treated, nor does it need to be effective in the majority of subjects being treated in a given group or population. An effective or sufficient dose means efficacy or sufficiency in a specific subject, not efficacy or sufficiency in a group or general population. As is typical for such methods, some subjects will show a greater response, a smaller response, or no response at all to a given treatment method or use.

[0296] 【0317】 The term "remission" means a detectable or measurable improvement in the disease or its symptoms, or in the underlying cellular response. Detectable or measurable improvements include subjective or objective reduction, decrease, inhibition, suppression, limitation, or control of the occurrence, frequency, severity, progression, or duration of the disease or complications caused by or associated with the disease, or improvement in the symptoms, underlying causes, or outcomes of the disease, or recovery from the disease. With respect to HemA, an effective dose is, for example, a dose that reduces the frequency or severity of acute bleeding episodes in the subject, or, for example, a dose that reduces clotting time as measured by a coagulation assay.

[0297] 【0318】 Accordingly, the pharmaceutical compositions of the present invention encompass compositions in which the active ingredient is contained in an amount effective to achieve the target therapeutic objective. Determining a therapeutically effective dose is well within the skill of those skilled in the art using the techniques and guidance provided in the present invention.

[0298] 【0319】 The therapeutic dose depends, in particular, on the patient's age and general health, the severity of the abnormal phenotype, and the strength of the regulatory sequence that modulates the expression level. Therefore, the therapeutically effective dose in humans falls within a relatively wide range, which can be determined by the physician based on the individual patient's response to vector-based therapy. Such doses may be administered alone or in combination with immunosuppressants or other drugs.

[0299] 【0320】Compositions such as pharmaceutical compositions can be delivered to a subject to enable transgene expression and, optionally, the production of encoded proteins. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to enable the recipient to produce a therapeutically effective amount of a coagulation factor to affect congestion in the subject.

[0300] 【0321】 The composition may be administered alone. In certain embodiments, recombinant AAV particles provide a therapeutic effect without the use of immunosuppressants. The therapeutic effect is optional and lasts for a certain period without the administration of immunosuppressants, for example, 2 to 4 days, 4 to 6 days, 6 to 8 days, 8 to 10 days, 10 to 14 days, 14 to 20 days, 20 to 25 days, 25 to 30 days, or 30 to 50 days or longer, for example, 50 to 75 days, 75 to 100 days, 100 to 150 days, 150 to 200 days or longer. Therefore, in certain embodiments, rAAV virus particles provide a therapeutic effect for a certain period without the administration of immunosuppressants.

[0301] 【0322】 The compositions of the present invention may be administered in combination with at least one other inactivating or therapeutic agent. In certain embodiments, the rAAV vector is administered prior to, substantially simultaneously with, or after the administration of the rAAV vector, in combination with one or more immunosuppressants. In certain embodiments, this may occur 1 to 12 hours, 12 to 24 hours, or 24 to 48 hours after the administration of the rAAV vector, or 2 to 4 days, 4 to 6 days, 6 to 8 days, 8 to 10 days, 10 to 14 days, 14 to 20 days, 20 to 25 days, 25 to 30 days, 30 to 50 days, or longer than 50 days after the administration of the rAAV vector. In the administration of immunosuppressants a certain period after administration of an rAAV vector, a decrease in encoded protein expression is observed after the initial expression level, after a certain period of time following the administration of the rAAV vector, for example, 20-25 days, 25-30 days, 30-50 days, 50-75 days, 75-100 days, 100-150 days, 150-200 days, or longer than 20 days.

[0302] 【0323】 In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid. In certain embodiments, the immunosuppressant is prednisone, cyclosporine (e.g., cyclosporine A), mycophenolate, rituximab, rapamycin, or derivatives thereof. In certain embodiments, the active ingredient comprises a stabilizing compound. Other immunosuppressants that may be used in the present invention include, but are not limited to, B-cell targeting antibodies, e.g., rituximab; proteasome inhibitors, e.g., bortezomib; mammalian targets of rapamycin (mTOR) inhibitors, e.g., rapamycin; tyrosine kinase inhibitors, e.g., ibrutinib; B-cell activator (BAFF) inhibitors; and proliferation-inducing ligand (APRIL) inhibitors.

[0303] 【0324】 The composition may be administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to physiological saline, buffered physiological saline, dextrose, and water. The composition may be administered to the patient alone or in combination with other active ingredients (e.g., cofactors) that affect congestion.

[0304] 【0325】The methods and uses of the present invention include delivery and administration by systemic, topical, or localized, or by any route, not limited to, injection or infusion. In vivo delivery of the pharmaceutical composition may generally be achieved by conventional syringe injection, but other delivery methods such as convection-enhanced delivery are envisioned (see, for example, U.S. Patent No. 5,720,720). For example, the composition may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly. Other methods of administration include oral and pulmonary administration, suppositories, and transdermal application. For example, a clinician specializing in the treatment of patients with blood coagulation or coagulation factor disorders may determine the optimal route for administration of the adenovirus-associated vector based on a number of criteria, including, but not limited to, the patient's condition and the objective of treatment (e.g., increasing GAA, enhancing or reducing blood coagulation).

[0305] 【0326】 The therapeutic methods according to the present invention encompass combination therapies that include the further use of one or more compounds, active agents, drugs, treatments, or other therapeutic regimens or protocols having a desired therapeutic, beneficial, additional, synergistic, or complementary activity or effect. Exemplary combination compositions and treatments include, but are not limited to, two additives, e.g., biologics (proteins), active agents (e.g., immunosuppressants), and drugs. Such biologics (proteins), active agents, drugs, treatments, and therapies may be administered or implemented prior to, substantially concurrently with, or after any other therapeutic methods according to the present invention, e.g., a treatment for a subject for lysosomal storage disorders such as Pompe disease, or a treatment for a subject for blood coagulation disorders such as HemA or HemB.

[0306] 【0327】Compounds, active substances, drugs, treatments, or other therapeutic regimens or protocols may be administered as a combination composition or separately, for example, simultaneously with, consecutively with, or sequentially (before or after) the delivery or administration of nucleic acids, vectors, recombinant vectors (e.g., rAAV), or recombinant viral particles. Accordingly, the present invention provides combinations of therapeutic methods according to the present invention in combination with any compound, active substance, drug, therapeutic regimen, therapeutic protocol, process, treatment, or composition described herein or known to those skilled in the art. Compounds, active substances, drugs, therapeutic regimens, therapeutic protocols, processes, treatments, or compositions may be administered or implemented prior to, substantially concurrently with, or after the administration of nucleic acids, vectors, recombinant vectors (e.g., rAAV), or recombinant viral particles administered to a patient or subject according to the present invention.

[0307] 【0328】 The present invention may be used for human and veterinary applications. Accordingly, suitable subjects include mammals such as humans, as well as non-human mammals. The term "subject" refers to animals, usually mammals, such as humans, non-human rodents (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), livestock (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, neonates, infants, young adults, and adult subjects. Subjects also include animal disease models, such as mouse models of blood coagulation disorders, such as HemA and others known to those skilled in the art, and other animal disease models.

[0308] 【0329】Subjects suitable for treatment according to the present invention include those who have an insufficient amount of functional gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX), are at risk of producing an insufficient amount of functional gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX), have a deficiency of functional gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX), or produce abnormal gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX), partially functional gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX), or non-functional gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX) that may lead to disease. Target subjects suitable for treatment according to the present invention also include subjects who have or are at risk of producing disease-causing abnormal or deficient (mutated) gene products (proteins), such that reducing the amount, expression, or function of the abnormal or deficient (mutated) gene product (protein) would lead to treatment of the disease, reduction of one or more symptoms, or remission of the disease. For example, target subjects include subjects who have abnormal or insufficient production of blood coagulation factors, or who do not produce blood coagulation factors, such as hemophilia patients (e.g., hemophilia A or hemophilia B), or subjects who have abnormal or insufficient GAA, or who do not have GAA, such as patients with Pompe disease.

[0309] 【0330】 The subjects include those who possess undetectable neutralizing antibodies against AAV. Furthermore, the subjects include those who possess neutralizing antibodies against AAV. Such subjects possess low-titer neutralizing antibodies against AAV.

[0310] 【0331】Subjects may be tested for an immune response, such as antibodies against AAV. Candidate subjects (e.g., subjects with hemophilia or Pompe disease) may be screened prior to treatment according to the method of the present invention. Subjects may also be tested for antibodies against AAV after treatment and, optionally, be monitored for a period after treatment. Subjects expressing antibodies may be treated with an immunosuppressant (e.g., prednisone) or administered one or more additional doses of AAV vector.

[0311] 【0332】 Subjects deemed negative for antibodies binding to AAV have a titer of less than 1:1. Subjects possessing antibodies binding to AAV have a titer higher than 1:1 but less than 1.5. Subjects may also have AAV antibody titers of 1.5 or higher. These antibody titers can be calculated, for example, by serial dilution of blood, plasma, or serum (or other body fluid) samples derived from the subjects. When the sample is measured by reporter activity in an in vitro cell-based assay, the first dilution that inhibits AAV transduction at 50% or higher is reported as the antibody titer.

[0312] 【0333】 Strategies to reduce (overcome) or evade humoral immunity to AAV in systemic gene transfer include administering high vector doses, using empty AAV capsids as decoys to adsorb anti-AAV antibodies, administering immunosuppressants to reduce, mitigate, inhibit, prevent or eradicate the humoral immune response to AAV, modifying the AAV capsid serotype or manipulating the AAV capsid to be less affected by neutralizing antibodies, using plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers, using delivery techniques such as balloon catheters followed by saline flushing (Mingozzi et al., 2013, Blood, 122:23-36), and immunoadsorption (US Patent Application Publication No. 2018 / 0169273 A1).

[0313] 【0334】Target subjects suitable for treatment according to the present invention also include subjects that have antibodies against AAV or are at risk of producing antibodies against AAV. rAAV vectors can be administered or delivered to such subjects using several techniques. For example, an empty capsid AAV (e.g., an AAV lacking a transgene) may be delivered and bind to AAV antibodies in the subject, thereby enabling an AAV vector to possess nucleic acids or nucleic acid variants to transform the target cells.

[0314] 【0335】 The ratio of the empty capsid to the rAAV vector can be approximately 2:1 to 50:1, or approximately 2:1 to 25:1, or approximately 2:1 to 20:1, or approximately 2:1 to 15:1, or approximately 2:1 to 10:1. The ratio can also be approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0315] 【0336】 The amount of empty AAV capsid administered can be calibrated based on the amount (titer) of AAV antibodies produced in a particular subject. The empty capsid can be any AAV serotype, e.g., AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8, LK03 (SEQ ID NO: 91), SPK (SEQ ID NO: 92).

