Optimized promoter sequences, intron-free expression constructs, and methods of use

Abstract

The present invention provides expression cassettes. In certain embodiments, the expression cassette comprises (a) a regulatory element having at least 90% the same sequence as any one of SEQ ID NO: 2-67, and (b) a nucleic acid sequence encoding a factor VIII protein (FVIII-BDD) with a B domain deletion, wherein the nucleic acid sequence of (a) 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 wherein there are no introns between the regulatory element and the nucleic acid sequence encoding FVIII-BDD, or wherein no more than 0-107 nucleotides of untranslated nucleic acid are between the regulatory element and the nucleic acid sequence encoding FVIII-BDD. In certain embodiments, the expression cassette contains a sequence element that replaces CpG at the same position with CpT, CpA, TpG, or ApG, or a nucleic acid sequence with reduced CpG.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 722,547, filed on August 24, 2018, U.S. Provisional Patent Application No. 62 / 725,096, filed on August 30, 2018, and U.S. Provisional Patent Application No. 62 / 784,116, filed on December 21, 2018. The entire contents of the above applications (including all text, tables, sequence listings, and figures) are incorporated herein by reference. Background Art

[0003] Gene therapy shows great promise in therapeutic applications involving abnormal protein function or activity, such as loss of protein function or activity due to gene deficiency or defect, or where suppression of abnormal protein expression is desired. Improvements in transgene expression, enhancer and promoter function that drive transgene expression, will enhance gene therapy therapeutic applications. The present invention addresses this need, among other things. Summary of the Invention

[0004] According to the present invention, an expression cassette comprising a nucleic acid sequence encoding a Factor VIII protein with a B domain deletion (FVIII-BDD) is provided.

[0005] In certain embodiments, the expression cassette comprises a sequence that is at least 98% identical to SEQ ID NO: 1, is at least 99% identical to SEQ ID NO: 1, comprises, or consists of SEQ ID NO: 1.

[0006] In certain embodiments, the expression cassette comprises a regulatory element operably linked to a nucleic acid sequence encoding a Factor VIII protein with a B domain deletion (FVIII-BDD), wherein no introns are present between the regulatory element and the nucleic acid sequence, and wherein the expression cassette comprises a sequence that is at least 91% identical to the sequence of SEQ ID NO: 1.

[0007] In certain embodiments, the expression cassette comprises (a) a regulatory element that is at least 90% identical to the sequence of any one of SEQ ID NOs: 2-67, and (b) a nucleic acid sequence encoding a Factor VIII protein with a B domain deletion (FVIII-BDD), wherein the nucleic acid sequence of (a) is at least 90% identical to the sequence of SEQ ID NO: 77, and wherein the regulatory element is operably linked to the nucleic acid sequence, and wherein no introns are present between the regulatory element and the nucleic acid sequence encoding FVIII-BDD.

[0008] In certain embodiments, the expression cassette comprises (a) a regulatory element that is at least 90% identical to the sequence of any one of SEQ ID NOs: 2-67, and (b) a nucleic acid sequence encoding a Factor VIII protein with a B domain deletion (FVIII-BDD), wherein the nucleic acid sequence is at least 90% identical to the sequence of SEQ ID NO: 77, and wherein the regulatory element is operably linked to the nucleic acid sequence, and wherein no more than 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 nucleotides of untranslated nucleic acid are between the regulatory element and the nucleic acid sequence encoding the FVIII-BDD.

[0009] In certain embodiments, the regulatory element in the expression cassette comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 2-67.

[0010] In certain embodiments, the regulatory elements in the expression cassette have the same total number of reduced CpGs as set forth in the sequence of any one of SEQ ID NOs: 4-21 or 24-67.

[0011] In certain embodiments, the regulatory element in the expression cassette comprises the sequence of any one of SEQ ID NOs: 2-21 or 24-67, which substitutes CpG with CpT, CpA, TpG, or ApG at the same position as set forth in the sequence of any one of SEQ ID NOs: 4-21 or 24-67.

[0012] In certain embodiments, the nucleic acid sequence in the expression cassette exhibits higher expression when compared to expression from an expression cassette having 108 or more nucleotides of (a) introns or (b) untranslated nucleic acid between the regulatory elements and the nucleic acid sequence.

[0013] In certain embodiments, the encoded FVIII-BDD in the expression cassette exhibits greater biological activity compared to expression from an expression cassette having 108 or more nucleotides of (a) introns or (b) untranslated nucleic acid between the regulatory element and the nucleic acid sequence.

[0014] In certain embodiments, biological activity is determined by reducing bleeding in a coagulation assay or a FVIII assay or a FVIII deficiency model.

[0015] In certain embodiments, the expression cassette is more efficiently packaged into an AAV vector when compared to an expression cassette of 108 or more nucleotides having (a) introns or (b) untranslated nucleic acid between the regulatory elements and the nucleic acid sequence.

[0016] According to the present invention, a nucleic acid sequence with reduced cytosine-guanine dinucleotides (CpG) of a regulatory element (promoter) is provided. Exemplary promoters include the TTR (transthyretin gene) promoter and the ApoE / hAAT (human apolipoprotein E gene / human alpha-1 antitrypsin gene) promoter. Exemplary promoters also include the fibrinogen gamma chain gene (FGG) promoter, the albumin promoter, and the serum amyloid A1 gene (SAA1) promoter. Exemplary promoters further include a TTR promoter fused to one or more of the hAAT promoter, the FGG promoter, the albumin promoter, and / or the SAA1 promoter (generating a hybrid promoter or promoter chimera).

[0017] CpG-reduced nucleic acid regulatory elements include variants that exhibit altered gene expression levels when transferred into cells compared to non-CpG-reduced regulatory elements. In certain embodiments, CpG-reduced regulatory elements can provide increased expression of a transgene or heterologous nucleic acid (such as a transgene encoding a protein such as a blood coagulation factor (e.g., FVIII)) in a mammal, and provide increased therapeutic efficacy in the context of gene transfer by increasing circulating levels of proteins such as blood coagulation factors and achieving hemostasis to obtain beneficial therapeutic results.

[0018] In certain embodiments, the nucleic acid sequence has at least one fewer CpG than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 2, 3, 22, and 23).

[0019] In certain embodiments, the nucleic acid sequence has at least 2 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 2, 3, 22, and 23).

[0020] In certain embodiments, the nucleic acid sequence has at least 3 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 2, 3, 22, and 23).

[0021] In certain embodiments, the nucleic acid sequence has at least 4 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 2, 3, 22, and 23).

[0022] In certain embodiments, the nucleic acid sequence has at least 5 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 22 and 23).

[0023] In certain embodiments, the nucleic acid sequence has at least 6 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 22 and 23).

[0024] In certain embodiments, the nucleic acid sequence has at least 7 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any of SEQ ID NOs: 22 and 23).

[0025] In certain embodiments, the nucleic acid sequence has at least 8 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any of SEQ ID NOs: 22 and 23).

[0026] In certain embodiments, the nucleic acid sequence has at least 9 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any of SEQ ID NOs: 22 and 23).

[0027] In certain embodiments, the nucleic acid sequence has at least 10 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any of SEQ ID NOs: 22 and 23).

[0028] In certain embodiments, the nucleic acid sequence has at least 11 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 22 and 23).

[0029] In certain embodiments, the nucleic acid sequence has at least 12 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any of SEQ ID NOs: 22 and 23).

[0030] In certain embodiments, the nucleic acid sequence has at least 13 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 22 and 23).

[0031] In certain embodiments, the nucleic acid sequence has at least 14 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 22 and 23).

[0032] In certain embodiments, the nucleic acid sequence has at least 15 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any one of SEQ ID NOs: 22 and 23).

[0033] In certain embodiments, the nucleic acid sequence has at least 16 fewer CpGs than a wild-type non-CpG reduced regulatory element (eg, any of SEQ ID NOs: 22 and 23).

[0034] In certain embodiments, the nucleic acid sequence has no more than 16 CpGs; has no more than 15 CpGs; has no more than 14 CpGs; has no more than 13 CpGs; has no more than 12 CpGs; has no more than 11 CpGs; has no more than 10 CpGs; has no more than 9 CpGs; has no more than 8 CpGs; has no more than 7 CpGs; has no more than 6 CpGs; has no more than 5 CpGs; has no more than 4 CpGs; has no more than 3 CpGs; has no more than 2 CpGs; or has no more than 1 CpG.

[0035] In certain embodiments, 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 certain embodiments, the nucleic acid sequence has no CpGs.

[0036] In certain embodiments, the nucleic acid sequence comprising SEQ ID NO: 22 or 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 any one of SEQ ID NO: 2, 3, 22, and 23 is modified to have 0 CpGs.

[0037] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C in each CpG site is modified to a T, optionally excluding the C in the C / EBP site.

[0038] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C in each CpG site is modified to a T, except that the first CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0039] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C in each CpG site is modified to a T, except that the second CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0040] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C in each CpG site is modified to a T, except that the third CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0041] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C in each CpG site is modified to a T, except that the fourth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0042] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the fifth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0043] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the sixth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0044] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the seventh CpG site remains unmodified.

[0045] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the eighth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0046] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the ninth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0047] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the tenth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0048] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the eleventh CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0049] In certain embodiments, the nucleic acid sequence in either of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the twelfth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0050] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the thirteenth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0051] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the fourteenth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0052] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the fifteenth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0053] In certain embodiments, the nucleic acid sequence in either of SEQ ID NOs: 22 and 23 is modified such that the C in each CpG site is modified to a T, except that the sixteenth CpG site remains unmodified, optionally excluding the C in the C / EBP site.

[0054] In certain embodiments, the nucleic acid sequence is modified such that at least the first CpG from the 5' end of any one of SEQ ID NOs: 2, 3, 22, and 23 is modified to not be a CpG.

[0055] In certain embodiments, the nucleic acid sequence is modified such that at least the second CpG from the 5' end of any one of SEQ ID NOs: 2, 3, 22, and 23 is modified to not be a CpG.

[0056] In certain embodiments, the nucleic acid sequence is modified such that at least the third CpG from the 5' end of any one of SEQ ID NOs: 2, 3, 22, and 23 is modified to not be a CpG.

[0057] In certain embodiments, the nucleic acid sequence is modified such that at least the fourth CpG from the 5' end of any one of SEQ ID NOs: 2, 3, 22, and 23 is modified to not be a CpG.

[0058] In certain embodiments, the nucleic acid sequence is modified such that at least the 5th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0059] In certain embodiments, the nucleic acid sequence is modified such that at least the sixth CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0060] In certain embodiments, the nucleic acid sequence is modified such that at least the 7th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0061] In certain embodiments, the nucleic acid sequence is modified such that at least the 8th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0062] In certain embodiments, the nucleic acid sequence is modified such that at least the ninth CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0063] In certain embodiments, the nucleic acid sequence is modified such that at least the 10th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0064] In certain embodiments, the nucleic acid sequence is modified such that at least the 11th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0065] In certain embodiments, the nucleic acid sequence is modified such that at least the 12th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0066] In certain embodiments, the nucleic acid sequence is modified such that at least the 13th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0067] In certain embodiments, the nucleic acid sequence is modified such that at least the 14th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0068] In certain embodiments, the nucleic acid sequence is modified such that at least the 15th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0069] In certain embodiments, the nucleic acid sequence is modified such that at least the 16th CpG from the 5' end of any one of SEQ ID NOs: 22 and 23 is modified to not be a CpG.

[0070] In certain embodiments, the cytosine of one or more CpGs in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified to thymine. In certain embodiments, the cytosine of one or more CpGs in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified to adenine.

[0071] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C in one or more CpGs is deleted.

[0072] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the G in one or more CpGs is deleted.

[0073] In certain embodiments, the nucleic acid sequence in any one of SEQ ID NOs: 2, 3, 22, and 23 is modified such that the C and G in one or more CpGs are deleted.

[0074] Exemplary CpG-reduced TTR promoters are set forth in SEQ ID NOs: 4-13.

[0075] Exemplary CpG-reduced hybrid promoters are set forth in SEQ ID NOs: 14-21.

[0076] Exemplary CpG-reduced ApoE / hAAT promoters are set forth in SEQ ID NOs: 24-67.

[0077] In certain embodiments, one or more CpG cytosines in a hybrid promoter comprising all or a portion of a TTR promoter fused to all or a portion of at least one of an hAAT promoter, and / or an FGG promoter, and / or an albumin promoter, and / or an SAA1 promoter are modified to thymine (CT) or adenine (GA). The TTR promoter can be fused to any one or combination of the aforementioned promoters in any 5' to 3' orientation.

[0078] In certain embodiments, the hybrid promoter has a 5'3' orientation of TTR / hAAT or hAAT / TTR. In certain embodiments, the hybrid promoter has a 5'3' orientation of TTR / FGG or FGG / TTR. In certain embodiments, the hybrid promoter has a 5'3' orientation of TTR / hAAT / albumin or hAAT / TTR / albumin or albumin / TTR / hAAT or TTR / albumin / hAAT, hAAT / albumin / TTR or albumin / hAAT / TTR, etc.

[0079] In certain embodiments, the hybrid promoter has a 5' 3' orientation, TTR / FGG / albumin or hAAT / TTR / FGG or FGG / TTR / hAAT or TTR / FGG / hAAT, etc.

[0080] In certain embodiments, the hybrid promoter has the TTR promoter fused to all or part of each of the hAAT promoter, the FGG promoter, the albumin promoter, and the SAA1 promoter. The TTR promoter can be fused to all or part of the aforementioned promoters in any 5' to 3' orientation and in any promoter order.

[0081] In certain embodiments, a nucleic acid sequence or polynucleotide of the invention (such as a CpG-reduced nucleic acid sequence) is operably linked to a transgene.

[0082] In certain embodiments, a nucleic acid sequence or polynucleotide of the invention (such as a modified SEQ ID NO: 2, 3, 22, or 23 as described herein) is operably linked to a transgene.

[0083] In certain embodiments, a nucleic acid sequence or polynucleotide of the invention (such as a modified SEQ ID NO: 2, 3, 22 or 23 as described herein) confers transcription on an operably linked transgene that is within about 5-100% of the transcription conferred by the unmodified SEQ ID NO: 2, 3, 22 or 23, or within about 50% of the transcription conferred by the unmodified SEQ ID NO: 2, 3, 22 or 23, or within about 25-50% of the transcription conferred by the unmodified SEQ ID NO: 2, 3, 22 or 23.