[0316] 【0337】 Alternatively, AAV vectors can be delivered by direct intramuscular injection (e.g., into one or more slow-twitch muscle fibers). In another alternative, AAV vectors can be delivered to the liver via the hepatic artery using a catheter introduced into the femoral artery. Furthermore, AAV vectors can be delivered directly to the liver using non-surgical means such as endoscopic retrograde cholangiopancreatography (ERCP), thereby bypassing the bloodstream and AAV antibodies. Other ductal systems, such as the submandibular gland duct, can also be used as gateways for delivering AAV vectors to subjects expressing or possessing existing anti-AAV antibodies.

[0317] 【0338】Administration to a subject or in vivo delivery may be carried out prior to the onset of adverse symptoms, conditions, or complications caused by or associated with the disease. For example, such subjects can be identified as candidate substances for the compositions, methods, and uses of the present invention using a screen (e.g., genetic). Thus, such subjects include those screened as positive for insufficient amounts or deficiencies of functional gene products (e.g., coagulation factors), or those that produce abnormal, partially functional, or non-functional gene products (e.g., coagulation factors).

[0318] 【0339】 Administration or in vivo delivery to a subject by the methods and uses of the present invention disclosed herein can be carried out within 1 to 2 hours, 2 to 4 hours, 4 to 12 hours, 12 to 24 hours, or 24 to 72 hours after the subject has been identified as having the disease to be targeted for treatment, having one or more symptoms of the disease, or has been screened and identified as positive as described herein, even if the subject does not have one or more symptoms of the disease. Naturally, the methods and uses of the present invention can be carried out within 1 to 7, 7 to 14, 14 to 21, or 21 to 48 or more days, months, or years after the subject has been identified as having the disease to be targeted for treatment, having one or more symptoms of the disease, or has been screened and identified as positive as described herein.

[0319] 【0340】As used herein, “unit dosage form” refers to physically separate units suitable as a unit dose for the subject to be treated, each unit containing a specified amount with an optional pharmaceutical carrier (excipient, diluent, vehicle, or filler) that, when administered in one or more doses, is calculated to produce the desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be contained in ampoules and vials, for example, and may contain liquid compositions or compositions in a freeze-dried or lyophilized state. For example, a sterile liquid carrier may be added prior to administration or in vivo delivery. Separate unit dosage forms may be contained in multi-dose kits or containers. Recombinant vectors (e.g., rAAV) sequences, recombinant virus particles, and their pharmaceutical compositions may be packaged in single-dose and multi-dose dosage forms for ease of administration and uniformity of dosing.

[0320] 【0341】 Subjects can be tested for the protein or activity levels of relevant gene products (e.g., GAA or blood coagulation factors, e.g., FVIII or FIX) to determine whether such subjects are suitable for treatment by the method of the present invention. For example, a candidate hemophilia A subject can be tested for FVIII levels or activity prior to treatment by the method of the present invention, and a candidate Pompe disease subject can be tested for GAA levels or activity prior to treatment according to the present invention. Subjects can also be tested for the protein or activity levels of FVIII or GAA after treatment by the method of the present invention. Such treated subjects can be monitored periodically after treatment for blood coagulation activity (with respect to HemA) or GAA activity (with respect to Pompe), for example, every 1 to 4 weeks, every 1 to 6 months, or every 1, 2, 3, 4, 5 years or longer.

[0321] 【0342】Subjects may be examined for one or more liver enzymes in relation to adverse responses, or to determine whether such subjects are suitable for treatment by the method of the present invention. For example, candidate hemophilia or Pompe subjects may be screened for the amount of one or more liver enzymes prior to treatment by the method of the present invention. Subjects may also be examined for the amount of one or more liver enzymes after treatment by the method of the present invention. Such treated subjects may be monitored regularly, for example every 1 to 4 weeks or every 1 to 6 months, after treatment for elevated liver enzymes.

[0322] 【0343】 Exemplary liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), but other enzymes that indicate liver damage can also be monitored. Normal levels of these enzymes in circulation are usually defined as a range with an upper limit; levels exceeding this are considered elevated and therefore indicate liver damage. The normal range is somewhat dependent on the standards used by the clinical laboratory performing the assay.

[0323] 【0344】 In one embodiment, subjects with bleeding disorders may be monitored for bleeding episodes to determine whether such subjects are suitable for or responding to the treatment according to the present invention, and / or the amount or duration of the response. Subjects may be monitored for bleeding episodes to determine whether such subjects require further treatment, such as subsequent AAV vector administration or immunosuppressant administration, or more frequent monitoring. Hemophilia subjects may be monitored for bleeding episodes prior to and after treatment according to the method of the present invention. Subjects may also be monitored for the frequency and severity of bleeding episodes during or after treatment according to the method of the present invention.

[0324] 【0345】In one embodiment, subjects suffering from Pompe disease or requiring GAA may be monitored by various tests, assays, and functional evaluations to demonstrate, measure, and / or evaluate the effectiveness of GAA, thereby determining whether such subjects are suitable for treatment according to the present invention, are responding to treatment, or require further treatment.

[0325] 【0346】 The present invention provides a kit comprising a packaging material and one or more components contained therein. The kit typically includes a label or packaging insert containing a description of the components contained therein, or instructions for the in vitro, in vivo, or ex vivo use of the components contained therein. The kit may contain a series of such components, such as nucleic acids, recombinant vectors, viral (e.g., AAV) vectors, or viral particles, and optionally a second active substance such as another compound, actionant, drug, or composition.

[0326] 【0347】 A kit refers to a physical structure that contains one or more components of a kit. Packaging materials can be made from materials commonly used for this purpose, such as paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc., that can be sterilized to preserve the components.

[0327] 【0348】The label or insert may include identification of clinical pharmacological information of the active ingredient(s) contained therein, including one or more components, dosage, mechanism of action, pharmacokinetics, and pharmacodynamics. The label or insert may include information identifying the manufacturer, lot number, place and date of manufacture, and expiration date. The label or insert may include information identifying the manufacturer, lot number, place and date of manufacture. The label or insert may include information about diseases for which the kit components may be used. The label or insert may include instructions for clinicians and subjects on how to use one or more of the kit components in a method, use, or treatment protocol or treatment regimen. The instructions may include instructions on dosage, frequency, or duration, and on how to perform any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0328] 【0349】 Labels or inserts may include information about any benefits that the components may provide, such as preventive or therapeutic benefits. Labels or inserts may include warnings to the subject or clinician about potential adverse side effects, complications, or reactions, such as situations in which the use of a particular composition is inappropriate. Adverse side effects or complications may also occur if the subject has been, is scheduled to be, or is currently receiving one or more other medications that may be incompatible with the composition, or if the subject has been, is scheduled to be, or is currently receiving another treatment protocol or regimen that may be incompatible with the composition, and therefore the instructions may include information about such incompatibility.

[0329] 【0350】Labels or inserts include paper or cardboard that is separate from or attached to the "printed material," such as components, kits, or packaging materials (e.g., boxes), or attached to ampoules, tubes, or vials containing kit components. Labels or inserts further include computer-readable media such as barcoded printed labels, optical discs such as discs, CD- or DVD-ROM / RAM, DVDs, MP3s, magnetic tapes, or energy storage media such as RAM and ROM, or hybrids thereof such as magnetic / optical storage media, FLASH media, or memory cards.

[0330] 【0351】 Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in carrying out or experimenting with the present invention, but suitable methods and materials are described herein.

[0331] 【0352】 All patents, patent applications, publications, and other references cited herein, including GenBank and ATCC citations, are incorporated by reference in their entirety. In case of any inconsistency, the specification, including its definitions, shall prevail.

[0332] 【0353】 Various terms relating to the biological molecules of this invention are used above, as well as throughout the specification and claims.

[0333] 【0354】 All features disclosed herein can be combined in any other combination. Each feature disclosed in the specification can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise clearly stated, the nucleic acids, vectors, plasmids, expression / recombinant vectors (e.g., rAAV) sequences, or recombinant viral particles of the disclosed features (e.g., CpG-reduced) are examples of equivalent or similar features.

[0334] 【0355】As used herein, the singular forms "a," "and," and "the" encompass the plural unless the context clearly indicates otherwise. Thus, for example, a reference to "nucleic acid" encompasses multiple such nucleic acids, a reference to "vector" encompasses multiple such vectors, and a reference to "virus" or "particle" encompasses multiple such viruses / particles.

[0335] 【0356】 As used herein, all numerical terms or numerical ranges include integers and values ​​or fractions of integers within such ranges unless circumstances clearly indicate otherwise. Thus, for example, a reference to identity of 80% or more includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0336] 【0357】 References to integers greater than or less than the reference number encompass any number greater than or less than the reference number, respectively. For example, a reference to numbers less than 100 encompasses numbers from 99, 98, 97, etc., down to the number 1(1), and a reference to numbers less than 10 encompasses numbers from 9, 8, 7, etc., down to the number 1(1).

[0337] 【0358】 As used herein, all numerical values ​​or numerical ranges, unless circumstances clearly indicate otherwise, encompass the values ​​and integer fractions within such ranges and the integer fractions within such ranges. Thus, for example, a reference to a numerical range such as 1 to 10 encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Similarly, a reference to a range of 1 to 50 encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. up to 50 (and including 50), as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0338] 【0359】A reference to a series of ranges encompasses ranges that combine the boundary values ​​of various ranges within that series. Therefore, for example, a reference to the series of ranges 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-850 is equivalent to a reference to the series of ranges 1-20, 1-30, 1-40, 1-50, 1-60, 10-30, 10-40, 10-50, 10- This range includes 60, 10-70, 10-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 50-75, 50-100, 50-150, 50-200, 50-250, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 150-250, 150-300, 150-400, 150-450, 150-500, etc.

[0339] 【0360】 The present invention is generally disclosed herein using positive language and describes numerous embodiments of the present invention. The present invention also encompasses embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols or procedures, is excluded in whole or in part. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Accordingly, the present invention generally does not encompass what is described herein, but nevertheless embodiments that are not obviously excluded in the present invention are disclosed herein.