[0084] In certain embodiments, a nucleic acid sequence or polynucleotide, such as a modified SEQ ID NO: 2, 3, 22, or 23 as described herein, is located 5' of the transgene.

[0085] In certain embodiments, the transgene encodes a blood coagulation or clotting protein.

[0086] In certain embodiments, the transgene encodes Factor IX (FIX), Factor VIII (FVIII), Factor VII (FVII), or Protein C.

[0087] In certain embodiments, the transgene encodes a Factor VIII having a sequence that is at least 95% identical to the sequence of SEQ ID NO:68.

[0088] In certain embodiments, the transgene is transcribed into an inhibitory RNA. In certain embodiments, the inhibitory RNA comprises antisense RNA, microRNA (miRNA), or small interfering RNA (siRNA).

[0089] In certain embodiments, the transgene encodes a therapeutic protein that is expressed in liver cells and secreted into the systemic circulation.

[0090] In certain embodiments, the therapeutic protein treats or prevents a neurodegenerative or central nervous system (CNS) disease.

[0091] In certain embodiments, the therapeutic protein is a protective ApoE isoform.

[0092] In certain embodiments, the therapeutic protein is the ApoE ε2 isoform.

[0093] In certain embodiments, the therapeutic protein treats or prevents an autoimmune disease or an allergic disease.

[0094] In certain embodiments, the therapeutic protein is a fusion protein comprising an undesired antigen and a leader sequence that drives secretion of the therapeutic protein from a cell.

[0095] In certain embodiments, the undesired antigen is the myelin oligodendrocyte glycoprotein (MOG) extracellular domain or a fragment thereof.

[0096] In certain embodiments, the expression cassette comprises a nucleic acid sequence or polynucleotide of the invention, such as a modified SEQ ID NO: 2, 3, 22, or 23 as described herein, operably linked to a transgene, wherein the nucleic acid sequence or polynucleotide is positioned upstream of the 5' end of the transgene, and optionally wherein there are no more than 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 nucleotides of untranslated nucleic acid sequence positioned between the nucleic acid sequence or polynucleotide and the 5' end of the transgene.

[0097] In certain embodiments, the expression cassette comprises a first nucleotide sequence having 95% or greater sequence identity to the sequence of any one of SEQ ID NOs: 4-21 or 24-67, wherein the first nucleotide sequence positioned upstream of the 5' end of the second nucleotide sequence has 95% or greater sequence identity to the sequence of SEQ ID NO: 77, and optionally wherein there are no more than 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 nucleotides of untranslated nucleic acid sequence positioned between the first and 5' ends of the second nucleotide sequence.

[0098] In certain embodiments, the expression cassette comprises the sequence of SEQ ID NO: 1 or a polynucleotide having at least 98% sequence identity to the sequence of SEQ ID NO: 1.

[0099] In certain embodiments, the expression cassette comprises a polynucleotide having at least 99% sequence identity to the sequence of SEQ ID NO: 1.

[0100] In certain embodiments, the expression cassette consists essentially of SEQ ID NO: 1.

[0101] In certain embodiments, the transgene or the second nucleotide sequence comprises a nucleic acid sequence encoding Factor VIII (FVIII) with a B domain deletion (FVIII-BDD), and the nucleic acid sequence encodes a FVII-BDD protein having FVIII blood clotting activity and having at least 90% sequence identity to the sequence of SEQ ID NO: 68.

[0102] In certain embodiments, the transgene or the second nucleotide sequence comprises a nucleic acid sequence encoding Factor VIII (FVIII) with a B domain deletion (FVIII-BDD), and the nucleic acid sequence has 90% or greater sequence identity to the sequence of SEQ ID NO: 77 and encodes a protein having FVIII blood clotting activity.

[0103] In certain embodiments, the untranslated nucleic acid sequence is not an intron or is intron-less.

[0104] In certain embodiments, the first nucleotide sequence comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 4-21 or 24-67.

[0105] In certain embodiments, the first nucleotide sequence comprises a nucleic acid sequence that is at least 95% identical to the sequence of any one of SEQ ID NOs: 4-21 or 24-67, and has the same total number of reduced CpGs as set forth in the sequence of any one of SEQ ID NOs: 4-21 or 24-67.

[0106] In certain embodiments, the first nucleotide sequence comprises a nucleic acid sequence that is at least 95% identical to the sequence of any one of SEQ ID NOs: 4-21 or 24-67 and CpG has been substituted with CpT, CpA, TpG, or ApG at the same position as set forth in the sequence of any one of SEQ ID NOs: 4-21 or 24-67.

[0107] In certain embodiments, the second nucleotide sequence exhibits higher expression when 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.

[0108] In certain embodiments, the second nucleotide sequence exhibits greater biological activity when 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.

[0109] In certain embodiments, biological activity is determined by a coagulation assay or reduced bleeding in a FVIII assay or a FVIII deficiency model.

[0110] In certain embodiments, the second nucleotide sequence is more efficiently packaged into an AAV vector when compared to a polynucleotide having 10<8 > or more nucleotides between the first nucleotide sequence and the 5' end of the second nucleotide sequence.

[0111] In certain embodiments, an adeno-associated viral (AAV) vector comprises a nucleic acid sequence or polynucleotide or expression cassette as described herein.

[0112] In certain embodiments, an AAV vector comprises one or more of: a) an AAV capsid; and b) one or more AAV inverted terminal repeats (ITRs), wherein the AAV ITRs flank the 5' or 3' end of the nucleic acid sequence, polynucleotide, and / or transgene.

[0113] In certain embodiments, the AAV vector further comprises an intron positioned within the flanking 5' or 3' ITR.

[0114] In certain embodiments, an intron or one or more ITRs are modified to have reduced CpG.

[0115] In certain embodiments, the AAV capsid serotype comprises a modified or variant AAV VP1, VP2, and / or VP3 capsid having 90% or greater 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; or a modified or variant AAV capsid having 90% or greater 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; NO:92 VP1, VP2 and / or VP3 sequence having 95% or greater sequence identity; or a capsid having 100% sequence identity to 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 sequence.

[0116] In certain embodiments, the ITRs comprise one or more ITRs of any of the following: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, or Rh74 AAV serotypes, or a combination thereof.

[0117] In certain embodiments, the AAV vector further comprises an ITR, a polyadenylation (polyA) signal, and / or an intron sequence.

[0118] In certain embodiments, an AAV vector as described herein is any pharmaceutical composition.

[0119] In certain embodiments, pharmaceutical compositions comprise a biocompatible carrier or excipient.

[0120] In certain embodiments, the pharmaceutical composition further comprises an empty AAV capsid.

[0121] In certain embodiments, the composition or pharmaceutical composition comprises a ratio of empty AAV capsid to AAV vector within or between about 100:1-50:1, about 50:1-25:1, about 25:1-10:1, about 10:1-1:1, about 1:1-1:10, about 1:10-1:25, about 1:25-1:50, or about 1:50-1:100.

[0122] In certain embodiments, the ratio of empty AAV capsids to AAV vectors in the composition or pharmaceutical composition is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0123] In certain embodiments, the compositions or pharmaceutical compositions described herein further comprise a surfactant.

[0124] In certain embodiments, methods of treating a human in need of gene therapy are provided.

[0125] In certain embodiments, humans are in need of blood coagulation or clotting factors.

[0126] In certain embodiments, a method of treating a human comprises (a) providing an expression cassette as described herein, a polynucleotide as described herein, an AAV vector as described herein, or a pharmaceutical composition as described herein; and (b) administering to the human an amount of the expression cassette, polynucleotide, AAV vector, or pharmaceutical composition, wherein the blood coagulation or coagulation factor is expressed in the human.

[0127] In certain embodiments, the human has hemophilia A or B.

[0128] In certain embodiments, the AAV vector is administered to a human intravenously, intraarterially, intracavitary, intramucosally, or via a catheter.

[0129] In certain embodiments, the blood coagulation or clotting factor is expressed at increased levels following administration.

[0130] In certain embodiments, the blood coagulation or clotting factor is expressed at greater than 1% of the level of blood coagulation or clotting factor found in humans that do not require blood coagulation or clotting factors.

[0131] In certain embodiments, blood coagulation or clotting factors are expressed at about 1%-40% of the level of blood coagulation or clotting factors found in humans that do not require blood coagulation or clotting factors.

[0132] In certain embodiments, blood coagulation or clotting factors are expressed at about 5%-30% of the level of blood coagulation or clotting factors found in humans who do not require blood coagulation or clotting factors.

[0133] In certain embodiments, the AAV vector is expressed in approximately 1×10 8 to about 1×10 14 The dosage range is 500 vector genomes per kilogram (vg / kg) of human body weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1 Human Factor IX (hFIX) levels measured by activity assay in mouse plasma 24 hours after hydrodynamic delivery of hFIX encoding constructs displaying CpG-reduced ApoE / hAAT regulatory elements labeled "CpG1" to "CpG22." SEQ ID NOs: 24-67 correspond to regulatory elements CpG1-ApoE / hAAT to CpG22-ApoE / hAAT with or without restriction enzyme sites, respectively, and are further described in Example 13. hFIX levels are expressed as a multiple of a reference plasmid containing non-CpG-reduced ApoE / hAAT (SEQ ID NO: 23).

[0135] Figure 2 Shown are human factor VIII (hFVIII) antigen levels measured by ELISA in mouse plasma 24 hours after hydrodynamic delivery of a CpG-reduced TTRm promoter hFVIII coding construct labeled "CpG1" to "CpG5." SEQ ID NOs: 4-21 correspond to promoters CpG1-TTRm to CpG5-TTRm with or without flanking restriction enzyme sites, respectively, 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 an hFVIII coding construct containing a non-CpG-reduced TTRm promoter (SEQ ID NO: 3).

[0136] Figure 3Shown are hFVIII antigen levels measured by ELISA in mouse plasma 24 hours after hydrodynamic delivery of CpG-reduced hybrid promoter hFVIII coding constructs labeled "Hybrid 6" through "Hybrid 9." SEQ ID NOs: 14-21 correspond to CpG-reduced promoters Hybrid 6 through Hybrid 9, respectively. Levels are expressed as a percentage of normal human plasma FVIII, where 100% = 150 ng / mL. "TTRm" refers to an hFVIII coding construct containing the non-CpG-reduced TTRm promoter (SEQ ID NO: 3).

[0137] Figure 4 Shown are hFVIII antigen levels measured by ELISA in mouse plasma 2, 4, and 8 weeks after delivery of AAV-encapsidated non-CpG-reduced (TTRm) and CpG-reduced Hybrid 6, 7, 8, and 9 promoter hFVIII constructs at a dose of 6.4e11 vector genomes (vg) / kg. Levels are expressed as a percentage of normal human plasma FVIII, where 100% = 150 ng / mL.

[0138] Figure 5 Shown are hFVIII antigen levels measured by ELISA in mouse plasma 8 weeks after delivery of AAV encapsidated non-CpG reduced (TTRm-hFVIII), Hybrid 7, and Hybrid 9 promoter hFVIII constructs at a dose range 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.

[0139] Figure 6 Shown is the expression of hFVIII mRNA in the liver, brain, testis, spleen, and kidney of mice 8 weeks after intravenous administration of AAV encapsidated non-CpG reduced (TTRm-hFVIII), Hybrid 7, and Hybrid 9 promoter hFVIII constructs at a dose of 6.4el lvg / kg. No hFVIII RNA was observed in any tissue except the liver, indicating the liver-specific nature of the activator. From left to right, the result sets for liver, brain, testis, spleen, and kidney are shown for TTRm-hFVIII, Hybrid7-hFVIII, and Hybrid9-hFVIII, respectively.

[0140] Figure 7Shown is a schematic comparison of an expression cassette with an intron, designated "AAV-WINT" (TTRm-intron-hFVIII-BDD), and an expression cassette without an intron, designated "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 human Factor VIII with a B domain deletion (hFVIII-BDD), which is not present in AAV-INTL. The codon-optimized nucleic acid sequence in these cassettes encoding hFVIII-BDD is set forth in SEQ ID NO: 77.

[0141] Figure 8 Shown are hFVIII levels as measured by ELISA performed on mouse plasma samples from the 6.4e9 vg / mouse dose of Study #1. Results are the mean of animals (n=5) in each treatment group. Error bars represent standard deviation.

[0142] Figure 9 Shown are hFVIII levels as measured by ELISA on mouse plasma samples from Study #1 at a dose of 1.6e10 vg / mouse. Results are the mean of animals in each treatment group (TTRm hFVIII, n=4: intronless TTRm hFVIII, n=5). Error bars represent standard deviation.

[0143] Figure 10 Shown are hFVIII levels as measured by ELISA performed on mouse plasma samples from Study #2. Results are the mean of animals (n=10) in each treatment group. Error bars represent standard deviation.

[0144] Figure 11 Shown are hFVIII levels as measured by ELISA performed on non-human primate (NHP) plasma samples from Study #1. Shown are hFVIII levels from low dose (2e12 V Figure 2 shows the results for individual monkeys from Group 1 (AAV-WINT, line with squares, n=2) and Group 2 (AAV-INTL, SEQ ID NO: 1, line with triangles, n=3) of the 100 mg / kg (2400 mmol / l) group. Animal P0001 from one low-dose AAV-WINT was removed due to positive neutralizing antibodies against AAV observed in samples taken 8 days before dosing.

[0145] Figure 12Shown are hFVIII levels as measured by ELISA on NHP plasma samples from Study #1. Results are shown for individual monkeys in Group 3 (AAV-WINT, line with squares, n=2) and Group 4 (AAV-INTL, SEQ ID NO: 1, line with triangles, n=3) of the high-dose (6e12 vg / kg) AAV-WINT group. One animal, P0101, from the high-dose AAV-WINT group was removed due to no hFVIII expression observed upon treatment.

[0146] Figure 13 Shown are hFVIII levels as detected by ELISA performed on NHP plasma samples from Study # 2. Shown are results for individual monkeys at a dose of 2e12 vg / kg for AAV-WINT (line with squares, n=5) and AAV-INTL (SEQ ID NO: 1, line with triangles, n=5).

[0147] Figure 14 The results of cell-based vector potency assays at three different multiplicities of infection (MOI) are shown. Cell supernatants were assessed for hFVIII activity by Chromogenix Coatest SP4 and are the average of two biological replicates assayed twice. Error bars represent standard deviations. "AAV-WINT" and "AAV-INTL" are original undiluted virus stock bottles, while "AAV-WINT dose" and "AAV-INTL dose" indicate diluted material for infusion.

[0148] Figure 15 Shows the Figure 14 The results are replotted as fold change at each MOI relative to AAV-WINT.

[0149] Figure 16 Evaluation of vector potency in an in vitro cell-based vector potency assay at three different MOIs is shown. Cell supernatants were assessed for hFVIII activity using a Chromogenix Coatest SP4 and are the average of two biological replicates assayed in duplicate. Error bars represent standard deviation. AAV-WINT and AAV-INTL (SEQ ID NO: 1) are original virus stock vials from two different batches.

[0150] Figure 17Comparison of in vitro hFVIII levels from supernatants of Huh7 cells transfected with independent plasmid DNA preparations (preps) of mTTR-intron-hFVIII-BDD and mTTR-hFVIII-BDD (SEQ ID NO: 1) using Chromogenix Coatest SP4 assays for expression cassettes is shown. Individual data points are shown as solid circles, with each bar representing the mean of two biological replicates (n=2) assayed in duplicate. Error bars represent standard deviations.

[0151] Figure 18 Shown with 5×10 11 Daily FVIII activity levels in four human subjects (Participants 1 (circles), 2 (squares), 3 (triangles), and 4 (diamonds)) infused with 100 vg / kg of an AAV-INTL hFVIII expression cassette (SEQ ID NO: 1) encapsidated in the LK03 AAV vector (SEQ ID NO: 91), referred to herein as LK03-INTL hFVIII-BDD.

[0152] Figure 19 Shown with 5×10 11 Weekly mean values ​​of FVIII activity levels in four human subjects (Participants 1 (circles), 2 (squares), 3 (triangles), and 4 (diamonds)) infused with LK03-INTL hFVIII-BDD at 4 vg / kg.

[0153] Figure 20 Shown with 5×10 11 Four-week block averages of FVIII activity levels in four human subjects (Participants 1 (circles), 2 (squares), 3 (triangles), and 4 (diamonds)) infused with LK03-INTL hFVIII-BDD at 4 vg / kg. DETAILED DESCRIPTION

[0154] Disclosed herein are intronless expression cassettes for the expression of factor VIII (FVIII) and FVIII with a deleted B domain (FVIII-BBD), e.g., human FVIII (hFVIII) and human FVIII-BDD (hFVIII-BDD). Researchers have reported that including introns in expression cassettes (including in AAV delivery vectors) can help increase 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 results in increased AAV vector potency and transgene (in this case, Factor VIII) expression levels in cell culture, mice, and non-human primates. The "intronless" expression cassette design is an improvement in vectors for treating blood clotting disorders such as hemophilia A and can provide therapeutic efficacy at lower vector doses, potentially providing benefits to patient safety and outcomes in addition to reducing manufacturing barriers such as cost and time.

[0155] Also disclosed herein are nucleic acid sequences having reduced CpGs compared to a reference wild-type mammalian (e.g., human) sequence and / or having less than 100% sequence identity to a reference wild-type mammalian (e.g., human) sequence. Nucleic acid sequences having reduced CpGs include one or more of the following promoters: TTR promoter, ApoE / hAAT promoter, FGG promoter, albumin promoter, and SAA1 promoter. Nucleic acid sequences having reduced CpGs include fusions or hybrids of the TTR promoter with at least one or more of the following promoters: ApoE / hAAT promoter, FGG promoter, albumin promoter, and SAA1 promoter.

[0156] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, for example, small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides include naturally occurring, synthetic and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single, double or triple stranded, linear or circular, and can have any length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0157] As used herein, the term "modification" or "variant" and grammatical variations thereof refer to a nucleic acid, polypeptide, or subsequence thereof that is different from a reference sequence. Thus, the modified and variant sequences may have substantially the same, higher, or lower expression, activity, or function as compared to the reference sequence, but retain at least some of the activity or function of the reference sequence. Specific examples of modifications or variants are C P G-reduced TTR promoter, ApoE / hAAT promoter, FGG promoter, albumin promoter and SAA1 promoter.

[0158] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered compared to the wild type. A nucleic acid or polynucleotide variant may refer to a sequence that has been codon-modified but still retains at least partial sequence identity with a reference sequence such as a wild-type sequence. A nucleic acid or polynucleotide encoding a protein may be genetically modified without changing the sequence of the encoded protein. Alternatively, a sequence may be genetically modified to encode a variant protein. For example, some codons of the nucleic acid variant will be changed (e.g., CpG reduction) without changing the amino acids of the protein encoded thereby.

[0159] An expression vector containing a promoter with reduced CpG content can show improvement compared to a promoter whose CpG content has not been reduced. When comparing expression, the CpG-reduced promoter is compared to a wild-type or non-CpG-reduced promoter.

[0160] The term "variant" or "modified" need not appear in every instance of reference to a CpG-reduced nucleic acid sequence herein. Likewise, the term "CpG-reduced nucleic acid" or its analogs may omit the term "variant" or "modified," but means that reference to a "CpG-reduced nucleic acid" includes variants at the gene level.

[0161] A specific embodiment of a variant is a CpG-reduced nucleic acid. CpG reduction can be achieved by changing a C or G nucleotide to a different nucleotide, such as changing a C to a T or a G to an A. CpG reduction can also be achieved by deleting a C nucleotide, or deleting a G nucleotide, or deleting both a C and a G nucleotide.

[0162] "Variant or modified" FVIII refers to FVIII or FVIII-BDD that has been genetically altered compared to unmodified wild-type FVIII or FVIII-BDD (SEQ ID NO: 68). Such variants can be referred to as "nucleic acid variants encoding Factor VIII (FVIII)."

[0163] "Variant factor VIII (FVIII)" may also refer to a modified FVIII protein such that 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 appropriate to compare it to wild-type FVIII that also has a B domain deletion.

[0164] Variant FVIII may comprise a portion of the B domain. Thus, FVIII-BDD comprises a portion of the B domain. Typically, in FVIII-BDD, most of the B domain is deleted.

[0165] Variant FVIII may comprise an "SQ" sequence, such as SFSQNPPVLKRHQR (SEQ ID NO: 69). Typically, such variant FVIII with SQ (FVIII / SQ) has a BDD, e.g., at least all or part of the BD is deleted. Variant FVIII, e.g., FVIII-BDD, may have all or part of an "SQ" sequence, i.e., all or part of SEQ ID NO: 69. Thus, for example, a variant FVIII-BDD with an SQ sequence (SFSQNPPVLKRHQR, SEQ ID NO: 69) may have all or only part of the amino acid sequence SFSQNPPVLKRHQR (SEQ ID NO: 69). For example, FVIII-BDD may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid residues of SFSQNPPVLKRHQR (SEQ ID NO: 69), inclusive. Thus, SFSQNPPVLKRHQR (SEQ ID NO: 69) having 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 carboxyl-terminal deletions are included in the variant FVIII proteins as described herein.

[0166] "Polypeptides," "proteins," and "peptides" encoded by "nucleic acid" or "polynucleotide" sequences include full-length native sequences (as with naturally occurring wild-type proteins), as well as functional subsequences, modified forms, or sequence variants, as long as the subsequences, modified forms, or variants retain a certain degree of function of the native full-length protein. For example, a nucleic acid encoding a FVIII protein (e.g., a CpG-reduced nucleic acid) may have a B domain deletion as described herein and retain coagulation function. In the methods and uses of the present invention, such polypeptides, proteins, and peptides encoded by the nucleic acid sequences may, but need not, be identical to endogenous proteins that are defective, underexpressed, or absent in the treated mammal.

[0167] For example, and without limitation, modifications include one or more nucleotide or amino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-250, 250-500, 500-750, 750-850 or more nucleotides or residues). As described herein, an example of a nucleic acid modification is CpG reduction.

[0168] Examples of amino acid modifications are conservative amino acid substitutions or deletions (e.g., subsequences or fragments) of a reference sequence, such as FVIII, such as FVIII with a B domain deletion. In certain embodiments, the modified or variant sequence retains at least a portion of the function or activity of the unmodified sequence.

[0169] All mammalian and non-mammalian forms of the nucleic acid, known or unknown, are expressly included, including other mammalian forms of CpG-reduced promoters herein.

[0170] The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV) or other vehicle that can be manipulated by inserting or incorporating nucleic acid. Vectors can be used for genetic manipulation (i.e., "cloning vectors") to introduce / transfer polynucleotides into cells and / or organs and transcribe or translate the polynucleotides inserted into the cells. An "expression vector" is a vector containing a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. The vector nucleic acid sequence generally contains at least one origin of replication and optional additional elements for propagation in the cell, such as heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), optional selectable markers, polyadenylation signals.

[0171] As disclosed herein, vectors without introns exhibit superior properties compared to the same vectors with synthetic introns. Accordingly, the present invention provides expression cassettes 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 wherein no more than 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 nucleotides of untranslated nucleic acid sequence are present between the regulatory element and the 5' end of the transgene.

[0172] The present invention also provides an expression cassette comprising a first nucleotide sequence having 95% or greater sequence identity to the sequence of any one of SEQ ID NOs: 2-67, wherein the first nucleotide sequence is positioned 5' upstream of a second nucleotide sequence having 95% or greater sequence identity to the sequence of SEQ ID NO: 77, and wherein no more than 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 nucleotides of untranslated nucleic acid sequence are between the first nucleotide sequence and the 5' end of the second nucleotide sequence.

[0173] The present invention further provides an expression cassette comprising a first nucleotide sequence having 95% or greater sequence identity to the sequence of any one of SEQ ID NOs: 2-67, wherein the first nucleotide sequence is positioned at the position corresponding to SEQ ID NOs: or more. The invention further comprises a method comprising: preparing an untranslated nucleic acid sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence having 95% or greater sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the sequence of any one of NOs: 71-88, wherein the first nucleotide sequence comprises ... second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a second nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising: a first nucleotide sequence comprising:

[0174] The present invention further provides an expression cassette having an untranslated (non-coding) nucleic acid positioned between the regulatory element and the transgene, wherein the untranslated nucleic acid is not an intron. Such an expression cassette can be referred to as an intronless cassette.

[0175] Introns are sequences with donor sites and splice acceptor sites that allow the cellular machinery to splice out untranslated nucleotide sequences during the process of RNA maturation into mRNA. As used herein, "intron-free" refers to an untranslated nucleic acid sequence that lacks donor and splice acceptor sites, but does not mean that the untranslated nucleic acid sequence does not contain other sites, such as restriction enzyme recognition / cleavage sites, Kozak sequences, transcription factor recognition / binding sites. In other words, intron-free does not mean that the nucleic acid sequence is completely free of any untranslated nucleic acid sequence.

[0176] AAV vectors are derived from adeno-associated viruses. AAV vectors are suitable for use as gene therapy vectors because they can penetrate cells and introduce nucleic acid / gene material so that the nucleic acid / gene material can be stably maintained in the cell. Because AAV is not associated with human pathogenic diseases, AAV vectors are able to deliver heterologous nucleic acid sequences (e.g., heterologous nucleic acid sequences encoding therapeutic proteins and inhibitory RNAs) to human patients without causing substantial AAV pathogenesis or disease.

[0177] The term "recombinant" as a modifier of vector, such as recombinant AAV (rAAV) vector, and of sequence, such as recombinant polynucleotides and polypeptides, means that the composition has been manipulated (i.e., engineered) in a manner not normally found in nature. Specific embodiments of recombinant AAV vectors would be those in which a nucleic acid not normally found in the wild-type AAV genome (heterologous sequence) is inserted into the viral genome. Examples would be those in which a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector with or without the 5', 3', and / or intronic regions with which genes are normally associated within the AAV genome. Although the term "recombinant" is not used throughout this document when referring to AAV vectors and sequences such as polynucleotides, recombinant forms including AAV vectors, polynucleotides, etc. are expressly included despite any such omission.

[0178] "rAAV vectors" are derived from the wild-type AAV genome by using molecular methods to remove all or a portion of the wild-type AAV genome and replace it with a non-natural (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for rAAV vectors, one or two inverted terminal repeat (ITR) sequences of the AAV genome are retained. Because all or a portion of the AAV genome has been replaced with a non-natural sequence relative to the AAV genomic nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence, rAAV is distinguished from the AAV genome. Therefore, the incorporation of non-natural (heterologous) sequences defines AAV as a "recombinant" AAV vector, which may be referred to as a "rAAV vector."

[0179] Recombinant AAV vector sequences can be packaged—referred to herein as "particles"—for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo. Where the recombinant vector sequences are encapsidated or packaged into AAV particles, the particles may also be referred to as "rAAV" or "rAAV particles" or "rAAV virions." Such rAAV, rAAV particles, and rAAV virions include proteins that encapsidate or package the vector genome. In the case of AAV, specific embodiments include capsid proteins.

[0180] "Vector genome" or conveniently abbreviated as "vg" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form rAAV particles. Where a recombinant plasmid is used to construct or manufacture 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 referred to as the "plasmid backbone" that is critical for the cloning and amplification of the plasmid, processes required for propagation and recombinant AAV vector production, but is not itself packaged or encapsidated into rAAV particles. Thus, "vector genome" refers to the nucleic acid packaged or encapsidated by rAAV.

[0181] "AAV helper functions" refers to AAV-derived coding sequences (proteins) that can be expressed to provide AAV gene products and AAV vectors, which in turn act in trans for productive AAV replication and packaging. Thus, AAV helper functions include both the major AAV open reading frames (ORFs), rep and cap. The Rep expression product has been shown to have many functions, including, among others: recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and transcriptional regulation of AAV (or other heterologous) promoters. The Cap expression product (capsid) provides the necessary packaging function. AAV helper functions serve to complement trans-acting AAV functions that are missing from the AAV vector genome.