[0340] 【0361】 Numerous embodiments of the present invention are described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention to suit various uses and conditions. Accordingly, the following embodiments are intended to illustrate the scope of the present invention, but in no event shall they limit the claimed scope of the invention. [Examples]

[0341] Example 1 :method 【0362】 NHP plasma hFVIII ELISA: 96-well plates were coated overnight with human-specific FVIII antibody, washed, blocked, and incubated with diluted NHP study plasma samples. A standard curve was created by incubating additional wells with a series of dilutions of recombinant B-domain deletion hFVIII, Xyntha® (Pfizer). The plates were washed and subsequently incubated with biotinylated human-specific FVIII detection antibody. The plates were incubated with horseradish peroxidase (HRP) conjugated streptavidin, treated with TMB substrate, read with a microplate reader, and absorbance at 450 nm was determined.

[0342] 【0363】 hFVIII ELISA of mouse plasma: The hFVIII ELISA of mouse plasma was performed essentially as described above for NHP, but the capture and detection antibodies used were different.

[0343] 【0364】 Cell-based assay to measure the potency of AAV vector encoding human FVIII transgene: Huh7 cells were measured in a 48-well dish at 5 × 10⁶ per well. 4 Cells were cultured on plates. After the study, the remaining undiluted stock vector and the vector diluted for drug administration were cultured in DMEM + 10% FBS + penicillin / streptavidin / L-glutamine to create a 10-fold dose curve (1 × 10⁻¹⁰ 6 ~1 × 10 3The medium was prepared within the MOI range. Existing medium was removed from the Huh7 cells and replaced with a medium containing virus particles. The cells were maintained at 37°C and 5% CO2 for 72 hours, and the supernatant was collected and stored in low-retention microtiter plates at -80°C until assayed for hFVIII activity. The supernatant was assayed with Coatest® SP4 factor VIII (Chromogenix) using a standard curve prepared by diluting recombinant B-domain deletion hFVIII, Xyntha® (Pfizer), in cell growth medium.

[0344] 【0365】 Cell-based assay to measure the protein expression efficiency of plasmids encoding the human FVIII transgene: Huh7 cells were expressed in a 48-well dish at 5 × 10⁶ per well. 4 Cells were cultured on plates in DMEM + 10% FBS + penicillin / streptomycin / L-glutamine. Plasmids were prepared using the Plasmid Giga kit (Qiagen) and transfected into cells at 250 ng per well using polyethyleneimine (PEI) Max. Cells were maintained at 37°C and 5% CO2 for 72 hours, and the supernatant was collected and stored in low-retention microtiter plates at -80°C until assayed for hFVIII activity. The supernatant was assayed with CoreTest® SP4 factor VIII (Chromogenix) using a standard curve prepared by diluting recombinant B-domain deletion hFVIII, Xyntha® (Pfizer), in cell growth medium.

[0345] Example 2 【0366】 The FIX construct regulatory element unit (SEQ ID NOs. 22 and 23) consists of a 321 bp intron of the apolipoprotein E (ApoE) gene and a 397 bp promoter of the human alpha-1 antitrypsin (hAAT) gene. Overall, this unit contains 16 CpGs.

[0346] Design of a promoter with reduced CpG 【0367】 Either cytosine or guanine in the CpG site was modified according to the consensus sequence. If no potential transcription factor binding site was found, cytosine (C) in the CpG dinucleotide was replaced with thymine (T). This maintained the pyrimidine-purine structure. In some cases, the C nucleotide or the entire CpG dinucleotide was deleted, and in some cases, guanine (G) in the CpG dinucleotide was replaced with alanine (A) or C.

[0347] 【0368】 Using this strategy, we generated 22 different sequences based on ApoE / hAAT regulatory elements. Sequences 24 to 67 below show sequences containing 5' and 3' adjacent restriction enzyme sites.

[0348] cloning 【0369】 Various promoters were synthesized and cloned upstream of the codon-optimized sequence (SEQ ID NO: 94) that encodes hFIX.

[0349] Mouse research 【0370】 The efficacy of the human alpha-1 antitrypsin (hAAT) gene promoter was evaluated by hydrodynamic delivery of the plasmid construct in 8-week-old male wild-type C57BL / 6 mice (Jackson Laboratories). Non-fasted plasma samples were collected in heparin by submandibular blood collection 24 hours after plasmid administration. Plasma was placed on ice and stored at -80°C until analysis. All animal studies were conducted in accordance with institutional guidelines and authorized protocols. Efficacy research 【0371】 The collected plasma was used to evaluate hFIX transgene expression.

[0350] 【0372】The activity level of human FIX was measured by the activated partial thromboplastin time (aPTT) assay. Sample plasma was mixed with human FIX-deficient plasma (George King Bio-Medical) and aPTT reagent (Trinity Biotech) in a 1:1:1 volume ratio, followed by an incubation period of 180 seconds at 37°C to perform the aPPT assay. Coagulation was initiated by the addition of 25 mM calcium chloride. The time to clot formation was measured using a Start 4 coagulation instrument (Diagnostica Stago). A standard curve was created using pooled normal plasma (George King Bio-Medical), starting with a 1:5 dilution in TBS pH 7.4 (48 μL + 192 μL), followed by a stepwise 1:2 dilution (120 μL + 120 μL). Using the human standard curve, the activity of each sample was calculated 17 weeks after AAV vector administration, and the activity in two untreated mice was also measured. The average FIX activity in untreated mice was calculated and then subtracted from that of treated samples to determine the external (i.e., human) activity attributable to the exogenous FIX protein. The data are shown in Figure 1.

[0351] Example 3 【0373】 The FVIII construct modulating element unit (SEQ ID NOs: 2 and 3) consists of a 225 bp TTR promoter. Overall, this unit contains 4 CpGs.

[0352] Design of a TTR promoter with reduced CpG 【0374】 Either cytosine or guanine in the CpG site was modified according to the consensus sequence. If no potential binding site was found, the cytosine in the CpG dinucleotide was replaced with thymine. This maintained the pyrimidine-purine structure. Using this strategy, five novel sequences were generated based on the TTRm (SEQ ID NO: 3) regulatory element. TTR sequences with and without restriction sites, and with reduced CpG levels, are shown in SEQ ID NOs: 4 to 13.

[0353] 【0375】Another set of four different short TTR hybrid promoters was designed. Five different liver-specific promoters were evaluated in silico for the presence of putative transcription factor binding sites within 1000 nucleotides of the transcription start site (TSS) for the native gene. Subsequently, TTR hybrid promoters were constructed by selecting specific regions from the original native promoters and constructing them in tandem. TTR hybrid promoter sequences with and without restriction enzyme sites are shown below as SEQ ID NOs: 14 to 21.

[0354] cloning 【0376】 Various promoters were synthesized and cloned upstream of the codon-optimized nucleotide sequence (SEQ ID NO: 77) encoding hFVIII-BDD.

[0355] Mouse research 【0377】 First, the efficacy of the TTR promoter was evaluated by hydrodynamic delivery in 8-week-old male wild-type C57BL / 6 mice (Jackson Laboratories). 24 hours after plasmid administration, non-fasted plasma samples were collected in heparin by submandibular blood collection. The plasma was placed on ice and stored at -80°C until analysis. For AAV delivery studies, the first 0.5 mL of blood was discarded, and the remaining sample was collected in EDTA and processed into plasma. All animal studies were conducted in accordance with institutional guidelines and authorized protocols.

[0356] hFVIII antigen levels in mouse plasma 【0378】The levels of hFVIII transgene product in mouse plasma were quantified using a sandwich-style ELISA as follows: First, the wells of a microtiter plate were coated with anti-hFVIII capture antibody (Green Mountain Antibodies, diluted to 2 μg / mL). The following day, the plate was washed four times and blocked at room temperature for 30 minutes (6% BSA, 0.2% Tween20 in PBS). Recombinant B-domain deletion hFVIII (XYNTHA Solofuse®) at known concentrations was added to the pooled mouse plasma and serially diluted (1:2) to create an 8-point standard curve ranging from 300 ng / mL to 2.34 ng / mL. The limit of quantification for the assay is 4.8 ng / mL. Three levels of quality control samples were prepared and placed on each plate to evaluate assay performance. After adding the sample to the wells, the plate was incubated at 37°C for 1 hour, followed by four washes. Biotinylated anti-hFVIII detection antibody (Green Mountain Antibodies, diluted to 1 μg / mL) was added to the plate at room temperature for 1 hour to conjugate to the captured hFVIII protein. After washing, a 1:5000 dilution of peroxidase conjugate streptavidin reagent (Thermo Fisher Scientific) was added to the plate at room temperature for 30 minutes to conjugate to the biotinylated anti-hFVIII antibody. After washing the plate to remove unbound conjugate antibody, the plate was incubated with 3,3',5,5'-tetramethylbenzidine substrate (TMB) at room temperature for 15 minutes to reveal peroxidase activity. The reaction was stopped with TMB stop solution, and the plate was read using an absorbance plate reader for optical density (OD). The obtained absorbance values ​​are proportional to the concentration of hFVIII present in the plasma sample. The data are shown in Figures 2 to 5.

[0357] RNA units and qPCR 【0379】Mouse tissues from the brain, testes, kidneys, spleen, and liver were collected, rinsed with DPBS, and cut / ground into multiple micropieces. Approximately 30 mg of each tissue was used for RNA isolation as described in the kit protocol (RNeasy plus Universal Mix Kit, Qiagen). RNA concentration was measured using a Nanodrop2000 instrument, and the samples were diluted to 150 ng / mL in nuclease-free water. DNase treatment was performed using a Trubo DNA-free kit (Invitrogens) as per the manufacturer's instructions. For the cDNA reaction, 200 ng of RNA was used according to the instructions for the High Capacity cDNA Reverse Transcription Kit (ABI). The cDNA sample was diluted 5-fold, and 20 ng of cDNA was used in the PCR reaction. Forward primer: 5'-TGAGGAGGCTGAAGACTATGA-3' (SEQ ID NO: 95), Reverse primer: 5'-CCACAGACCTGATCTGAATGAA-3' (SEQ ID NO: 96), and Probe: 5'-56-FAM-TGGATGTGG / ZEN / TGAGGTTTGATGATGACA-3IABkFQ-3' (SEQ ID NO: 97) and Sequence ID 99 Quantitative real-time PCR was performed using the mouse actB gene (Integrated DNA Technologies). The mouse actB gene functioned as a housekeeping gene for normalization. The data are shown in Figure 6.