[0182] For example, "AAV auxiliary structure" generally refers to a nucleic acid sequence comprising a nucleotide sequence that provides an AAV function deleted from an AAV vector, which will be used to produce a transducing AAV vector for delivering a nucleic acid sequence of interest to a subject by gene therapy. AAV auxiliary structures are typically used to provide transient expression of AAV rep and / or cap genes to supplement AAV vector replication and the missing AAV functions necessary for encapsidation. Auxiliary structures generally lack AAV ITR and may neither replicate nor package themselves. AAV auxiliary structures may be in the form of plasmids, phages, transposons, cosmids, viruses or virions. A large number of AAV auxiliary structures have been described, such as plasmids pAAV / Ad and pIM29+45 encoding Rep and Cap expression products (see, for example, Samulski et al. (1989) J. Virol. 63: 3822-3828; and McCarty et al. (1991) J. Virol. 65: 2936-2945). Numerous other vectors encoding Rep and / or Cap expression products have been described (see, eg, US Patent Nos. 5,139,941 and 6,376,237).

[0183] The term "helper function" refers to non-AAV-derived viral and / or cellular functions that AAV replication depends on. The term includes proteins and RNAs required for AAV replication, including those involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid packaging. Virus-based helper functions can be derived from any of the known helper viruses, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

[0184] "Helper function vector" generally refers to a nucleic acid molecule comprising a polynucleotide sequence that provides an auxiliary function. These sequences can be on an auxiliary function vector and transfected into a suitable host cell. The auxiliary function vector can support the production of rAAV virus particles in the host cell. The auxiliary function vector can be in the form of a plasmid, a phage, a transposon or a cosmid. In addition, for auxiliary function, a complete complement of adenoviral genes is not required. For example, it has been reported that adenovirus mutants that cannot perform DNA replication and late gene synthesis are allowed to be used for AAV replication (Ito et al., (1970) J. Gen. Virol. 9: 243; Ishibashi et al., (1971) Virology 45: 317). Similarly, it has been shown that mutants in the E2B and E3 regions support AAV replication, thereby indicating that the E2B and E3 regions are likely not involved in providing auxiliary functions (Carter et al., (1983) Virology 126: 505). Adenoviruses that are defective in the E1 region or have a deleted E4 region cannot support AAV replication. Thus, the E1A and E4 regions appear to be required, directly or indirectly, 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 mutants include: E1B (Laughlin et al. (1982), supra; Janik et al., (1981), supra; 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, in 1 CRC Handbook of Parvoviruses (P. Tijssen ed., 1990); E3 (Carter et al., (1983), supra); and E4 (Carter et al., (1983), supra; Carter (1995)). Studies of helper function provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, but E1B55k may be required for AAV virion production, whereas E1B19k is not (Samulski et al., (1988) J. Virol. 62:206-210). In addition, International Publication No. WO 97 / 17458 and Matshushita et al., (1998) Gene Therapy 5:938-945 describe helper function vectors encoding various adenovirus genes. Exemplary helper function vectors include adenovirus VA RNA coding region, adenovirus E4ORF6 coding region, adenovirus E2A 72kD coding region, adenovirus E1A coding region, and adenovirus E1B region without a complete E1B55k coding region. These helper function vectors are described in, for example, International Publication No. WO 01 / 83797.

[0185] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from another AAV serotype. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies and one AAV compared to another AAV. Differences in cross-reactivity are typically attributed to differences in capsid protein sequences / antigenic determinants (e.g., differences in VP1, VP2, and / or VP3 sequences between AAV serotypes).

[0186] Under the traditional definition, a serotype means that the target virus has been tested for neutralizing activity against sera of all existing and characterized serotypes, and no antibodies that can neutralize the target virus have been found. Due to the discovery of more naturally occurring virus isolates and / or the generation of capsid mutants, there may or may not be serological differences from any currently existing serotype. Therefore, in the case where a new virus (e.g., AAV) has no serological differences, the new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, mutant viruses with capsid sequence modifications have not yet been serologically tested for neutralizing activity to determine whether they belong to another serotype according to the traditional serotype definition. Therefore, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically different viruses (e.g., AAV) and serologically different viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0187] rAAV vectors include any strain or serotype. For example, and without limitation, rAAV vector genomes or particles (capsids, such as VP1, VP2 and / or VP3) can be based on any AAV serotype, such as, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10 or AAV-2i8. These vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other. For example, and without limitation, rAAV plasmids or vector genomes or particles (capsids) based on a serotype genome can be consistent with one or more of the capsid proteins of the packaging vector. Additionally, the rAAV plasmid or vector genome may be based on an AAV serotype genome that differs 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 a different AAV serotype, e.g., 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 can comprise the AAV2 ITRs in addition to the capsid from a different serotype, such as, for example, AAV1, 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 variants thereof. Thus, the rAAV vector includes gene / protein sequences that are identical to those characteristic of a particular serotype, as well as mixed serotypes, also known as pseudotypes.

[0188] In certain embodiments, the rAAV vector comprises or consists of a capsid sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to 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 certain embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, or -rh10 ITR.

[0189] In certain embodiments, rAAV vectors include AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, and AAV-2i8 variants thereof (e.g., ITR and capsid variants, such as amino acid insertions, additions, substitutions, and deletions), for example, as described in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670), and US 2013 / 0059732 (U.S. Application No. 13 / 594,773, disclosing LK01, LK02, LK03 (SEQ ID NO: 91), etc.).

[0190] rAAV (e.g., 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 recombinant techniques known to those skilled in the art to include one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAV ITR sequences. Such AAV vectors typically retain at least one functional flanking ITR sequence, as required for rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will include sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0191] As used herein, the phrase "bona fide AAV vector" or "bona fide rAAV vector" refers to an AAV vector that contains heterologous nucleic acid capable of infecting target cells. The phrase excludes empty AAV vectors (no heterologous nucleic acid) and AAV vectors that do not contain a complete insert (e.g., a heterologous nucleic acid segment) or those AAV vectors that contain host cell nucleic acid.

[0192] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0193] Nucleic acids include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA).

[0194] Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can have any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0195] A "heterologous" nucleic acid sequence refers to a polynucleotide that is inserted into an AAV plasmid or vector for the purpose of vector-mediated transfer / delivery of the polynucleotide into a cell. A heterologous nucleic acid sequence is different from, i.e., non-natural relative to, an AAV nucleic acid. Once transferred / delivered into a cell, the heterologous nucleic acid sequence contained in the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous polynucleotide transferred / delivered in the cell contained in the vector does not need to be expressed. Although the term "heterologous" is not always used herein when referring to nucleic acid sequences and polynucleotides, even in the absence of the modifier "heterologous", a reference to a nucleic acid sequence or polynucleotide is meant to include heterologous nucleic acid sequences and polynucleotides, even if omitted.

[0196] "Transgene" is used herein for convenience to refer to a nucleic acid that is to be introduced or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. The terms transgene and heterologous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0197] In cells with a transgene, the transgene has been introduced / transferred via a plasmid or AAV vector, and the cell is "transduced" or "transfected." The terms "transduction" and "transfection" refer to the introduction of a molecule, such as a nucleic acid, into a host cell (e.g., HEK293) or a cell or organ of an organism. The transgene may or may not be integrated into the genomic nucleic acid of the recipient cell.

[0198] "Nucleic acid," "polynucleotide," "heterologous nucleic acid," "transgene," and "reduced CpG nucleic acid sequence" include the full-length sequence as well as functional subsequences, so long as the subsequence retains some functionality of the full-length sequence. Nucleic acid, polynucleotide, heterologous nucleic acid, transgene, and reduced CpG nucleic acid sequence.

[0199] "Polypeptides," "proteins," and "peptides" encoded by a "nucleic acid sequence," such as a heterologous nucleic acid sequence, include the full-length sequence of the naturally occurring protein as well as functional subsequences, modifications, or sequence variants, so long as the subsequence, modification, or variant retains some functionality of the full-length protein. Such polypeptides, proteins, and peptides encoded by a nucleic acid sequence may, but need not, be identical to an endogenous protein that is defective or underexpressed or absent in the treated mammal.

[0200] For example, "host cell" indicates a microorganism, yeast cell, insect cell, and mammalian cell that can be used as or has been used as a recipient of an AAV vector plasmid, an AAV helper structure, an auxiliary function vector, or other transfer DNA. The term includes the progeny of the original cell that has been transfected. Therefore, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It should be understood that the progeny of a single parent cell may not necessarily have exactly the same morphology or genome or total DNA complement as the original parent cell due to natural, accidental, or deliberate mutations. Exemplary host cells include human embryonic kidney (HEK) cells, such as HEK293.

[0201] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell refers to a genetic change in a cell after an exogenous molecule, such as a nucleic acid (e.g., a transgene), has been incorporated into the cell. Thus, a "transduced" cell is a cell or its progeny into which an exogenous nucleic acid has been introduced. These cells can be propagated (cultured), and the introduced protein can be expressed or the nucleic acid can be transcribed, or a vector such as rAAV can be produced by the cell. For gene therapy uses and methods, the transduced cells can comprise an organ or tissue and, in turn, can be within a subject.

[0202] As used herein, the terms "stable" or "stably integrated" in reference to a cell mean that a nucleic acid sequence, such as a selectable marker or a heterologous nucleic acid sequence, or a plasmid or vector has been inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.), or is maintained extrachromosomally in a recipient cell or host organism, and has remained chromosomally or extrachromosomally for a period of time.

[0203] A "cell line" refers to a cell population capable of continuous or prolonged growth and division in vitro under appropriate culture conditions. A cell line may, but need not, be a clonal population derived from a single progenitor cell. In a cell line, spontaneous or induced changes may occur in the karyotype during storage or transfer of such a clonal population, as well as during long-term passage in tissue culture. Therefore, daughter cells derived from a cell line may not be exactly identical to the parent cell or culture. An exemplary cell line suitable for the purification method of the present invention is HEK293.

[0204] "Expression control element" refers to a nucleic acid sequence that affects the expression of an operably linked nucleic acid. Control elements include expression control elements as described herein, such as promoters and enhancers. The rAAV vector may include one or more "expression control elements". Typically, such elements are included to promote appropriate heterologous polynucleotide transcription and (if appropriate) translation (e.g., one or more of promoters, enhancers, intronic splicing signals, maintenance of the appropriate reading frame of genes that permit in-frame translation of mRNA, and termination codons). Such elements typically act in cis, referred to as "cis-acting" elements, but may also act in trans.

[0205] Expression control can be achieved at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements (e.g., CpG-reduced TTR, ApoE / hAAT, FGG, albumin, and SAA1 promoters, and fusions / hybrids thereof) can be positioned adjacent to the transcribed sequence or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500 or more nucleotides from the polynucleotide), or even at a considerable distance from the 5' or 3' end. However, due to the length limitations of rAAV vectors, expression control elements will typically be within 1 to 1000 nucleotides of the transcribed nucleic acid.

[0206] Functionally, the expression of an operably linked nucleic acid can be at least partially controlled by an element (e.g., a promoter, an enhancer, etc.) such that the element regulates the transcription of the nucleic acid and, optionally, the translation of the transcript. A specific embodiment of an expression control element is a promoter, which is typically located 5' to the transcribed sequence. A promoter typically increases expression from the operably linked nucleic acid compared to the amount expressed in the absence of the promoter (if present).

[0207] Examples of promoters include TTR and ApoE / hAAT promoters, including CpG-reduced versions and hybrid forms of the TTR promoter disclosed herein. Other examples of promoters include ApoE / hAAT, FGG, albumin, and SAA1 promoters, including CpG-reduced versions and hybrid forms thereof.

[0208] As used herein, "enhancer" may refer to a sequence that is positioned adjacent to a nucleic acid sequence such as a heterologous nucleic acid sequence. An enhancer element is typically located upstream (5') of a promoter element and acts, and may also be located downstream (3') of a sequence or within a sequence. Thus, an enhancer element may be located upstream or downstream, for example, within 100 base pairs, 200 base pairs, or 300 or more base pairs of a heterologous nucleic acid such as a selectable marker and / or encoding a therapeutic protein or polynucleotide sequence. An enhancer element typically increases the expression of an operably linked nucleic acid above that provided by the promoter element.

[0209] The term "operably linked" means that regulatory sequences necessary for expression of a nucleic acid sequence are placed in appropriate positions relative to the sequence to achieve expression of the nucleic acid sequence. This same definition is sometimes applied to the configuration of a nucleic acid sequence and transcriptional control elements (e.g., promoters, enhancers, and termination components) in an expression vector (e.g., rAAV vector).

[0210] In embodiments where an expression control element is operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two DNA sequences operably linked means that the two DNA sequences are arranged in a relationship (cis or trans) such that at least one of the two DNA sequences is able to exert a regulatory effect on the other sequence.

[0211] Thus, additional elements of the vector include, but are not limited to, expression control (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) flanking sequences (e.g., heterologous sequences) such as one or more copies of AAV ITR sequences, or introns.

[0212] Other elements include, for example, filler or stuffer polynucleotide sequences, such as to improve packaging and reduce the presence of foreign nucleic acids. AAV vectors typically accept insertions of DNA ranging in size from about 4kb to about 5.2kb or slightly more. Therefore, for shorter sequences, filler or stuffer are included so that the length is adjusted to the normal size of the acceptable viral genome sequence of the vector being packaged into the rAAV particle. In certain embodiments, the filler / stuffer nucleic acid sequence is an untranslated (non-protein coding) fragment of nucleic acid. For nucleic acid sequences less than 4.7Kb, filler or stuffer polynucleotide sequences have a total length between about 3.0-5.5Kb or about 4.0-5.0Kb or about 4.3-4.8Kb when merging with the sequence (for example, inserting into the vector).

[0213] When the wild-type heterologous nucleic acid or transgene is too large to be packaged within an AAV vector particle, the heterologous nucleic acid can be provided in a modified, fragmented, or truncated form for packaging in and delivery by an AAV vector so as to ultimately provide a functional protein or nucleic acid product, such as a therapeutic protein or nucleic acid product.

[0214] In certain embodiments, the heterologous nucleic acid encoding a protein (eg, a therapeutic protein) will be provided in a modified or truncated form, or the heterologous nucleic acid will be provided in multiple structures, delivered by separate and multiple AAV vectors.

[0215] In certain embodiments, the heterologous nucleic acid is provided as a truncated variant that maintains the function of the encoded protein (e.g., a therapeutic protein), including removal of portions not necessary for function, such that the size of the encoding heterologous polynucleotide is reduced for packaging in an AAV vector.