[0358] Example 4 【0380】The expression of factor VIII was increased by modifying the elements within the expression cassette that contribute to transgene expression. An intron-free version of the B-domain deletion hFVIII expression AAV vector (AAV-INTL) was created and compared with the intron-containing version (AAV-WINT). The AAV-INTL hFVIII expression cassette (SEQ ID NO: 1) contains the same elements as the AAV-XINT hFVIII expression cassette, except that AAV-WINT hFVIII has a synthetic intron (SEQ ID NO: 93) positioned between the TTRm promoter and the transgene encoding B-domain deletion human factor VIII (Figure 7).

[0359] Example 5 【0381】 To evaluate the efficacy of an intron-free cassette (TTRm hFVIII intron-free; SEQ ID NO: 1) compared to an intron-containing cassette (TTRm hFVIII) in mammals, approximately 8-week-old male C57BL / 6 mice (Jackson Laboratories) were intravenously injected into the lateral tail vein at doses of 6.4e9 or 1.6e10 vg / mouse with AAV capsid-forming cassettes. Plasma was collected at several time points as shown (Figures 8 and 9) and evaluated for circulating hFVIII levels by hFVIII ELISA.

[0360] 【0382】 Determination of hFVIII levels demonstrated that TTRm hFVIII without introns showed a significant increase in potency compared to TTRm hFVIII with introns (Figures 8 and 9), and this effect was observed at both the dose and time point tested (Study #1). These results were repeated in Study #2, a subsequent study using 1.6e10 vg / mouse in 10 mice in each group (Figure 10). Study #2 confirmed that TTRm hFVIII without introns (AAV-INTL) was more potent than vectors containing synthetic introns (AAV-WINT), and that these differences persisted for at least 8 weeks.

[0361] Example 6 :Efficacy in NHP, Study 1 【0383】 The efficacy of AAV vectors AAV-INTL and AAV-WINT was compared in NHP (Study #1). Twelve male cynomolgus monkeys (Macaca fascicularis) aged 24 to 50 months, weighing 2 kg to 6 kg and negative for AAV neutralizing antibodies, were divided into four randomized groups and administered either a single dose of AAV-WINT or AAV-INTL intravenously, based on the treatment groups shown in Table 1. Subsequently, plasma samples were obtained weekly to determine circulating hFVIII levels.

[0362] [Table 1]

[0363] 【0384】 Plasma levels of hFVIII in monkeys administered either AAV-WINT or AAV-INTL were determined by ELISA at weekly intervals throughout the 8-week study. At either of the doses examined in this study, 2e12 vg / kg (Figure 11) or 6e122 vg / kg (Figure 12), a 2- to 4-fold increase in circulating hFVIII levels based on peak circulating values ​​was observed at all time points. As expected, the inventors observed a loss of expression 2-3 weeks after treatment, indicating the expression of inhibitory antibodies against BDD hFVIII. The results of the first study demonstrate that AAV-INTL shows increased efficacy compared to AAV-WINT in NHP.

[0364] Example 7 :Efficacy in NHP, Study 2 【0385】The second study (Study #2) attempted in NHP to confirm the increased vector potency of AAV-INTL compared to AAV-WINT. Ten male cynomolgus monkeys (Macaca fascicularis), aged 24 to 50 months, weighing 2 to 6 kg, and negative for AAV neutralizing antibodies, were divided into two randomized groups and administered either AAV-WINT or AAV-INTL intravenously as a single dose (2 e12 vg / kg). Subsequently, plasma samples were obtained weekly to determine the level of circulating hFVIII.

[0365] 【0386】 Plasma hFVIII levels in macaques treated with either AAV-WINT or AAV-INTL were determined by ELISA at weekly intervals throughout the 8-week study. At the doses examined in this study, 2e12 cells vg / kg, a 2- to 7-fold increase in circulating hFVIII levels was observed at all time points, based on peak circulating values ​​(Figure 13). As previously observed in NHP, loss of transgene expression was observed 2-3 weeks after treatment, due to the expression of inhibitory antibodies against BDD hFVIII. Results from Study #2 confirmed that AAV-INTL shows increased efficacy compared to AAV-WINT in NHP.

[0366] Example 8 Determination of vector efficacy 【0387】 To confirm that the correct vector was administered to each group of NHP at the appropriate concentration, the potency of the drug formulation was determined by qPCR, and the presence or absence of synthetic introns in the stock vector was assayed by a genotyping PCR assay that allowed for differences between AAV-WINT and AAV-INTL. Cell-based potency assays were used to directly evaluate the vector potency of both the undiluted stock vector and the drug formulation at 2e12 vg / kg.

[0367] 【0388】The potency of human liver cell lines was determined by transduction with a series of vector dilutions and evaluation of secreted BDD hFVIII in the supernatant using an hFVIII activity assay (Chromogenix Coatest SP4). At all MOIs, AAV-INTL showed increased potency compared to AAV-WINT (Figure 14). In particular, the stock virus and diluted formulations showed similar potency within the vector group, further confirming that the potency of the drug formulations in 2e12 vg / kg groups was appropriately prepared. Furthermore, at each MOI, AAV-INTL showed approximately a four-fold increase in potency compared to AAV-WINT (Figure 15). These values ​​are consistent with the increased potency of AAV-INTL observed in NHP Studies 1 and 2.

[0368] Example 9 : Lot comparison 【0389】 The first and second NHP studies used different lots of AAV-WINT and AAV-INTL vectors. To assess whether the vector potency was equivalent between the different lots, in vitro assays were used to evaluate potency (Figure 16). The results of these comparisons show minimal batch-to-batch variability, and that AAV-INTL remained approximately 4 to 5 times more potent than AAV-WINT.

[0369] Example 10 Determination of expression cassette efficiency 【0390】 To study the mechanism of increased potency in vivo, the transcription efficiency in the absence of viral transduction was determined. Human liver cell lines were transfected with plasmids containing expression cassettes (SEQ ID NO: 1) comprising AAV-WINT, AAV-INTL, TTRm-intron-BDD-hFVIII, and TTRm-BDD-hFVIII without introns, respectively. The supernatants from these cells were assayed for hFVIII levels using a human FVIII activity assay (Chromogenix Coatest SP4).

[0370] 【0391】A comparison of three independent DNA preparations (TTRm-intron-BDD-hFVIII) and two independent DNA preparations (TTRm-BDD-fHVIII without introns, SEQ ID NO: 1) showed similar hFVIII levels, with a tendency toward decreased expression upon intron removal (Figure 17). While we do not wish to be constrained by any theory, the data suggest that a non-transcriptional mechanism drives the increased potency of AAV-INTL over AAV-WINT.

[0371] Example 11 Data discussion 【0392】 At corresponding doses, AAV-INTL demonstrated increased potency and expression of BDD-hVIII compared to AAV-WINT in cell cultures, mouse, and NHP models. Mechanistically, the increased potency did not appear to be attributable to increased transcription of the FVIII transgene from the expression cassette, but rather to possible viral packaging efficiency or alternative mechanisms. These results suggest that intron-free expression cassettes may exhibit increased potency in human clinical trials and offer benefits to patient safety and efficacy.

[0372] Example 12: Results of human clinical trials 【0393】 A single-dose study was conducted in four men (N=4) with hemophilia A, and the results are summarized in Table 4. All four participants received a single injection of the AAV-INTL hFVIII-BDD expression cassette (SEQ ID NO: 1), which is capsid-formed in the LK03 AAV vector (SEQ ID NO: 91), referred to herein as "LK03-INTL hFVIII-BDD," at a dose of 5 × 10⁻¹⁴. 11 It was administered at a dose of one VG / kg.

[0373] [Table 2]

[0374] 【0394】The LK03-INTL hFVIII-BDD vector was observed to drive FVIII expression in all four participants (Figures 18-19).

[0375] 【0395】 Example 13: Array

[0376] [Table 3] TIFF0007874125000004.tif216149 TIFF0007874125000005.tif206149 TIFF0007874125000006.tif215149 TIFF0007874125000007.tif119149

[0377] The complete nucleic acid sequence and description of the AAV-INTL expression cassette (5'-ITR, TTRm, hFVIII-BDD, poly(A), and 3'ITR) (SEQ ID NO: 1) (Table 3):

[0378] [Table 4]

[0379] Wild-type TTR promoter (SEQ ID NO: 2). The four underlined nucleotides are modified in the following mutant TTR promoter (SEQ ID NO: 3). gtgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatt tgtg taggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggaggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctg

[0380] Mutant TTR promoter (4 nucleotide changes, underlined) "TTRm" (SEQ ID NO: 3): gtgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatt Nact taggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggaggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctg

[0381] Nucleic acid sequence of CpG1-TTRm (SEQ ID NO: 4). In CpG1, all Cs in all four CpGs have been changed to T (double underlined). When the sequence was cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites are located at the 5' and 3' ends, respectively. SEQ ID NO: 5 is a CpG1-TTRm with these restriction enzyme sites (underlined).

[0382] Sequence ID 4: [ka]

[0383] Sequence ID 5: [ka]

[0384] Nucleic acid sequence of CpG2-TTRm (SEQ ID NO: 6). In CpG2, the Cs in the 2nd, 3rd, and 4th CpGs have been changed to T (double underlined). When the sequence was cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 7 is a CpG2-TTRm containing these restriction enzyme sites (underlined).

[0385] Sequence ID 6: [ka]

[0386] Sequence ID 7: [ka]

[0387] Nucleic acid sequence of CpG3-TTRm (SEQ ID NO: 8). In CpG3, the Cs in the 1st, 3rd, and 4th CpGs have been changed to T (double underlined). When the sequence was cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites are located at the 5' and 3' ends, respectively. SEQ ID NO: 9 is a CpG3-TTRm with these restriction enzyme sites (underlined).

[0388] Sequence ID 8: [ka]

[0389] Sequence ID 9: [ka] Nucleic acid sequence of CpG4-TTRm (SEQ ID NO: 10). In CpG4, the Cs in the 1st, 2nd, and 4th CpGs have been changed to T (double underlined). When the sequence was cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 11 is a CpG4-TTRm with these restriction enzyme sites (underlined).

[0390] Sequence ID 10: [ka]

[0391] Sequence ID 11: [ka]

[0392] Nucleic acid sequence of CpG5-TTRm (SEQ ID NO: 12). In CpG5, the Cs in the 1st, 2nd, and 3rd CpGs have been changed to T (double underlined). When the sequence was cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 13 is a CpG5-TTRm having these restriction enzyme sites (underlined).

[0393] Sequence ID 12: [ka]

[0394] Sequence ID 13: [ka]

[0395] Nucleic acid sequence of the hybrid 6 promoter (TTR / hAAT / albumin hybrid) (SEQ ID NO: 14). In a single CpG dinucleotide, G is replaced with A (double underlined). When the sequence is cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites are located at the 5' and 3' ends, respectively. (Italics = TTR, underline = albumin, bold = hAAT.) SEQ ID NO: 15 is a hybrid 6 promoter with these restriction sites. 【0396】 [ka] 【0397】 Nucleic acid sequence of the hybrid 7 promoter (TTR / hAAT / hybrid) (SEQ ID NO: 16). In both CpG dinucleotides, C is replaced with T (double underlined). When the sequence is cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites are located at the 5' and 3' ends, respectively. (Italics = TTR, bold = hAAT.) SEQ ID NO: 17 is a hybrid 7 promoter with these restriction sites. 【0398】 [ka] 【0399】 Nucleic acid sequence (SEQ ID NO: 18) of the hybrid 8 promoter (TTR / FGG (fibrinogen gamma chain gene promoter) / albumin promoter hybrid). In a single CpG dinucleotide, G is changed to A (double underlined). When the sequence is cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites are located at the 5' and 3' ends, respectively. (Underlined = albumin, underlined italicized = FGG, italicized = TTR.) SEQ ID NO: 19 is a hybrid 8 promoter with these restriction enzyme sites. 【0400】 [ka] 【0401】 Nucleic acid sequence (SEQ ID NO: 20) of the hybrid 9 promoter (TTR / FGG / hAAT / SAA1 hybrid). In all three CpG dinucleotides, C has been changed to T (double underlined). When the sequence was cloned into the FVIII expression cassette, the MluI(acgcgt) and PmeI(gtttaaac) restriction sites are located at the 5' and 3' ends, respectively. (Italics = TTR, bold = hAAT, underlined italics = FGG, underlined bold = SAA1.) SEQ ID NO: 21 is a hybrid 9 promoter with these restriction sites. 【0402】 [ka] 【0403】 Nucleic acid sequence of an ApoE / hAAT regulatory element with unreduced CpG (SEQ ID NO: 22). This sequence contains total CpG (double underlined). The C / EBP (CCAAT / enhancer-binding protein) site is underlined. 【0404】 [ka] 【0405】 The nucleic acid sequence (SEQ ID NO: 23) of an ApoE / hAAT regulatory unit with unreduced CpGs, flanked at the 5' and 3' ends by the ApaI restriction site. The sequence contains a total of 16 CpGs (indicated by double underlines). The ApaI restriction site was used when the sequence was cloned into the FIX expression cassette. 【0406】 [ka] 【0407】 Nucleic acid sequence of CpG1-ApoE / hAAT (SEQ ID NO: 24). All Cs have been changed to Ts, except in the 7th CpG, where G is changed to A. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 25 is CpG1-ApoE / hAAT with these restriction sites (underlined). 【0408】 [ka] 【0409】 Nucleic acid sequence of CpG2-ApoE / hAAT (SEQ ID NO: 26). All Cs were changed to Ts, except for the first CpG, which remained unchanged, and the seventh CpG, where G was changed to A. Unchanged "c"s are shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 27 is CpG2-ApoE / hAAT with these restriction sites (underlined). 【0410】 [ka] 【0411】 Nucleic acid sequence of CpG3-ApoE / hAAT (SEQ ID NO: 28). All Cs were changed to Ts, except that the second CpG remained unchanged, and in the seventh CpG, the G was changed to A. Unchanged "c"s are shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 29 is CpG3-ApoE / hAAT with these restriction sites (underlined). 【0412】 [ka] 【0413】 Nucleic acid sequence of CpG4-ApoE / hAAT (SEQ ID NO: 30). All Cs were changed to Ts, except that the third CpG remained unchanged, and in the seventh CpG, the G was changed to A. Unchanged "c"s are shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 31 is CpG4-ApoE / hAAT with these restriction sites (underlined). 【0414】 [ka] 【0415】 Nucleic acid sequence of CpG5-ApoE / hAAT (SEQ ID NO: 32). All Cs were changed to Ts, except for the 4th CpG, which remained unchanged, and in the 7th CpG, the G was changed to A. The unchanged CpG (4th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 33 is CpG5-ApoE / hAAT with these restriction sites (underlined). 【0416】 [ka] 【0417】 Nucleic acid sequence of CpG6-ApoE / hAAT (SEQ ID NO: 34). All Cs were changed to Ts, except that the 5th CpG remained unchanged, and in the 7th CpG, the G was changed to A. The unchanged CpG (5th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 35 is CpG6-ApoE / hAAT with these restriction sites (underlined). 【0418】 [ka] 【0419】 Nucleic acid sequence of CpG7-ApoE / hAAT (SEQ ID NO: 36). All Cs were changed to Ts, except that the 6th CpG remained unchanged, and in the 7th CpG, the G was changed to A. The unchanged CpG (6th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 37 is CpG7-ApoE / hAAT with these restriction sites (underlined). 【0420】 [ka] 【0421】 Nucleic acid sequence of CpG8-ApoE / hAAT (SEQ ID NO: 38). All Cs have been changed to Ts, except that the 7th site remains unchanged. The unchanged CpG (7th site) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 39 is CpG8-ApoE / hAAT with these restriction sites (underlined). 【0422】 [ka] 【0423】 Nucleic acid sequence of CpG9-ApoE / hAAT (SEQ ID NO: 40). All Cs were changed to Ts, except for the 8th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (8th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 41 is CpG9-ApoE / hAAT with these restriction sites (underlined). 【0424】 [ka] 【0425】 Nucleic acid sequence of CpG10-ApoE / hAAT (SEQ ID NO: 42). All Cs were changed to Ts, except for the 9th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (9th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 43 is CpG10-ApoE / hAAT with these restriction sites (underlined). 【0426】 [ka] 【0427】 Nucleic acid sequence of CpG11-ApoE / hAAT (SEQ ID NO: 44). All Cs were changed to Ts, except for the 10th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (10th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 45 is CpG11-ApoE / hAAT with these restriction sites (underlined). 【0428】 [ka] 【0429】 Nucleic acid sequence of CpG12-ApoE / hAAT (SEQ ID NO: 46). All Cs were changed to Ts, except for the 11th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (11th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 47 is CpG12-ApoE / hAAT with these restriction sites (underlined). 【0430】 [ka] 【0431】 Nucleic acid sequence of CpG13-ApoE / hAAT (SEQ ID NO: 48). All Cs were changed to Ts, except for the 12th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (12th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 49 is CpG13-ApoE / hAAT with these restriction sites (underlined). 【0432】 [ka] 【0433】 Nucleic acid sequence of CpG14-ApoE / hAAT (SEQ ID NO: 50). All Cs were changed to Ts, except for the 13th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (13th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 51 is CpG14-ApoE / hAAT with these restriction sites (underlined). 【0434】 [ka] 【0435】 Nucleic acid sequence of CpG15-ApoE / hAAT (SEQ ID NO: 52). All Cs were changed to Ts, except for the 14th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (14th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 53 is CpG15-ApoE / hAAT with these restriction sites (underlined). 【0436】 [ka] 【0437】 Nucleic acid sequence of CpG16-ApoE / hAAT (SEQ ID NO: 54). All Cs were changed to Ts, except for the 15th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (15th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 55 is CpG16-ApoE / hAAT with these restriction sites (underlined). 【0438】 [ka] 【0439】 Nucleic acid sequence of CpG17-ApoE / hAAT (SEQ ID NO: 56). All Cs were changed to Ts, except for the 16th CpG, which remained unchanged, and the G in the 7th CpG was changed to A. The unchanged CpG (16th) is shown in bold. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 57 is CpG17-ApoE / hAAT with these restriction sites (underlined). 【0440】 [ka] Nucleic acid sequence of CpG18-ApoE / hAAT (SEQ ID NO: 58). The 5th, 7th, 8th, 10th, and 11th CpGs have been removed, and the remaining CpGs have had their Cs changed to Ts. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 59 is CpG18-ApoE / hAAT with these restriction sites (underlined). 【0441】 [ka] 【0442】 Nucleic acid sequence of CpG19-ApoE / hAAT (SEQ ID NO: 60). In CpGs 1-5 and 8-11, C is changed to T, and C is removed from CpG 7. The remaining CpGs (6th and 12-16th) remain unchanged (bold). When the sequence is cloned into a FIX expression cassette, the ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 61 is CpG19-ApoE / hAAT with these restriction sites (underlined). 【0443】 [ka] 【0444】 Nucleic acid sequence of CpG20-ApoE / hAAT (SEQ ID NO: 62). This is the result of multiple deletions in a region that does not contain a putative transcription factor binding site. In the only remaining CpG, G was changed to A (the 7th CpG in SEQ ID NO: 22). When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 63 is CpG20-ApoE / hAAT with these restriction enzyme sites (underlined). 【0445】 [ka] 【0446】 Nucleic acid sequence of CpG21-ApoE / hAAT (SEQ ID NO: 64). In all CpGs, C is changed to T, except for the 5th and 6th CpGs, where G is changed to C and A, respectively. When the sequence is cloned into a FIX expression cassette, the ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 65 is CpG21-ApoE / hAAT with these restriction sites (underlined). 【0447】 [ka] 【0448】 Nucleic acid sequence of CpG22-ApoE / hAAT (SEQ ID NO: 66). In all CpGs, C was changed to T, except for the 5th CpG, which remained unchanged (bold), and in the 6th CpG, G was changed to A. When the sequence was cloned into the FIX expression cassette, the ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 67 is CpG22-ApoE / hAAT with these restriction sites (underlined). 【0449】 [ka] 【0450】 The amino acid sequence of FVIII-BDD (SQ sequence bold / underlined) (SEQ ID NO: 68). [ka] 【0451】 SQ sequence (sequence number 69). SFSQNPPVLKRHQR 【0452】 Wild-type FVIII-BDD cDNA (SEQ ID NO: 70). 