[0216] In certain embodiments, the heterologous nucleic acid will be provided in split AAV vectors, each of which provides nucleic acid encoding a different portion of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of the protein (e.g., a therapeutic protein) that assemble and function in the cell.

[0217] In certain embodiments, the heterologous nucleic acid is provided by dual AAV vectors using overlapping, trans-splicing, or hybrid trans-splicing dual vector technology. In certain embodiments, two overlapping AAV vectors that are combined in a cell are used to generate a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0218] The terms "identity", "homology" and grammatical variations thereof mean that two or more of the entities mentioned are identical when they are "aligned" sequences. Thus, for example, when two polypeptide sequences are identical, they have the same amino acid sequence at least in the region or portion mentioned. When two polynucleotide sequences are identical, they have the same polynucleotide sequence at least in the region or portion mentioned. Identity can be within a defined region (region or domain) of a sequence. A "region" or "region" of identity refers to a portion of two or more of the entities mentioned that are identical. Thus, when two protein or nucleic acid sequences are identical in one or more sequence regions or regions, they share identity within that region. An "aligned" sequence refers to a plurality of polynucleotide or protein (amino acid) sequences that typically contain corrections for missing or extra bases or amino acids (gaps) compared to a reference sequence.

[0219] In some embodiments, the length of the sequence sharing the percentage of identity is 2, 3, 4, 5 or more adjacent nucleic acids or amino acids, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 adjacent nucleic acids or amino acids. In some embodiments, the length of the sequence sharing the percentage of identity is 21 or more adjacent nucleic acids or amino acids, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 adjacent nucleic acids or amino acids. In some embodiments, the length of the sequence sharing the percentage of identity is 41 or more adjacent nucleic acids or amino acids, for example, 42, 43, 44, 45, 46, 47, 48, 49, 50 adjacent nucleic acids or amino acids. In certain embodiments, the length of sequences that share identity is 50 or more contiguous nucleic acids or amino acids, e.g., 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. contiguous nucleic acids or amino acids.

[0220] As described herein, nucleic acid variants of CpG-reduced promoters, such as those including hybrid forms thereof, will differ from the wild type but may exhibit sequence identity to the wild type promoter. In CpG-reduced promoters, including hybrid forms thereof, at the nucleotide sequence level, the CpG-reduced activator will typically be at least about 70% identical, more typically at least about 75% identical, and even more typically about 80%-90% identical to the wild type activator. For example, the CpG-reduced promoter may have 70%-99% identity to the wild type promoter. Thus, the CpG-reduced promoter may have 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-99%, or 75%-99% identity to the wild type promoter.

[0221] At the amino acid sequence level, variants such as variant FVIII or hFVIII-BDD proteins will be at least about 70% identical, more typically about 75% identical, or about 80% identical, even more typically about 85% identical, or about 90% or more identical to the reference sequence. In certain embodiments, variants such as variant FVIII or hFVIII-BDD proteins have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the reference sequence of, for example, a wild-type FVIII protein with or without the B domain.

[0222] The terms "homologous" or "homology" mean that two or more referenced entities share at least partial identity within a given region or portion. A "region, area, or domain" having homology or identity means that a portion of two or more referenced entities share homology or are identical. Thus, when two sequences are identical within one or more sequence regions, they share identity in these regions. "Substantial homology" means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structure or function (e.g., biological function or activity) of a reference molecule or a related / corresponding region or portion of a reference molecule with which it shares homology.

[0223] The degree of consistency (homology) between two sequences or " percent consistency " can be determined using computer programs and / or mathematical algorithms. For purposes of the present invention, the comparison of nucleotide sequences is performed using the 9.1 version GCG Wisconsin Package available from the Genetics Computer Group (Madison, Wisconsin). For convenience, the default parameters specified by the program (gap formation penalty=12, gap extension penalty=4) are intended to be used herein for comparing sequence identity. Alternatively, the gap alignment with default parameters is compared using the National Center for Biotechnology Information (National Center for Biotechnology Information) (can be found on the world wide web ncbi.nlm.nih.gov / blast / ; Altschul et al., 1990, J Mol Biol 215:403-410) Blastn 2.0 program provided can be used to measure consistency and similarity between nucleotide sequences and amino acid sequences. For polypeptide sequence comparisons, the BLASTP algorithm is often used in combination with a scoring matrix such as PAM100, PAM250, BLOSUM62, or BLOSUM50. 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 structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0224] In certain embodiments, a "therapeutic protein" is a peptide or protein that can alleviate or reduce symptoms caused by insufficient amounts, absence, or deficiency of a protein in a cell or subject. A "therapeutic" protein encoded by a transgene can provide benefit to a subject, for example, to correct a genetic defect, to correct a gene (loss of expression or function), or the like.

[0225] For example, and without limitation, heterologous nucleic acids suitable for use in accordance with the present invention for encoding gene products (e.g., therapeutic proteins) include those that are useful for treating diseases or disorders including, but not limited to, the following: "hemostasis" or blood coagulation (bleeding) disorders, such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, hemophilia B patients with inhibitory antibodies, any of the blood clotting factors: VII, VIII, IX, X, XI, V, XII, II, von Willebrand Factor, IX, XII, XIII ... Deficiency of the factor (FV / FVIII), combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; anemia; bleeding, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) associated with trauma or injury; excessive anticoagulation associated with heparin, low molecular weight heparin, pentoses, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors); and platelet disorders such as Bernard Soulier syndrome, Glanz mannthrom basthenia, and storage pool deficiency. In certain embodiments, the subject suffers from a blood coagulation disorder. In certain embodiments, the subject has hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any of the following coagulation factors: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency.

[0226] In certain embodiments, the subject has a disease or disorder including, for example, but not limited to, a lung disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), a thalassemia, a blood disorder (e.g., anemia), a neurodegenerative disorder (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS)), a neurological disorder (e.g., epilepsy), a lysosomal storage disease (e.g., aspartyldiabetes, Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), glycogen storage disease type III (GSDIII; Corinomyces disease), disease));Monosialoganglioside 2 (GM2)-gangliosidosis type I (Tay Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (salivary gland diseases I and II), mucolipidosis type II (I-cell disease), mucolipidosis type III (pseudo-Hurler disease), and mucolipidosis type IV, mucopolysaccharidoses (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Maroteaux-Lamy and Sly diseases) disease), Niemann-Pick disease types A / B, C1, and C2, and Schindler disease types I and II), inflammatory disorders (e.g., hereditary angioedema (HAE)), copper or iron accumulation disorders (e.g., Wilson's or Menkes disease), lysosomal acid lipase deficiency, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic diseases or disorders (e.g., glycogen storage disease, methylmalonic acidemia, ornithine transcarbamylase deficiency, hypophosphatsia, very long chain acylglycerol deficiency, β-CoA dehydrogenase deficiency (VLCAD), galactosemia), autoimmune diseases (e.g., multiple sclerosis, type I diabetes, celiac disease, neuromyelitis optica (NMO), immune thrombocytopenia (ITP), idiopathic thrombocytopenic purpura), Addison's disease, myasthenia gravis), solid organ diseases (e.g., brain, liver, kidney, heart) or infectious viral (e.g., hepatitis B and C, human immunodeficiency virus (HIV), etc.), bacterial or fungal diseases.

[0227] In certain embodiments, the subject suffers from 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 hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's 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).

[0228] Apolipoprotein E (ApoE) is the main cholesterol carrier involved in lipid transport and brain damage repair. It shows that human ApoE isoforms differently affect the clearance or synthesis of amyloid-β (Aβ) in vivo. The epsilon4 (ε4) allele of ApoE is associated with an increased risk of Alzheimer's disease (AD), and the presence of the ApoE ε2 allele appears to reduce the risk of AD and is a protective ApoE isoform. As used herein, the term "protective ApoE isoform" refers to an ApoE isoform that reduces one or more symptoms or indications (e.g., physical, physiological, biochemical, histological, behavioral) of Alzheimer's disease. Protective ApoE isoforms also refer to ApoE isoforms that can reduce the risk of Alzheimer's disease by at least 5% (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more).

[0229] In certain embodiments, the present invention provides methods for delivering a protective ApoE isoform (e.g., ApoE ε2) to the CNS of a subject (e.g., a mammal) by delivering or administering to a non-CNS cell, organ, or tissue of the subject (e.g., not to the cerebrospinal fluid (CSF) or the brain).

[0230] In certain embodiments, rAAV particles comprising an AAV capsid protein and a vector comprising a nucleic acid encoding a protective ApoE isoform (e.g., ApoE ε2) are inserted between paired AAV inverted terminal repeats (ITRs) in a manner effective to transduce non-CNS cells (e.g., liver cells) in a subject (e.g., a mammal), such that the non-CNS cells (e.g., liver cells) secrete the protective ApoE isoform into the systemic circulation (vascular structures or 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, such as brain parenchyma).

[0231] In certain embodiments, the present invention provides vectors, expression cassettes, or nucleic acids encoding protective ApoE isoforms (eg, ApoE e2) that are expressed in the liver or liver cells.

[0232] In certain embodiments, the heterologous nucleic acid encodes a protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor (TGF), fibroblast growth factor ( ... Growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activins, inhibins, bone morphogenic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophins, lectins, axon guidance factor-1 and axon guidance factor-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0233] In certain embodiments, the heterologous nucleic acid encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor alpha and beta, interferon alpha, beta and gamma, stem cell factor, flk-2 / flt3 ligand, 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 and class II MHC molecules.

[0234] In certain embodiments, the heterologous nucleic acid encodes CFTR (cystic fibrosis transmembrane regulator protein), blood coagulation (clotting) factors, (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, beta-globulin, alpha-globulin, spectrin, alpha-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, beta-25 glucocerebrosidase, sphingomyelinase, Lysosomal hexuronidase dehydrogenase, branched-chain ketoacid dehydrogenase, hormone, growth factor, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor alpha and beta, cytokines, interferon alpha, interferon beta, interferon gamma, 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 protein, tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Heber-Lindau) Hippel-Lindau (VHL), adenomatous polyposis coli (APC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroideremia), LCA5 (LCA-lebercilin), ornithine ketoate aminotransferase (gyratory atrophy), retinoschisis 1 (X-linked retinoschisis), USH1C (Usher's Syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, Niemann-Pick Cl (NPC1), VPC2, saposin, one or more zinc finger nucleases for genome editing, and one or more donor sequences for use as repair templates for genome editing.

[0235] In certain embodiments, the protein encoded by the 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 effector nuclease (TALEN). In certain embodiments, the gene editing nuclease comprises a functional type II CRISPR-Cas9.

[0236] Other heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) that can be used with the present invention and that can optionally be expressed in the liver or liver cells (e.g., hepatocytes) and provide benefits include, for example, but are not limited to: GAA (acid alpha-glucosidase) for the treatment of Pompe disease; ATP7B (copper-transporting ATPase 2) for the treatment of Wilson's disease; alpha-galactosidase (GLA) for the treatment of Fabry disease; ASS1 (arginine succinate synthetase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of Gaucher disease type 1; beta-hexosaminidase A for the treatment of Tay-Sachs disease; SERPING1 (C1 protease inhibitor; C1 esterase inhibitor (C1EI)) for the treatment of hereditary angioedema (HAE); glucose-6-phosphatase for the treatment of glycogen storage disease type 1 (GSDI); and glucose-6-phosphatase for the treatment of glycogen storage disease type III (GSD). III; Koch 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-α, interferon-β, and interferon-γ for the treatment of various immune disorders, viral infections, and cancers; interleukins (ILs), including any of IL-1 to IL-36 and their corresponding receptors, for the treatment of various inflammatory diseases or immune deficiencies; chemokines, including chemokine (CXC motif) ligand 5 (CXCL5), for the treatment of immune disorders; and cytokines for the treatment of diseases such as Crohn's disease. disease); granulocyte colony-stimulating factor (G-CSF) for the treatment of various human inflammatory diseases; 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 chemoattractant 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); and mucopolysaccharidosis I (MPS) α-L-iduronidase for treating familial hypercholesterolemia (FH); ornithine transcarbamylase (OTC) for treating OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia lyase (PAL) for treating phenylketonuria (PKU); lipoprotein lipase for treating lipoprotein lipase deficiency; apolipoprotein for treating apolipoprotein (Apo) AI deficiency; low-density lipoprotein receptor (LDL-R) for treating familial hypercholesterolemia (FH); albumin for treating hypoalbuminemia; lecithin cholesterol transacylase (LCAT); carbamyl synthetase I;Arginine succinate synthetase; arginine succinate resolvase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine P-synthase for the treatment of homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl-CoA carboxylase; methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; liver phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; and dystrophin.

[0237] In certain embodiments, the subject has an autoimmune disease or disorder (e.g., multiple sclerosis, anti-MAG peripheral neuropathy, type 1 diabetes, Graves' disease, rheumatoid arthritis, proteoglycan-induced arthritis (PGIA), or myasthenia gravis); an allergy or an allergic disease.

[0238] Mature myelin oligodendrocyte glycoprotein (MOG) is associated with the lipid bilayer. MOG is characterized by an IgV-like extracellular domain, a single bypass transmembrane protein, a membrane-associated domain, and a cytoplasmic tail. The extracellular IgV-like domain is herein referred to as micro-MOG (mMOG). MOG is primarily found in the membranes of oligodendrocytes and contributes little to the final composition of the myelin sheath. Autoimmune responses to MOG have been linked to the development and etiology of multiple sclerosis.

[0239] In certain embodiments, the therapeutic protein is a fusion protein comprising an undesired antigen and a leader sequence for cellular secretion.

[0240] In certain embodiments, the therapeutic protein is a fusion protein comprising the extracellular domain of MOG or a fragment thereof and a leader sequence for cellular secretion.

[0241] In certain embodiments, the expression cassette comprises regulatory elements operably linked to a nucleic acid encoding a fusion protein comprising an undesired antigen and a leader sequence for secretion into a cell.

[0242] In certain embodiments, the undesirable antigen comprises an autoantigen, a self-antigen, or a protein or peptide that has structural similarity or sequence identity to an autoantigen or a self-antigen. In certain embodiments, the protein or peptide that has structural similarity or sequence identity to an autoantigen or a self-antigen is a microbial protein or peptide. In certain embodiments, the undesirable antigen comprises an allergen.