【0453】 A nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 71) atgcagattgagctgtctacctgcttcttcctgtgcctgctgaggttctgcttctctgct accaggaggtactacctgggggctgtggagctgagctgggattacatgcagtctgacctg ggggagctgcctgtggatgccaggtttccccccagggtgcccaagagcttccccttcaat acctctgtggtgtataagaagaccctgtttgtggagttcactgatcatctgttcaacatt gctaaacccaggcccccctggatggggctgctgggccctaccatccaggctgaggtgtat gacactgtggtgatcactctgaagaacatggctagccatcctgtgtctctgcatgctgtg ggggtgagctactggaaggcttctgagggggctgagtatgatgatcagactagccagagg gagaaggaggatgacaaggtgttccctgggggctctcacacctatgtctggcaggtgctg aaggagaatggccccatggcctctgatcctctgtgtctgacctatagctacctgagccat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgtagggag gggagcctggccaaggagaagacccagaccctgcacaagttcattctgctgtttgctgtg tttgatgagggcaagagctggcattctgaaaccaagaacagcctgatgcaggacagggat gctgcctctgctagggcctggcccaagatgcacactgtgaatgggtatgtcaataggtct ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggc accacccctgaggtgcacagcatctttctggagggccacaccttcctggtgaggaatcac agacaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg gacctgggccagtttctgctgttctgccacatctctagccaccagcatgatggcatggag gcctatgtgaaggtggactcctgccctgaggagccccagctgaggatgaagaataatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgagatttgat gatgacaattctcccagcttcattcagatcaggtctgtggccaagaagcatcccaagacc tgggtgcactacattgctgctgaggaggaggactgggactatgcccccctggtgctggcc cctgatgacaggagctataagagccagtacctgaataatggcccccagaggattgggagg aagtataagaaggtgaggttcatggcctatactgatgaaaccttcaagaccagagaggcc atccagcatgagtctgggatcctggggcccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggccagcaggccctacaacatctaccctcatggcatcact gatgtgaggcctctgtacagcagaaggctgcccaagggggtgaagcatctgaaggacttc cccattctgcctggggagattttcaagtacaagtggactgtgactgtggaggatggccca accaagtctgaccctaggtgcctgactaggtactacagcagctttgtgaatatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aacaggagctggtacctgactgagaacattcagaggtttctgcccaaccctgctggggtg cagctggaggaccctgaattccaggcctctaacatcatgcacagcattaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacattctgagc attggggcccagactgacttcctgtctgtgttcttctctggctacacctttaagcacaag atggtgtatgaggataccctgaccctgtttcctttctctggggagactgtgttcatgagc atggagaaccctggcctgtggatcctgggctgccacaactctgacttcaggaacaggggg atgactgctctgctgaaggtgagcagctgtgataagaacactggggactactatgaggac agctatgaggacatctctgcctatctgctgagcaagaataatgctattgagcccaggagc ttctctcagaacccccctgtgctgaagaggcaccagagggagatcaccagaactactctg cagtctgaccaggaggagattgactatgatgacaccatctctgtggagatgaagaaggag gattttgatatttatgatgaggatgaaaaccagagccccaggagctttcagaagaagact aggcactatttcattgctgctgtggagaggctgtgggactatggcatgtcttctagcccc catgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttc caggagttcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcac ctggggctgctgggcccttatatcagggctgaggtggaggataacatcatggtgaccttc aggaaccaggccagcaggccctacagcttctactctagcctgatcagctatgaggaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacttat ttctggaaggtgcagcaccatatggcccccaccaaggatgagtttgattgcaaagcctgg gcctacttctctgatgtggacctggagaaggatgtgcactctgggctgattggccccctg ctggtgtgccacaccaacactctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagactaagagctggtacttcactgagaacatg gagaggaactgcagggccccctgcaatatccagatggaggaccccacctttaaggaaaat tataggtttcatgccattaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgagcatgggcagcaatgagaacattcacagc atccacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tataatctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctg gtgtattctaacaagtgtcagacccccctgggcatggcctctggccatatcagggacttc cagatcactgcctctggccagtatgggcagtgggcccccaagctggccaggctgcattac tctggcagcatcaatgcctggagcaccaaggagccattcagctggattaaggtggacctg ctggctccaatgattatccatggcatcaagacccagggggccaggcagaagtttagcagc ctgtacatctctcagtttatcatcatgtactctctggatggcaaaaagtggcagacctac aggggcaattctactggcactctgatggtgttctttggcaatgtggacagctctgggatc aagcacaacatctttaacccccctatcattgccaggtacattaggctgcaccccacccat tacagcatcaggagcaccctgaggatggagctgatgggctgtgatctgaacagctgcagc atgcccctgggcatggagagcaaggctatctctgatgcccagattactgccagcagctac ttcaccaatatgtttgccacctggagccccagcaaggccaggctgcacctgcagggcagg tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgaccacccagggggtgaagagcctgctgactagcatg tatgtgaaggagttcctgatcagcagcagccaggatggccatcagtggaccctgttcttc cagaatggcaaggtgaaggtgttccagggcaatcaggacagcttcacccctgtggtgaac agcctggacccccccctgctgaccagatacctgaggatccacccccagagctgggtgcat cagattgccctgaggatggaggtgctggggtgtgaggcccaggacctgtactga 【0454】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 72) atgcagattgagctgtctacctgctttttcctgtgtctgctgaggttctgcttctctgcc actaggaggtactacctgggggctgtggagctgtcttgggattacatgcagtctgatctg ggggagctgcctgtggatgccaggtttcctcccagggtgcccaagtctttccccttcaat acctctgtggtgttaagaagaccctgtttgtggagtttactgatcacctgttcaacatt gccaagcccaggcccccttggatgggcctgctggggcccaccatccaggctgaggtgtat gacactgtggtgatcaccctgaagaacatggcctctcaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgaggggctgagtatgatgaccagaccagccagagg gagaaggaggatgataaggtgttccctgggggagccaacacttatgtgtggcaggtgctg aaggaatggcccaatggcctctgatcccctgtgcctgacctattcttacctgagccat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggctctctggctaaggaagagacccagaccctgcacaagttcatcctgctgtttgctgtg tttgatgagggaagagctggcactctgagaccaagaacagcctgatgcaggacagggat gctgcccttgccagggcctggcccaaaatgcacactgtgaatggctatgtgaataggagc ctgcctggcctgattggctgccacaggaagtctgtgtattggcatgtgattggcatgggc accacccctgaggtgcactctatcttcctggagggccatactttcctggtgaggaatcat aggcaggccagcctggagattagccccattacctttctgactgcccagaccctgctgatg gacctgggccagttcctgctgttttgccacatcagctctcaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaagaacaatgag gaggctgaggattatgatgatgatctgactgattctgaaatggatgtggtgaggtttgat gatgacaatagcccctctttcatccagatcaggtctgtggccaagaagcatcctaagacc tgggtgcactacattgctgctgaggaggaggactgggactatgctcccctggtgctggcc cctgatgacaggtcttacaagagccagtacctgaacaatggcccccagagaattgggagg aagtataagaaggtgagattcatggcttacactgatgagaccttcaagactagggaggcc atccagcatgagtctggcattctgggccccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggcctctaggccctacaatatttacccccatgggatcact gatgtgaggcccctgtacagcaggaggctgcctaagggggtgaagcatctgaaggacttc cccatcctgcctggggagatcttcaagtataagtggactgtgactgtggaagatggcccc accaagtctgaccctaggtgcctgaccaggtactactcttcttttgtgaacatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag agggggaaccagattatgtctgacaagaggaatgtgattctgttctctgtgtttgatgag aacaggagctggtatctgactgagaacatccagaggttcctgcccaatcctgctggggtg cagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatgggtatgtg tttgattctctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggctcagactgatttcctgtctgtgttcttttctggctacacctttaagcataag atggtgtatgaggacactctgaccctgtttcccttctctggggagactgtgtttatgagc atggagaaccctggcctgtggatcctgggctgccacaactctgatttcaggaacaggggc atgactgctctgctgaaggtgtcttcttgtgacaagaacactggggactattatgaggac agctatgaggacatctctgcctacctgctgagcaagaacaatgctattgagcccagatct ttcagccagaacccccctgtgctgaagaggcaccagagggagatcactaggaccaccctg cagtctgaccaggaggagattgactatgatgacactatctctgtggagatgaagaaggag gactttgatatctatgatgaggatgagaaccagtctcccaggagcttccagaaaaagacc aggcactacttcattgctgctgtggagaggctgtgggactatggcatgtcttctagcccc catgtgctgaggaacagggcccagtctgggtctgtgccccagttcaagaaggtggtgttc caggagttcactgatgggagcttcacccagcctctgtacaggggggagctgaatgagcac ctggggctgctgggcccttatattagggctgaggtggaggacaacatcatggtgactttc aggaatcaggcctctaggccctatagcttctacagctctctgatcagctatgaggaggat cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggctcctaccaaggatgagtttgactgcaaggcctgg gcctacttttctgatgtggacctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgtcataccaacaccctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagaccaagagctggtactttactgagaacatg gagaggaattgcagagccccttgcaacatccagatggaggacccaaccttcaaagagaac tacaggttccatgccatcaatgggtacatcatggacaccctgcctggcctggtgatggct caggaccagaggatcaggtggtatctgctgagcatgggcagcaatgagaatatccatagc attcacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tataacctgtaccctggggtgtttgagactgtggagatgctgccaagcaaggctgggatt tggagggtggagtgcctgattggggagcacctgcatgctggcatgtctaccctgttcctg gtgtactccaataagtgccagacccccctgggcatggcctctggccacatcagggacttc cagatcactgcctctggccagtatgggcagtgggccccaaagctggccaggctgcactat tctgggagcatcaatgcttggagcaccaaggagcctttcagctggattaaggtggatctg ctggcccccatgatcattcatggcatcaaaacccagggggctagacagaagttttctagc ctgtacatcagccagttcatcatcatgtacagcctggatggcaagaagtggcagacttac aggggcaatagcactggcaccctgatggtgttttttggcaatgtggacagctctggcatc aagcacaacatctttaacccccccattattgccaggtatatcaggctgcatcccacccac tattctattaggtctactctgagaatggagctgatgggctgtgacctgaacagctgtagc atgcccctggggatggagagcaaggctatctctgatgcccagatcactgccagctcttat ttcaccaatatgtttgccacctggtctccctctaaggccaggctgcacctgcagggcagg agcaatgcttggaggccccaggtgaataaccccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgactagcatg tatgtgaaggagttcctgatcagcagcagccaggatgggcatcagtggactctgttcttc cagaatggcaaggtgaaggtcttccaggggaaccaggatagcttcactcctgtggtgaac tctctggacccccccctgctgactaggtatctgaggatccacccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtattga 【0455】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 73) atgcagattgaactgtctacttgtttcttcctgtgcctgctgaggttttgcttctctgct actaggaggtactatctgggggctgtggagctgtcttgggactatatgcagtctgacctg ggggagctgcctgtggatgctaggtttccccccagggtgcccaagagcttcccctttaac acctctgtggtgtataagaagactctgtttgtggagttcactgaccatctgttcaacatt gccaagccaaggcccccctggatgggcctgctgggccccaccatccaggctgaggtgtat gacactgtggtgattactctgaagaacatggccagccatcctgtgagcctgcatgctgtg ggggtgtcttactggaaggcctctgagggggctgagtatgatgaccagacctctcagagg gagaaggaggatgacaaggtgttccctggggctctcatacctatgtgtggcaggtcctg aggagagaatgggcccatggcctctgaccccctgtgcctgacctactcttatctgtctcat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggctaaggaagaagacccagactctgcacaagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagggat gctgccctctgctagggcctggcccaagatgcacactgtgaatgggtatgtgaacaggagc ctgccaggcctgattggctgccataggaagtctgtgtattggcatgtgattgggatgggg actacccctgaggtccacagcattttcctggaggggcatacctttctggtgaggaaccac aggcaggcctctctggagatctctcccattactttcctgactgcccagaccctgctgatg gacctgggccagttcctgctgttctgccacatcagcagccaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaaaaacaatgag gaggctgaggattatgatgatgacctgactgattctgagatggatgtggtgaggtttgat gatgataacagccccagcttcatccagattaggtctgtggccaagaagcatcccaagacc tgggtgcactacattgctgctgaggaggaggattgggactatgctcctctggtgctggcc cctgatgacaggagctacaagagccagtacctgaataatggcccccagaggattggcagg aagtataagaaggtgaggttcatggcctacactgatgagacctttaagaccagggaggcc atccagcatgaatctgggatcctgggccccctgctgtatggggaggtgggggacaccctg ctgattatctttaagaaccaggctagcaggccctacaacatttacccccatggcattact gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaggatttc cccattctgcctggggagatctttaagtacaaatggactgtgactgtggaggatggccct actaagtctgatcccaggtgtctgaccagatactacagcagctttgtgaatatggagagg gacctggcttctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag aggggcaatcagattatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aacagaagctggtacctgactgagaacatccagaggttcctgcccaaccctgctggggtg cagctggaggaccctgagttccaggctagcaatatcatgcacagcattaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctattggtacattctgagc attggggcccagactgatttcctgtctgtgttcttttctggctacaccttcaagcacaag atggtgtatgaggatactctgaccctgtttcccttctctggggagactgtgttcatgagc atggagaaccctggcctgtggatcctgggctgtcacaactctgacttcaggaacaggggc atgactgccctgctgaaggtgagctcttgtgataagaacactggggactactatgaggac tcttatgaggacatctctgcctacctgctgagcaagaacaatgctattgagcccaggagc ttctctcagaatccccctgtgctgaagaggcatcagagggagatcactaggactaccctg cagtctgaccaggaagagattgactatgatgacaccatctctgtggaaatgaagaaggag gactttgatatctatgatgaggatgaaaaccagagccccaggagcttccagaagaagacc aggcattacttcattgctgctgtggagaggctgtgggactatgggatgagctcttctccc catgtgctgaggaatagggctcagtctggctctgtcccacagttcaagaaggtggtgttt caggagttcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcat ctgggcctgctggggccctacatcagggctgaggtggaggataacattatggtgactttc aggaaccaggcctctaggccctacagcttctacagcagcctgatcagctatgaggaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagactaagacctat ttctggaaggtgcagcatcacatggctcccactaaagatgagtttgactgcaaggcctgg gcctacttctctgatgtggatctggagaaggatgtgcattctgggctgattggccctctg ctggtctgccatactaacaccctgaatcctgcccatggcaggcaggtgactgtgcaggag tttgccctgttctttaccatctttgatgagaccaagtcttggtacttcactgagaacatg gagaggaactgcagggccccctgtaacatccagatggaggaccccacctttaaggagaac tacaggttccatgccatcaatggctacatcatggacactctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgtctatgggctctaatgagaacattcattct atccacttctctggccatgtgtttactgtgaggaagaaggaggagtacaagatggccctg tacaatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatc tggagggtggagtgcctgattggggaacacctgcatgctggcatgagcaccctgttcctg gtctatagcaataagtgccagacccccctggggatggcctctgggcatatcagagacttc cagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactac tctggcagcattaatgcctggagcaccaaggagcccttctcttggatcaaggtggacctg ctggctcccatgatcatccatgggatcaagacccagggggccaggcagaagttcagcagc ctgtacatctctcagttcatcatcatgtactctctggatggcaagaagtggcagacctac aggggcaatagcactgggaccctgatggtgttctttgggaatgtggacagctctggcatc aagcacaatatcttcaacccccccatcattgccaggtacatcagactgcaccccactcat tacagcatcaggagcactctgaggatggagctgatgggctgtgacctgaatagctgctct atgcccctgggcatggagagcaaggccatttctgatgcccagattactgcctcttcttac