[0243] In certain embodiments, the allergen comprises a plant, insect, or animal allergen. In certain embodiments, the undesired antigen comprises myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), proteolipid protein (PLP), or a subsequence thereof.

[0244] In certain embodiments, MOG lacks all or a portion of its transmembrane domain. In certain embodiments, MOG comprises or consists of amino acids 1-117 of mature MOG. In certain embodiments, a subsequence of MOG is a subsequence of its extracellular domain or a subsequence of its transmembrane domain. In certain embodiments, MOG comprises or consists of amino acids 35-55, 118-132, 181-195, or 186-200 of mature MOG. In certain embodiments, MOG comprises or consists of amino acids 1-20, 11-30, 21-40, 31-50, etc. of mature MOG.

[0245] In certain embodiments, the present invention provides a method for suppressing, reducing or inhibiting a cell-mediated or antibody-mediated immune response to an undesired antigen in a mammal. In a certain embodiment, the method comprises providing an expression cassette, particle, pharmaceutical composition or LNP composition as described herein; and administering to the mammal an amount of the expression cassette, particle, pharmaceutical composition or LNP composition, wherein the fusion protein is expressed in the mammal sufficient to suppress, reduce or inhibit a cell-mediated or antibody-mediated immune response to the undesired antigen.

[0246] In certain embodiments, the present invention provides methods for inducing tolerance to an undesirable antigen in a mammal. In certain embodiments, the method comprises providing an expression cassette, particle, pharmaceutical composition, or LNP composition as described herein; and administering to the mammal an amount of the expression cassette, particle, pharmaceutical composition, or LNP composition, wherein expression of the fusion protein in the mammal is sufficient to induce tolerance to the undesirable antigen.

[0247] In certain embodiments, the present invention provides methods of treating a subject (e.g., a human) in need of a fusion protein. In certain embodiments, the method comprises providing an expression cassette, particle, or pharmaceutical composition or LNP composition as described herein; and administering to the subject (e.g., a human) an amount of the expression cassette, particle, pharmaceutical, or LNP composition, wherein the fusion protein is expressed in the subject (e.g., a human).

[0248] 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.

[0249] In certain embodiments, the subject (eg, human) has multiple sclerosis, anti-MAG peripheral neuropathy, type 1 diabetes, Grave's disease, rheumatoid arthritis, proteoglycan-induced arthritis (PGIA), or myasthenia gravis.

[0250] As used herein, "unwanted antigens" are self-antigens or autoantigens that can induce, provide, enhance and / or stimulate immune tolerance to the antigen itself or a protein comprising all or a portion of the antigen and / or contain, inhibit, reduce and / or alleviate the immune response to the antigen itself or a protein comprising all or a portion of the antigen. Unwanted antigens as used herein also include allergens or allergenic antigens that can induce, provide, enhance and / or stimulate immune tolerance to allergens and allergens and allergenic antigens that contain, inhibit, reduce and / or alleviate the immune response to allergens or entities comprising allergens.

[0251] As described herein, undesirable antigens also include alloantigens or transplant antigens or trace tissue-compatible antigens, which can cause rejection of cells, tissues or organs after they are transplanted into a subject. Subjects typically recognize transplanted cells, tissues or organs as foreign cells, tissues or organs and develop an immune response against the cells, tissues or organs. Therefore, the methods of the present invention are directed to preventing or reducing rejection of cells, tissues or organs after they have been transplanted into a subject.

[0252] While not wishing to be bound by any theory or specific mechanism, it is believed that undesirable antigens act by binding to or activating T regulatory cells (Tregs), thereby preventing, curbing, inhibiting, reducing, alleviating, or otherwise downregulating the immune response. This binding to or activation of Tregs can in turn lead to immune tolerance to self-antigens or autoantigens.

[0253] As used herein, a "leader" sequence is an amino acid sequence that, when attached to a protein, provides for or promotes secretion of a connexin from a cell in which the protein is expressed. A leader sequence, as used herein, may also be referred to as a secretory sequence. Such leader and secretory sequences are intended to provide for or promote cellular secretion, but may not always promote secretion if they are attached to a protein that has a signal sequence that prevents protein secretion.

[0254] In certain embodiments, the undesired antigen comprises an autoimmune disease protein or a subsequence thereof. Autoimmune disease proteins include any antigen (such as a protein, a subsequence thereof, or a peptide) that contributes to the initiation and / or progression of an autoimmune disease. Because the sequence or structure of a protein from another organism mimics a self-antigen or autoantigen, such autoimmune disease proteins can be derived from other organisms, such as microorganisms.

[0255] In certain embodiments, the autoimmune disease protein is myelin oligodendrocyte glycoprotein (MOG, e.g., for multiple sclerosis), myelin basic protein (MBP, e.g., for multiple sclerosis), proteolipid protein (PLP, e.g., for multiple sclerosis), myelin associated glycoprotein (MAG, e.g., for anti-MAG peripheral neuropathy), insulin (e.g., for type 1 diabetes), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, e.g., for type 1 diabetes), preproinsulin (e.g., for 1 diabetes mellitus), glutamic acid decarboxylase (GAD, e.g., for type 1 diabetes mellitus), tyrosine phosphatase autoantigen (e.g., for type 1 diabetes mellitus), insulinoma antigen-2 (e.g., for type 1 diabetes mellitus), islet cell antigen (e.g., for type 1 diabetes mellitus); thyroid stimulating hormone (TSH) receptor (e.g., for Grave's disease), thyroid stimulating hormone receptor (e.g., for Grave's disease), chondroitin sulfate proteoglycan 1 (e.g., for rheumatoid arthritis), CD4+ T cell epitope (e.g., GRVRVNSAY (SEQ ID NO: 98)), (e.g., for proteoglycan-induced arthritis (PGIA) or rheumatoid arthritis) or acetylcholine receptor (e.g., for myasthenia gravis).

[0256] In certain embodiments, the autoimmune disease protein is a mammalian myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), proteolipid protein (PLP), or a subsequence thereof. In some embodiments, the autoimmune disease protein is a human protein, such as human myelin basic protein (MBP), human proteolipid protein (PLP), human myelin oligodendrocyte glycoprotein (MOG), or a subsequence thereof.

[0257] Other heterologous nucleic acids suitable for use in encoding gene products according to the present invention include, for example, but are not limited to, reporters or detectable markers such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein, cyan fluorescent protein, enhanced GFP, enhanced YFP, photoactivated GFP, Discosoma species fluorescent protein (dsRed), mFruit, mCherry, TagRFP, eqFP611, photoswitchable fluorescent proteins (e.g., Dronpa and EosFP), chloramphenicol acetyltransferase, Halo tag fusion proteins, alkaline phosphatase, horseradish peroxidase, and β-galactosidase.

[0258] In certain embodiments, the heterologous nucleic acid comprises inhibitory DNA or encodes 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.

[0259] In certain embodiments, the heterologous nucleic acid encodes an inhibitory nucleic acid. In certain embodiments, the inhibitory nucleic acid is selected from the group consisting of: siRNA, antisense molecules, miRNA, RNAi, ribonucleases, and shRNA. In certain embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease selected from the group consisting of: huntingtin (HTT) gene, a gene associated with dentatorubral pallidum Lewy body atrophy (atrophin 1, ATN1), androgen receptor on chromosome X for spinobulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Ca v2.1P / Q pressure-sensitive calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 opposite chain (ATXN8OS), serine / threonine protein phosphatase 2A55 kDa regulatory subunit Bβ isoform in spinocerebellar disorders (types 1, 2, 3, 6, 7, 8, 12, 17), FMR1 (Fragile Mental Retardation 1) in fragile X syndrome, FMR1 (Fragile Mental Retardation 1) in fragile X-associated tremor / dysregulation syndrome, FMR1 (Fragile Mental Retardation 2) in XE fragile mental retardation, or AF4 / FMR2 family member 2; myotonic protein kinase (MT-PK) in myotonic dystrophy; Friedreich's ataxia ataxia; mutants of the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene in HIV infection; HIV TAR, human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, delayed graft function kidney in acute kidney injury or renal transplantation. transplant) or acute renal failure with renal injury; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta type 9 (PSMB9), in metastatic melanoma; LMP7, also known as proteasome subunit beta type 8 (PSMB8), in metastatic melanoma; MECL1, also known as proteasome subunit beta type 10 (PSMB10), in metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin Axin in solid tumors, inhibitor of apoptosis B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase (PLK) in liver tumors. kinase) 1 (PLK1), diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, β-catenin in familial adenomatous polyposis; β2-adrenergic receptor, glaucoma; RTP801 / Reddl, also known as DNA damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration;Vascular endothelial growth factor receptor 1 (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase-2 in non-arteritic ischemic optic neuropathy; keratin 6A N17K mutant protein in congenital hyperonychia; influenza A virus genome / gene 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; genes (allele-specific silencing) in primary dystonia: silencing of the pathogenic allele of torsin A (TOR1A); pan-class I specific in transplantation. I) and HLA- alleles; and mutations in the rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP).

[0260] Nucleic acid molecules, vectors (such as clones, expression vectors (e.g., vector genomes)), and plasmids can be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information enables the preparation of nucleic acid molecules in a variety of ways. For example, heterologous nucleic acids comprising vectors or plasmids can be prepared using various standard cloning, recombinant DNA techniques via cellular expression or in vitro translation, and chemical synthesis techniques. The purity of a polynucleotide can be determined by sequencing, gel electrophoresis, and the like. 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 features, such as using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers that can anneal to the nucleic acid sequence of interest; (4) computer searching of sequence databases for related sequences; and (5) differential screening of subtracted nucleic acid libraries.

[0261] Nucleic acids of the present invention can be maintained as DNA in any suitable cloning vector. In certain embodiments, the clone is maintained in a plasmid cloning / expression vector such as pBluescript or pBluescriptII (Stratagene, LaJolla, CA), which is propagated in suitable Escherichia coli (E. coli) host cells. Alternatively, the nucleic acid can be maintained in a vector suitable for expression in mammalian cells.

[0262] Methods known in the art for producing rAAV virions include, for example, transfection using an AAV vector and an AAV helper sequence in combination with co-infection using one or more AAV helper viruses (e.g., adenovirus, herpes virus, or vaccinia virus) or transfection using a recombinant AAV vector, an AAV helper vector, and a helper function vector. Methods for producing rAAV virions are described, for example, and not limited to, in U.S. Patent Nos. 6,001,650 and 6,004,797. Following recombinant rAAV vector production (i.e., production of the vector in a cell culture system), rAAV virions can be obtained from host cells and cell culture supernatants and purified as described herein.

[0263] Methods for determining the infectious titer of rAAV vectors containing a transgene are known in the art (see, e.g., Zhen et al., (2004) Hum. Gene Ther. (2004) 15:709). Methods for detecting empty capsids and AAV vector particles with packaged genomes are also known (see, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128).

[0264] To determine degraded / denatured capsids, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, such as a gradient gel, and then the gel is run until the sample is separated and blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein (see, e.g., Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. The binding between the primary and secondary antibodies is semi-quantitatively detected to determine the amount of capsid.

[0265] rAAV vectors and other compositions, agents, drugs, and biological agents (proteins) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly suitable for administration and delivery to a subject in vivo or ex vivo.

[0266] The term "isolated" when used as a modifier of a composition means that the composition is artificially prepared or completely or at least partially separated from its naturally occurring in vivo environment. Typically, an isolated composition is substantially free of one or more materials with which it is normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0267] The term "isolated protein" or "isolated and purified protein" is sometimes used herein with respect to proteins. The term primarily refers to a protein produced by expressing a nucleic acid molecule. Alternatively, the term may refer to a protein that has been sufficiently separated from other proteins with which it is naturally associated so as to be in a "substantially pure" form.

[0268] The term "isolated" does not exclude compositions or artificially prepared compositions herein, for example, rAAV and / or pharmaceutical preparations. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrids / chimeras, multimers / oligomers, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in artificially prepared host cells.

[0269] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the target compound (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75% by weight, or about 90-99% by weight of the target compound. Purity is determined by methods appropriate to the target compound (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0270] When referring to a particular nucleotide sequence or amino acid sequence, the phrase "consisting essentially of" refers to a sequence that possesses the characteristics of the given sequence. For example, when referring to a nucleic acid or amino acid sequence, the phrase encompasses the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence.

[0271] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the subject receiving the composition and that can be administered without undue toxicity.

[0272] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to biologically acceptable formulations, whether gaseous, liquid, solid, or mixtures thereof, that are suitable for one or more routes of administration, in vivo delivery, or exposure. A "pharmaceutically acceptable" or "physiologically acceptable" composition is one that is not biologically or otherwise undesirable, e.g., one that can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, in the administration of nucleic acids, vectors, viral particles, or proteins to a subject.

[0273] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars, and ethanol. Pharmaceutically acceptable salts may also be included, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, and the like may be present in such vehicles.

[0274] Pharmaceutical compositions may be provided as salts and may be formed using a variety of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like.

[0275] Salts tend to be more soluble in water or other protic solvents than the corresponding free base forms. In other cases, the formulation can be a lyophilized powder that can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol at a pH range of 4.5 to 5.5, combined with a buffer prior to use.

[0276] 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, dispersants and suspending media, coatings, isotonic and absorption-promoting or -delaying agents that are compatible with drug administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterials, antivirals and antifungals) may also be incorporated into the compositions.

[0277] The pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery as described herein or known to those skilled in the art. Thus, the pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0278] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions of the active compound, which preparations are generally sterile and isotonic with the blood of the intended recipient. Illustrative examples include, for example, but not limited to, water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal oils, vegetable oils, or synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as, for example, but not limited to, sodium carboxymethylcellulose, sorbitol, or polydextrose.

[0279] Alternatively, suspensions of the active compound may be prepared as oily injection suspensions as desired. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

[0280] Cosolvents and adjuvants can be added to the formulation, examples of which include, but are not limited to, those containing hydroxyl groups or other polar groups, for example, alcohols such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Examples of adjuvants include, but are not limited to, surfactants such as soy lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinyl pyrrolidone.

[0281] After the pharmaceutical composition is prepared, it can be placed in an appropriate container and labeled for treatment. Such labeling may include the amount, frequency, and method of administration.