ttcactaatatgtttgccacctggagccccagcaaggccaggctgcatctgcaggggagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagactatgaaggtgactggggtgaccactcagggggtgaagagcctgctgaccagcatg tatgtgaaggagttcctgatctcttctagccaggatgggcaccagtggaccctgtttttc cagaatgggaaggtgaaggtgtttcagggcaatcaggacagctttactcctgtggtgaac agcctggacccccccctgctgactaggtacctgaggattcacccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga 【0456】 FVIII-BDDををダーする CpGががんますまするまするますますますすすすすすすがたしたしたしたしたしたしたしたしたしたしたしたしたしたしい atgcagattgagctgtctacctgcttctttctgtgcctgctgaggttctgtttctctgcc actaggaggtattatctggggctgtggagctgtcctgggactacatgcagtctgatctg ggggagctgcctgtggatgccaggttccctcccagggtgcccaagtctttccctttcaat acctctgtggtgtacaagaagactctgtttgtggagtttactgatcacctgtttaacatt gccaagcccaggcccccctggatggggctgctgggccccaccatccaggctgaggtgtat gacactgtggtgattactctgaagaatatggcttctcaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgaggggctgagtatgatgaccagaccagccagagg gagaaggaggatgacaaggtgttccctggggcagccacacttatgtgtggcaggtgctg aaggagaatggcccaatggcctctgaccccctgtgcctgacctacagctatctgagccat gtggatctggtgaaggatctgaactctggcctgattggggccctgctggtgtgcagggag ggctctctggccaaggagaagactcagactctgcacaagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagaccaagaactctctgatgcaggatagggat gctgcttctgccagggcctggcccaagatgcacactgtgaatgggtatgtgaataggagc ctgcctgggctgattgggtgtcacaggaagtctgtgtactggcatgtgattggcatgggc accactcctgaggtgcacagcatctttctggagggccacacttttctggtgaggaatcac aggcaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg gatctgggccagttcctgctgttttgccatatcagcagccatcagcatgatgggatggag gcttatgtgaaggtggactcttgccctgaggagcctcagctgaggatgaagaataatgaa gaggctgaggactatgatgatgatctgactgactctgagatggatgtggtgaggtttgat gatgacaacagccccagctttatccagattaggtctgtggccaagaagcaccccaagacc tgggtgcattacattgctgctgaggaagaggattgggactatgcccccctggtgctggcc cctgatgacaggagctacaagtctcagtacctgaacaatggccctcagaggattggcagg aagtacaagaaggtgaggttcatggcttacactgatgagaccttcaagaccagggaggcc attcagcatgaatctgggatcctgggccccctgctgtatggggaggtgggggacaccctg ctgattattttcaagaaccaggccagcaggccctacaacatttatcctcatggcattact gatgtgagacccctgtacagcaggaggctgcctaagggggtgaagcacctgaaggacttc cccatcctgcctggggagatcttcaagtacaagtggactgtgactgtggaggatggcccc actaagtctgaccccaggtgcctgactaggtactactccagctttgtgaacatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag aggggcaaccagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgag aataggtcttggtacctgactgagaacatccagaggtttctgcctaatcctgctggggtg cagctggaggatcctgagttccaggcctctaacattatgcacagcatcaatgggtatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggcccagactgactttctgtctgtgttcttctctggctacacctttaagcataag atggtgtatgaggacaccctgactctgttccccttctctggggagactgtgttcatgagc atggagaacccaggcctgtggatcctgggctgccacaactctgatttcaggaataggggc atgactgccctgctgaaggtgagcagctgtgataagaacactggggactattatgaggat agctatgaggacatctctgcctacctgctgagcaagaacaatgccattgagcccaggagc ttcagccagaatcctcctgtgctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaatcagagccccaggagcttccagaagaagact agacactactttattgctgctgtggagaggctgtgggactatggcatgagctcttctccc catgtgctgagaaacagggcccagtctggctctgtgccccagttcaagaaggtggtcttc caggagttcactgatggctctttcacccagcctctgtatagaggggagctgaatgagcac ctgggcctgctgggcccttacatcagggctgaggtggaggacaatatcatggtgaccttc aggaaccaggctagcaggccctactctttctacagcagcctgatcagctatgaggaggac cagaggcagggggctgagcctaggaagaattttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggctcccactaaggatgagtttgactgcaaggcctgg gcctacttttctgatgtggacctggagaaggatgtgcattctggcctgattggccccctg ctggtctgccacaccaatactctgaaccctgctcatgggagacaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagaccaagtcctggtactttactgagaacatg gagaggaattgcagggccccttgcaacatccagatggaggaccccaccttcaaggaaaat tataggttccatgccatcaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggatcaggtggtatctgctgtctatgggctctaatgagaacatccacagc atccatttctctggccatgtgttcactgtgaggaagaaggaggagtataagatggctctg tacaacctgtaccctggggtctttgagactgtggagatgctgcccagcaaggctggcatt tggagggtggagtgcctgattggggaacacctgcatgctgggatgagcaccctgttcctg gtgtactctaacaagtgccagaccccactgggcatggcttctggccacatcagggatttc cagattactgcctctggccagtatggccagtgggctcccaagctggctaggctgcactac tctgggagcatcaatgcctggtctactaaggagcctttctcttggatcaaagtggacctg ctggcccctatgatcatccatgggatcaagactcagggggccaggcagaagttcagcagc ctgtacatctctcagttcatcattatgtacagcctggatggcaagaagtggcagacctac aggggcaacagcactggcaccctgatggtgttctttgggaatgtggacagctctgggatt aagcacaacatctttaacccccccatcattgccaggtatatcaggctgcaccctacccac tacagcattaggagcaccctgaggatggagctgatgggctgtgacctgaacagctgcagc atgcccctggggatggagagcaaggccatttctgatgctcagatcactgcttctagctac ttcactaacatgtttgccacctggtctcccagcaaggctagactgcacctgcaggggagg agcaatgcctggaggccccaggtgaataatcccaaggagtggctgcaggtggatttccag aaaaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgaccagcatg tatgtgaaggagttcctgatcagcagcagccaggatgggcatcagtggaccctgttcttt cagaatgggaaggtgaaggtgtttcagggcaatcaggacagcttcacccctgtggtgaac agcctggacccccccctgctgaccaggtacctgaggatccacccccagagctgggtgcat cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga 【0457】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 75) atgcagattgagctgtctacttgcttcttcctgtgcctgctgaggttctgcttctctgcc actaggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctg ggggagctgcctgtggatgccaggtttcctcccagggtgcctaagagcttcccccttcaac acctctgtggtgtcaagaagaactctgtttgtggagtttactgatcatctgttcaacatt gccaagcccaggcctccttggatggggctgctgggccccaccatccaggctgaggtgtat gacactgtggtgattaccctgaagaatatggccagccatcctgtgagcctgcatgctgtg ggggtgagctattggaaggcctctgaggggctgagtatgatgatcagactagccagagg gagaaggaggatgacaaggtgttccctgggggagccatacctatgtgtggcaggtgctg aaggaatggccccatggcctctgaccctctgtgcctgactatagatctacctgagccat gtggatctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggccaaggaagagaactcagaccctgcacaagttcatcctgctgtttgctgtg tttgatgaggggaagtcctggcactctgagactaagaacagcctgatgcagtagtagggat gctgcttctgccagggcctggcctaagatgcacactgtgaatggctatgtgaataggagc ctgcctggcctgattggctgccataggaagtctgtgtactggcatgtgattgggatgggc accacccctgaggtgcactctattttcctggagggccatactttcctggtgaggaaccat aggcaggccagcctggagatcagccccatcactttcctgactgcccagactctgctgatg gacctgggccagttcctgctgttctgccacatcagcagccatcagcatgatggcatggag gcttatgtgaaggtggacagctgccctgaggagcctcagctgaggatgaagaataatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaactctccctctttcatccagatcaggtctgtggccaagaagcaccctaagacc tgggtgcactacattgctgctgaggaggaggattgggactatgcccccctggtgctggcc ccagatgacaggagctacaagtcccagtacctgaacaatggcccccagaggattggcagg aagtacaagaaggtgaggttcatggcttatactgatgagactttcaagaccagggaggcc atccagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctg ctgattatcttcaagaaccaggcttctaggccctacaatatctaccctcatggcatcact gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcatctgaaggatttc cccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggcccc actaagtctgaccccaggtgcctgaccaggtattacagcagctttgtgaacatggagagg gatctggcttctgggctgattggccccctgctgatctgctacaaggagtctgtggaccag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aataggagctggtacctgactgagaacatccagaggtttctgcccaatcctgctggggtg cagctggaggatcctgagtttcaggcctctaatatcatgcacagcatcaatggctatgtg tttgactctctgcagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagc attggggcccagactgactttctgtctgtgtttttttctggctacaccttcaagcacaag atggtgtatgaggatactctgactctgttccctttttctggggagactgtgttcatgtct atggagaaccctgggctgtggattctgggctgccacaattctgacttcaggaacagaggc atgactgctctgctgaaggtgagcagctgtgacaagaacactggggactactatgaggac tcttatgaggacatttctgcctacctgctgagcaagaacaatgccattgagcccagaagc ttttctcagaacccccctgtgctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgaccaggaggagattgactatgatgatactatttctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaaccagagccccaggtctttccagaagaagact aggcactactttattgctgctgtggagaggctgtgggactatgggatgtctagctctcct catgtgctgaggaacagggcccagtctggctctgtgccccagtttaaaaaggtggtgttc caggaattcactgatggcagctttacccagcctctgtacaggggggagctgaatgagcac ctggggctgctggggccttacattagggctgaggtggaggacaacatcatggtgaccttc aggaatcaggccagcaggccctactctttctacagcagcctgatctcttatgaggaggac cagaggcagggggctgaacccaggaagaactttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggctcccaccaaggatgagtttgattgcaaggcctgg gcttacttctctgatgtggatctggagaaggatgtgcactctgggctgattggccccctg ctggtgtgccacaccaacactctgaaccctgcccatggcagacaggtgactgtgcaggag tttgccctgttcttcactatctttgatgagactaagagctggtacttcactgagaacatg gagaggaattgcagggccccttgcaacatccagatggaggaccccacctttaaggagaac tacaggtttcatgccattaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgtctatggggagcaatgagaacatccacagc attcacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tacaacctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctgggatc tggagggtggagtgcctgattggggagcacctgcatgctgggatgagcaccctgttcctg gtgtatagcaacaagtgccagacccccctgggcatggcctctggccacatcagagacttt cagattactgcctctggccagtatgggcagtgggcccccaagctggccaggctgcactat tctggctctattaatgcctggagcactaaggagcccttcagctggattaaggtggacctg ctggctcccatgatcatccatggcatcaagactcagggggccaggcagaagttctcttct ctgtacatcagccagttcattatcatgtactccctggatggcaagaagtggcagacctat aggggcaacagcactggcaccctgatggtgttctttgggaatgtggacagctctggcatc aagcataatatcttcaatccccccatcattgctaggtacatcaggctgcaccccacccac tactctattaggtctaccctgaggatggagctgatgggctgtgacctgaacagctgcagc atgcctctgggcatggagagcaaagccatctctgatgcccagatcactgccagcagctac tttaccaacatgtttgctacttggagccccagcaaggccaggctgcacctgcaggggagg tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagactatgaaggtgactggggtgaccacccagggggtgaagagcctgctgacctctatg tatgtgaaggagttcctgattagcagcagcccaggatggccaccagtggaccctgtttttc cagaatgggaaggtgaaggtgtttcaggggaaccaggacagcttcactcctgtggtgaac tctctggacccccccctgctgaccaggtatctgaggatccaccctcagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga 【0458】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 76) 【0459】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 77) 【0460】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 78) 【0461】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 79) 【0462】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 80) 【0463】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 81) 【0464】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 82) 【0465】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 83) 【0466】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 84) 【0467】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 85) 【0468】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 86) 【0469】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 87) 【0470】 Nucleic acid variant with reduced CpG encoding FVIII-BDD (SEQ ID NO: 88) 【0471】 FVIII V3 cDNA (SEQ ID NO: 89) 【0472】 FVIII CO3 cDNA (SEQ ID NO: 90) 【0473】 AAV-LK03 VP1 Capsid (SEQ ID NO: 91) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPD PQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPAD VFMVPN AELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL 【0474】 AAV-SPK VP1 capsid (SEQ ID NO: 92) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVP DPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFP ADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKD NVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL 【0475】 Nucleic acid sequence of introns in AAV-WINT (SEQ ID NO: 93) AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTGACACTGACATCCACTTTTTCTTTTTCTCCACAG 【0476】 FIX (Sequence ID 94) 【0477】 [Related applications]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 722,547 filed on 24 August 2018, U.S. Provisional Patent Application No. 62 / 725,096 filed on 30 August 2018, and U.S. Provisional Patent Application No. 62 / 784,116 filed on 21 December 2018. The entire contents of the aforementioned applications, including all texts, tables, sequence listings and drawings, are incorporated herein by reference.