[0282] Pharmaceutical compositions and delivery systems suitable for use in the compositions, methods and uses of the present invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003)20 th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990)18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996)12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001)11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, NY, pp. 253-315).

[0283] In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the present invention are delivered or administered via AAV vector particles. In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the present invention can be delivered or administered via other types of viral particles, including retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, Epstein-Barr virus, vaccinia virus, and human cytomegalovirus particles.

[0284] In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the invention are delivered or administered using a non-viral delivery system. Non-viral 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 bombardment, ultrasound, and magnetofection.

[0285] In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the invention are delivered in the form of naked DNA, minicircles, transposons, or capped linear duplex DNA.

[0286] In certain embodiments, the nucleic acids, polynucleotides, and expression cassettes of the invention are delivered or administered in the form of AAV vector particles or other viral particles that are further encapsulated or complexed using liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0287] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles that are suitable for delivering AAV and have nanoscale dimensions, i.e., about 10 nm to about 1000 nm or about 50 to about 500 nm or about 75 to about 127 nm. Without being bound by theory, it is believed that LNPs provide nucleic acids, polynucleotides, expression cassettes, or AAV vectors in the presence of partial or complete shielding of the immune system. Shielding enables delivery of nucleic acids, polynucleotides, expression cassettes, or AAV vectors to tissues or cells while avoiding inducing a substantial immune response to nucleic acids, polynucleotides, expression cassettes, or AAV vectors in vivo. Shielding also enables repeated administration without inducing a substantial immune response to nucleic acids, polynucleotides, expression cassettes, or AAV vectors in vivo (e.g., in a subject such as a human being). Shielding can also improve or increase delivery efficiency in vivo.

[0288] The pI (isoelectric point) of AAV is in the range of about 6 to about 6.5. Therefore, AAV carries a small amount of negative charge on its surface. Thus, for LNPs, it may be beneficial to include cationic lipids such as, for example, amino lipids. Exemplary amino lipids are described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, No. 8,999,351, No. 8,722,082, No. 8,642,076, No. 8,569,256, No. 8,466,122, and No. 7,745,651 and U.S. Patent Publication Nos. 2016 / 0213785, No. 2016 / 0199485, No.2015 / 0265708, No.2014 / 0288146, No.2013 / 0123338, No.2013 / 0116307, No.2013 / 0064894, No.2012 / 0172411, and No.2010 / 0117125

[0289] The terms "cationic lipid" and "amino lipid" are used interchangeably herein to include those lipids and salts thereof having one, two, three or more fatty acid or fatty alkyl chains and a pH titratable amino group (e.g., an alkylamino or dialkylamino group). Cationic lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and are positively charged at a pH above the pKa. P The cationic lipids are substantially neutral at a pH of 100 to 150 K. The cationic lipids may also be titratable cationic lipids. In certain embodiments, the cationic lipids comprise: a protonatable tertiary amine (e.g., pH titratable) group; a C18 alkyl chain, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and an ether bond, ester bond, or ketal bond between the head group and the alkyl chain.

[0290] Cationic lipids may include, but are not limited to, 1,2-dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 1,2-di-γ-linoleyl-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linoleyl-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA, also known as DLin-C2K- DMA, XTC2 and C2K), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleoylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yl ester (DLin-M-C3-DMA, also known as MC3), salts thereof 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)-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0291] Still other cationic lipids may include, but are not limited to, 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl 1,2-Dilinoleyl-3-dimethylaminopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(l-(2,3-dioleylamino)propane N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2, 3-Dioleyloxy-N-[2(spermine-formamido)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), dioctadecylamidoglycinylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentenyloxy)-3-dimethyl-1-(cis, cis- 9',1-2'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or a mixture thereof.

[0292] A variety of commercially available cationic lipid preparations can be used, such as (including DOTMA and DOPE, available from GIBCO / BRL) and (Contains DOSPA and DOPE, available from GIBCO / BRL).

[0293] In certain embodiments, the cationic lipid may be present in an amount from about 10% by weight of the LNP to about 85% by weight of the lipid nanoparticle or from about 50% by weight of the LNP to about 75% by weight of the LNP.

[0294] Sterol can give LNP mobility.As used herein, " sterol " refers to any naturally occurring sterol in plant (phytosterol) or animal (zoosterol) source and non-naturally occurring synthetic sterol, and it is all characterized by the existence of the hydroxyl at the 3-position of steroid A-ring.Sterol can be any sterol conventionally used in the field of liposome, lipid vesicle or lipid granule preparation, is most generally cholesterol.Phytosterol can comprise campesterol, sitosterol and stigmasterol.Sterol also comprises lipid modified through sterol, such as the lipid described in U.S. Patent application case disclosure 2011 / 0177156.In certain embodiments, sterol can exist with the amount of the LNP of about 5 % by weight to the lipid nanoparticle of about 50 % by weight or the LNP of about 10 % by weight to the LNP of about 25 % by weight.

[0295] LNP can comprise neutral lipids. Neutral lipids can be included in any lipid substance that exists in the form of uncharged or neutral zwitterions at physiological pH. Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. The selection of neutral lipids is generally guided by, in particular, considering particle size and necessary stability. In certain embodiments, the neutral lipid component can be a lipid with two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0296] Lipids with a variety of acyl chain groups of different chain lengths and degrees of saturation are available or can be separated or synthesized by known techniques. In certain embodiments, lipids containing saturated fatty acids and carbon chain lengths in the range of C14 to C22 can be used. In certain embodiments, lipids with mono- or di-unsaturated fatty acids and carbon chain lengths in the range of C14 to C22 are used. In addition, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or any related phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups (such as serine and inositol).

[0297] In certain embodiments, the neutral lipid may be present in an amount from about 0.1% by weight of the lipid nanoparticles to about 75% by weight of the LNPs or from about 5% by weight of the LNPs to about 15% by weight of the LNPs.

[0298] The LNP-encapsulated nucleic acid, expression cassette, and AAV vector can be incorporated into a pharmaceutical composition (e.g., a pharmaceutically acceptable carrier or excipient). Such pharmaceutical compositions are particularly suitable for administering and delivering the LNP-encapsulated nucleic acid, expression cassette, and AAV vector to a subject in vivo or ex vivo.

[0299] LNP formulations can be combined with additional components which may include, for example, but not limited to, polyethylene glycol (PEG) and sterols.

[0300] The term "PEG" refers to polyethylene glycol, a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight; for example, PEG 2000 has an average molecular weight of approximately 2,000 daltons, and PEG 5000 has an average molecular weight of approximately 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, but not limited to, the following functional PEGs: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-trifluoroethanesulfonate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0301] In certain embodiments, PEG can be a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted with an alkyl, alkoxy, acyl, or aryl group. In certain embodiments, PEG can be substituted with a methyl group at the terminal hydroxyl position. In certain embodiments, PEG can have an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons, or about 750 daltons. PEG can be optionally substituted with an alkyl, alkoxy, acyl, or aryl group. In certain embodiments, the terminal hydroxyl group can be substituted with a methoxy or methyl group.

[0302] PEG-modified lipids include, for example, but not limited to, PEG-dialkoxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397. Applicable PEG-modified lipids (or lipid-polyethylene oxide conjugates) may have multiple "anchor" lipid moieties to fix the PEG moiety on the surface of the lipid vesicle. Examples of suitable PEG-modified lipids include, for example, but not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) described in U.S. Patent No. 5,820,873, PEG-modified dialkylamines, and PEG-modified diacyloxypropan-3-amines. In certain embodiments, the PEG-modified lipids may be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, PEG may be in an amount from about 0.5% by weight of the LNP to about 20% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.

[0303] In addition, LNP can be PEG-modified and sterol-modified LNP. The LNP merged with the additional component can be identical or independent LNP. In other words, the identical LNP can be PEG-modified and sterol-modified, or alternatively, the first LNP can be PEG-modified and the second LNP can be sterol-modified. Optionally, the first and second modified LNPs can be merged.

[0304] In certain embodiments, prior to 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 75 nm to about 125 nm. In certain embodiments, the LNP-encapsulated nucleic acid, expression vector, or AAV vector may have a size in the range of about 10 nm to 500 nm.

[0305] An "effective amount" or "sufficient amount" refers to an amount that, in single or multiple doses, alone or in combination with one or more other compositions (therapeutic or immunosuppressive agents, e.g., drugs (e.g., prednisone)), treatments, protocols, or therapeutic regimen agents, provides a detectable response of any duration (long or short) in or to a subject, provides an expected or desired result or benefit to any measurable or detectable degree or for any duration (e.g., minutes, hours, days, months, years, or cured) in or to a subject.

[0306] The dosage may vary and depends on the type, onset, progression, severity, frequency, duration or likelihood of the disease being treated, the desired clinical endpoint, the subject's previous or concurrent treatment, general health, age, sex, race or immune competence, and other factors as will be understood by those skilled in the art. The dosage, amount, frequency or duration may be proportionally 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. Those skilled in the art will understand the factors that may affect the dosage and timing required to provide an amount sufficient to provide therapeutic or preventive benefit.

[0307] The dosage required to achieve a therapeutic effect, for example, expressed as vector genomes per kilogram of body weight (vg / kg), will vary based on several factors, including, but not limited to, the route of administration, the level of heterologous polynucleotide expression required to achieve a 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 skilled in the art can determine the rAAV / vector genome dosage range for treating a patient with a specific disease or condition based on these and other factors.

[0308] Typically, the dose will be at least 1 × 10 8 or higher vector genomes / kg subject body weight (vg / kg) (e.g., 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1×10 14 or higher vector genomes / kg subject body weight (vg / kg) to achieve therapeutic effect. The AAV dose for mice is 1×10 10 -1×10 11 vg / kg, and the AAV dose for dogs was 1×10 12 -1×10 13 The dose can be less, for example, less than 6×10 12 vector genomes / kilogram (vg / kg). More specifically, 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 dose.

[0309] For Pompe disease, an effective amount would be, for example, an amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, reduces lysosomal changes in a subject's body tissues, or improves muscle tone and / or muscle strength and / or respiratory function in a subject. For example, an effective amount can be determined by determining the kinetics of GAA uptake by myoblasts from plasma. A myoblast GAA uptake rate (K uptake) of about 141-147 nM can be considered effective (see, e.g., Maga et al., J. Biol. Chem. 2012). In animal models, GAA activity levels in plasma greater than about 1,000 nmol / hr / mL (e.g., about 1,000 to about 2,000 nmol / hr / mL) have been observed to be therapeutically effective.

[0310] Taking hemophilia B as an example, it is generally believed that to achieve a therapeutic effect, a clotting factor concentration greater than 1% of that found in normal subjects is required to change the severe disease phenotype to a moderate disease phenotype. The severe phenotype is characterized by joint damage and life-threatening bleeding. To convert the moderate disease phenotype to a mild disease phenotype, a clotting factor concentration greater than 5% of normal is believed to be required.

[0311] The diagnosis and disease severity classification of hemophilia A and B are based on the results of factor VIII and factor IX activity assays, respectively. The two main assays for assessing factor activity are an OSA based on the activated partial thromboplastin time (aPTT) and a chromogenic substrate assay (CSA) using an enzymatic chromogenic substrate reaction based on factor Xa. Such assays are well known in the art and are further described in Adcock et al., 2018, Int. J. Lab. Hem., 40: 621-629.

[0312] In normal humans, FVIII levels are approximately 150-200 ng / mL of plasma, but can be less (e.g., in the range of about 100-150 ng / mL) or more (e.g., in the range of about 200-300 ng / mL) and still be considered normal due to functional coagulation as determined, for example, by the aPTT one-stage clotting assay. Thus, a therapeutic effect can be achieved by expressing FVIII or hFVIII-BDD such that the total amount of FVIII in a subject / human is greater than 1% of the FVIII present in a normal subject / human, e.g., 1% of the 100-300 ng / mL.

[0313] The dose of rAAV vector can be at a level, generally at the lower end of the dose spectrum, such that there is no substantial immune response against FVIII or the AAV vector. More specifically, doses up to but less than 6 × 1012 vg / kg, for example, about 5×10 11 to about 5×10 12 vg / kg, or more specifically, about 5×10 11 vg / kg or about 1×10 12 vg / kg.

[0314] In certain embodiments, the rAAV vector dose is in an amount that delivers a safe and effective amount of FVIII and provides a therapeutic benefit to a subject with hemophilia A who has inhibitory antibodies against FVIII (hemophilia A with inhibitors).

[0315] An "effective amount" or "sufficient amount" of a dosage for treatment (e.g., to ameliorate or provide a therapeutic benefit or improvement) is generally effective to provide a measurable degree of response to one, more or all of the adverse symptoms, consequences or complications of the disease, one or more of which are, for example, caused by or associated with the disease, although reduction, diminution, inhibition, arrest, limitation or control of the progression or worsening of the disease is a desirable result.

[0316] An effective amount or sufficient amount may, but need not, be provided in a single administration, may require multiple administrations, and may, but need not, be administered alone or in combination with another composition (e.g., medicament), treatment, trial protocol, or therapeutic regimen. For example, the amount may be proportionally increased according to the needs of the subject, the type of disease being treated, the state, and the severity or side effects (if any) of the treatment. In addition, if administered in a single dose or multiple doses without a second composition (e.g., another drug or medicament), treatment, trial protocol, or therapeutic regimen, an effective amount or sufficient amount need not be effective or sufficient because an additional dose, quantity, or duration greater than or exceeding the dosage, or an additional composition (e.g., drug or medicament), treatment, trial protocol, or therapeutic regimen may be included to be considered effective or sufficient in a given subject. It is believed that an effective amount also includes an amount that results in a reduction in the use of another treatment, therapeutic regimen, or trial protocol (e.g., administering a recombinant coagulation factor protein (e.g., FVIII) for the treatment of coagulation disorders (e.g., hemophilia A or hemophilia A with inhibitory antibodies against FVIII, also referred to as hemophilia A with inhibitors)).

[0317] Therefore, the method and uses of the present invention also include methods and uses that result in reducing the need or use of another compound, medicament, medicine, treatment regimen, treatment trial scheme, method or therapy.For example, for blood coagulation diseases, if the frequency of administering recombinant coagulation factor protein is less or the dosage is reduced or eliminated in a given subject to supplement the lack or defective (abnormal or mutated) endogenous coagulation factor in the subject, then the method or uses of the present invention has therapeutic benefit.Therefore, according to the present invention, there is provided a method and uses that reduces the need or use of another treatment or therapy.