Claims

[Claim 1] It is a polynucleotide containing regulatory elements, The regulatory element is a polynucleotide comprising the nucleic acid sequence of sequence number 58. [Claim 2] The polynucleotide according to claim 1, wherein the regulatory element includes the sequence of sequence number 59. [Claim 3] An expression cassette comprising a polynucleotide according to any one of claims 1 to 2, wherein the regulatory element is operably linked to an introduced gene. [Claim 4] The expression cassette according to claim 3, wherein the regulatory element is located at the 5' position of the introduced gene. [Claim 5] The expression cassette according to claim 4, comprising a polyadenylated sequence on the 3' side of the introduced gene. [Claim 6] The expression cassette according to claim 4 or 5, wherein the introduced gene encodes a therapeutic protein that is expressed in liver cells and secreted into the systemic circulation. [Claim 7] The expression cassette according to claim 6, wherein the therapeutic protein treats or prevents neurodegenerative diseases or central nervous system (CNS) diseases. [Claim 8] The expression cassette according to claim 6, wherein the therapeutic protein is a blood coagulation or coagulation factor protein. [Claim 9] The expression cassette according to claim 8, wherein the blood coagulation or coagulation factor protein is factor IX (FIX), factor VIII (FVIII), factor VII (FVII), or protein C. [Claim 10] An adeno-associated virus (AAV) vector comprising an expression cassette according to any one of claims 3 to 9. [Claim 11] a) AAV capsid, and b) Two AAV terminal inversion sequences (ITRs) adjacent to the 5' and 3' ends of the expression cassette, AAV ITRs The AAV vector according to claim 10, including the following: [Claim 12] The AAV vector according to claim 11, further comprising an intron located within the adjacent 5' or 3' ITR. [Claim 13] The AAV vector according to claim 12, wherein at least one of the introns or one or more ITRs is modified to have a reduced CpG. [Claim 14] The AAV vector according to any one of claims 11 to 13, wherein the AAV capsid has 95% or more sequence identity with respect to the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8, Sequence ID No. 91 or Sequence ID No. 92 VP1, VP2 and / or VP3 sequences. [Claim 15] The AAV vector according to any one of claims 11 to 13, wherein the AAV capsid includes the sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8 VP1, VP2 and / or VP3, or the sequence of SEQ ID NO: 91 or SEQ ID NO:

92. [Claim 16] The AAV vector according to any one of claims 11 to 15, wherein the ITR comprises one or more ITRs of any of the following: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74 AAV serotypes, or combinations thereof. [Claim 17] A pharmaceutical composition comprising a plurality of AAV vectors according to any one of claims 10 to 16 in a biocompatible carrier or excipient. [Claim 18] The pharmaceutical composition according to claim 17, further comprising an empty AAV capsid. [Claim 19] The pharmaceutical composition according to claim 18, wherein the ratio of the empty AAV capsid to the AAV vector is within or between 100:1 to 50:1, 50:1 to 25:1, 25:1 to 10:1, 10:1 to 1:1, 1:1 to 1:10, 1:10 to 1:25, 1:25 to 1:50, or 1:50 to 1:100.

Images