[0318] An effective or sufficient amount need not be effective in every treated subject, nor in the majority of treated subjects in a given group or population. An effective or sufficient amount is one that is effective or sufficient in a particular subject, not in a group or population in general. As is typical of such methods, some subjects may show a greater response, or a lesser or no response, to a given treatment method or use.

[0319] The term "improvement" refers to a detectable or measurable improvement in a subject's disease or its symptoms or underlying cellular responses. Detectable or measurable improvement includes subjectively or objectively reducing, lowering, inhibiting, suppressing, limiting or controlling the onset, frequency, severity, progression or duration of a disease, or complications caused by or associated with the disease, or ameliorating the symptoms, underlying causes or consequences of the disease, or reversing the disease. For hemophilia A, an effective amount would be, for example, an amount that reduces the frequency or severity of acute bleeding episodes in a subject, or, for example, an amount that reduces clotting time as measured by, for example, a coagulation assay.

[0320] Therefore, the pharmaceutical compositions of the present invention include compositions containing effective amounts of active ingredients to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capabilities of a skilled physician using the techniques and guidance provided herein.

[0321] The therapeutic dose will depend on, among other factors, the age and general condition of the subject, the severity of the abnormal phenotype, and the strength of the control sequences regulating expression levels. Thus, the therapeutically effective amount for humans will fall within a relatively wide range that can be determined by a physician based on the individual patient's response to vector-based therapy. Such doses may be alone or in combination with immunosuppressants or drugs.

[0322] Compositions such as pharmaceutical compositions can be delivered to a subject to allow for expression of the transgene and, optionally, production of the encoded protein. In certain embodiments, the pharmaceutical composition comprises sufficient genetic material to enable the recipient to produce a therapeutically effective amount of a blood coagulation factor to affect hemostasis in the subject.

[0323] The composition can be administered alone. In certain embodiments, the recombinant AAV particles provide a therapeutic effect in the absence of an immunosuppressant. The therapeutic effect optionally persists for a period of time, such as 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, or 30-50 days or longer, such as 50-75 days, 75-100 days, 100-150 days, 150-200 days or longer, without the need for administering an immunosuppressant. Therefore, in certain embodiments, the rAAV viral particles provide a therapeutic effect without the need for administering an immunosuppressant over a period of time.

[0324] The compositions of the present invention can be administered in combination with at least one other inert or therapeutic agent. In certain embodiments, before, substantially simultaneously or after the administration of rAAV vector, rAAV vector is co-administered with one or more immunosuppressants. In certain embodiments, after the administration of rAAV vector, for example 1-12, 12-24 or 24-48 hours, or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 or more than 50 days. If after rAAV vector, after the initial expression level for a period of time (for example 20-25, 25-30, 30-50, 50-75, 75-100, 100-150, 150-200 or more than 200 days) the protein expression encoded is reduced, then after a period of time after the administration of rAAV vector, immunosuppressants are administered.

[0325] 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 (cyclosporine) (e.g., cyclosporine A), mycophenolate mofetil, rituximab (rituximab), rapamycin or its derivatives. In certain embodiments, the reagent includes a stabilizing compound. Other immunosuppressants that can be used according to the present invention include, for example, but not limited to, B cell targeting antibodies, such as rituximab; proteasome inhibitors, such as bortezomib; mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin; tyrosine kinase inhibitors, such as ibrutinib; B cell activating factor (BAFF) inhibitors; and proliferation-inducing ligand (APRIL) inhibitors.

[0326] The composition can be administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose, and water. The composition can be administered to a patient alone or in combination with other agents that affect hemostasis (e.g., cofactors).

[0327] The methods and uses of the present invention include delivery and administration systemically, regionally or locally or by any route, such as, but not limited to, by injection or infusion. The delivery of the pharmaceutical composition in vivo can usually be accomplished by injection using a conventional syringe, although other delivery methods are envisioned, such as convection-enhanced administration (see, for example, U.S. Patent No. 5,720,720). For example, the composition can be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatically, via the portal vein or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application. For example, a clinician who specializes in treating patients with blood coagulation or coagulation factor disorders can determine the best way to administer adenovirus-associated vectors based on many criteria (including but not limited to: the patient's condition and therapeutic objectives (e.g., increasing GAA, enhancing or reducing blood coagulation, etc.)).

[0328] Therapeutic methods according to the present invention include combination therapies that include the additional use of any compound, agent, drug, treatment or other therapeutic therapy or one or more of the regimens having the desired therapeutic, beneficial, cumulative, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include, for example, but are not limited to, a second active agent, such as a biologic (protein), agent (e.g., immunosuppressant) and a drug. Such biologic (protein), agent, drug, treatment and therapy may be administered or performed prior to, substantially simultaneously with, or after any other therapeutic method according to the present invention, such as a method for treating a lysosomal storage disease such as Pompeii or a method for treating a blood coagulation disease such as HemA or HemB in a subject.

[0329] The compound, agent, drug, treatment or other therapeutic regimen or regimen can be administered as a combination composition or separately (such as in parallel or consecutively or sequentially) from the delivery or administration of a nucleic acid, vector, recombinant vector (e.g., rAAV) or recombinant viral particle (before or after). The present invention thus provides a combination of the methods of treatment according to the present invention and any compound, agent, drug, therapeutic treatment, treatment regimen, process, treatment or composition combination as described herein or known to those skilled in the art. The compound, agent, drug, therapeutic treatment, treatment regimen, process, treatment or composition can be administered or performed before, substantially simultaneously with or after the administration of a nucleic acid, vector, recombinant vector (e.g., rAAV) or recombinant viral particle administered to a patient or subject according to the present invention.

[0330] The present invention can be used for human and veterinary medical applications. Therefore, suitable subjects include mammals, such as humans, and non-human mammals. The term "subject" refers to an animal, typically a mammal, such as humans, non-human primates (ape, gibbon, gorilla (gorilla), chimpanzee (chimpanzee), orangutan (orangutan), macaque), livestock (dogs and cats), farm animals (poultry, such as chickens and ducks, horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, teenagers and adult subjects. Subjects include animal disease models, such as other animal models of mice and blood coagulation diseases (such as HemA and other diseases known to those skilled in the art).

[0331] The subject that is suitable for treatment according to the present invention includes having or being at the risk of producing insufficient amount or having insufficient functional gene product (for example GAA or blood coagulation factor, such as FVIII or FIX), or producing abnormal, partially functional or non-functional gene product (for example, GAA or blood coagulation factor, such as FVIII or FIX) that may cause disease.The subject that is suitable for treatment according to the present invention also includes those having or being at the risk of producing abnormal or defective (mutation) gene product (protein) that causes disease, so that reducing the quantity, expression or function of this abnormal or defective (mutation) gene product (protein) will lead to the treatment of disease, or alleviate one or more symptoms or improve disease.For example, target subject includes the subject that coagulation factor produces abnormal, insufficient or lacking, such as hemophiliac (for example, hemophilia A or hemophilia B); Or there is abnormal, insufficient or absent subject that GAA produces, such as the subject with Pompe disease.

[0332] Subjects include those that do not have detectable neutralizing antibodies against AAV. Subjects also include those that have neutralizing antibodies against AAV. Such subjects may have low titers of neutralizing antibodies against AAV.

[0333] The subject can be tested for an immune response, e.g., antibodies against AAV. Candidate subjects (e.g., hemophilia or Pompe disease subjects) can be screened prior to treatment according to the methods of the invention. The subject can also be tested for anti-AAV antibodies after treatment and, optionally, monitored for a period of time after treatment. Subjects who develop antibodies can be treated with an immunosuppressant (e.g., prednisone) or one or more additional amounts of the AAV vector can be administered.

[0334] A subject considered negative for antibodies that bind to AAV has a titer of less than 1:1. A subject with antibodies that bind to AAV may have a titer greater than 1:1 but less than 1:5. A subject may also have an AAV antibody titer equal to or greater than 1:5. For example, such an antibody titer can be calculated by performing a serial dilution of a blood, plasma, or serum (or other bodily fluid) sample from the subject, and the first dilution of the sample that inhibits AAV transduction by 50% or more, as measured by reporter gene activity in an in vitro cell-based assay, is reported as the antibody titer.

[0335] Strategies to reduce (overcome) or avoid humoral immunity to AAV in systemic gene transfer include administering higher vector doses, using empty AAV capsids as bait to absorb anti-AAV antibodies, administering immunosuppressive drugs to reduce, decrease, inhibit, prevent, or eradicate humoral immune responses to AAV, changing the AAV capsid serotype or engineering AAV capsids to be less susceptible to neutralizing antibodies, using plasma exchange cycles to absorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers, using delivery techniques such as balloon catheters followed by saline flushes (Mingozzi et al., 2013, Blood, 122:23-36), and immune absorption (U.S. Patent Application Publication No. US 2018 / 0169273 A1).

[0336] Subjects suitable for treatment according to the present invention also include those who have or are at risk of developing anti-AAV antibodies. Several techniques can be used to administer or deliver rAAV vectors to such subjects. For example, empty capsid AAV (i.e., AAV lacking a transgene) can be delivered to bind to AAV antibodies in the subject, thereby allowing the AAV vector carrying the nucleic acid or nucleic acid variant to transform the subject's cells.

[0337] The ratio of empty capsids to rAAV vectors can be between about 2:1 and about 50:1, or between about 2:1 and about 25:1, or between about 2:1 and about 20:1, or between about 2:1 and about 15:1, or between about 2:1 and about 10:1. The ratio can also be about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0338] The amount of empty capsid AAV to be 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, such as 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).

[0339] Alternatively or in addition, AAV vectors can be delivered by direct intramuscular injection (e.g., into the slow-twitch fibers of one or more muscles). In another alternative, a catheter introduced into the femoral artery can be used to deliver AAV vectors to the liver via the hepatic artery. Non-surgical methods, such as endoscopic retrograde cholangiopancreatography (ERCP), can also be used to deliver AAV vectors directly to the liver, thereby bypassing the blood and AAV antibodies. Other ductal systems, such as ducts of the submandibular gland, can also be used as ports for delivering AAV vectors to subjects who have developed or have pre-existing anti-AAV antibodies.

[0340] Administration or in vivo delivery to a subject can be performed before adverse symptoms, symptoms, complications, etc. caused by or associated with the disease occur. For example, screening (e.g., genetic) can be used to identify such subjects as candidates for the compositions, methods, and uses of the present invention. Thus, such subjects include those screened for insufficient or lacking a positive number of functional gene products (e.g., blood coagulation factors), or subjects that produce abnormal, partially functional, or non-functional gene products (e.g., blood coagulation factors).

[0341] Administration or in vivo delivery to a subject according to the methods and uses of the present invention disclosed herein can be implemented within 1-2, 2-4, 4-12, 12-24, or 24-72 hours after the subject has been identified as having a target disease, or having one or more symptoms of the disease, or even if the subject does not have one or more disease symptoms but has been screened and identified as positive as described herein. Of course, the methods and uses of the present invention can be implemented 1-7, 7-14, 14-21, 21-48, or more days, months, or years after the subject has been identified as having a target disease, or having one or more symptoms of the disease, or has been screened and identified as positive as described herein.

[0342] As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unit dosage for a subject to be treated; each unit contains a predetermined quantity, optionally associated with a pharmaceutical carrier (excipient, diluent, solvent or filler), which is calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms can be in, for example, ampoules and vials, which can include liquid compositions, or compositions in a freeze-dried or lyophilized state; sterile liquid carriers, for example, can be added prior to in vivo administration or delivery. Individual unit dosage forms can be contained in a multi-dose kit or container. Recombinant vector (e.g., rAAV) sequences, recombinant viral particles and pharmaceutical compositions thereof can be packaged as a single unit dosage form or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0343] The protein or activity level of the relevant gene product (e.g., GAA or blood coagulation factors, such as FVIII or FIX) of the subject can be tested to determine whether these subjects are suitable for treatment according to the method of the present invention. For example, the FVIII amount or activity of the candidate hemophilia A subject can be tested before treatment according to the method of the present invention; the GAA amount or activity of the candidate Pompeii subject can be tested before treatment according to the present invention. The FVIII or GAA protein amount or activity of the subject can also be tested after treatment according to the method of the present invention. The blood coagulation activity (for HemA) or GAA activity (for Pompeii) of the subject treated in this way can be monitored periodically (e.g., every 1-4 weeks, 1-6 months or 1, 2, 3, 4, 5 years or more) after treatment.

[0344] The adverse effects of one or more liver enzymes in a subject can be tested or to determine whether such subject is suitable for treatment according to the methods of the present invention. For example, the amount of one or more liver enzymes in a candidate hemophilia or Pompeii subject can be screened prior to treatment according to the methods of the present invention. The amount of one or more liver enzymes in a subject can also be tested after treatment according to the methods of the present invention. Subjects treated in this manner can be monitored periodically (e.g., every 1-4 weeks or 1-6 months) following treatment for elevated liver enzymes.

[0345] Exemplary liver enzymes include alanine transaminase (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 the circulation are generally defined as a range with an upper limit, above which the enzyme levels are considered elevated and, therefore, indicative of liver damage. Normal ranges depend in part on the standards used by the clinical laboratory performing the assay.

[0346] In certain embodiments, bleeding episodes in subjects with bleeding disorders can be monitored to determine whether such subjects meet the treatment conditions according to the present invention or respond to the treatment, and / or the amount or duration of the response. Bleeding episodes in subjects can be monitored to determine whether such subjects require additional treatment, such as subsequent AAV vector administration or administration of an immunosuppressant, or more frequent monitoring. Bleeding episodes in subjects with hemophilia can be monitored before and after treatment according to the methods of the present invention. The frequency and severity of bleeding episodes in subjects can also be tested during or after treatment according to the methods of the present invention.

[0347] In certain embodiments, subjects with Pompe disease or in need of GAA can be monitored by various tests, assays, and functional assessments to demonstrate, measure, and / or assess the therapeutic efficacy of GAA to determine whether such subjects are eligible for or responsive to treatment or require additional treatment according to the present invention.

[0348] The present invention provides kits having packaging materials and one or more components. The kits typically include a label or package insert that includes a description of the components or instructions for use of the components in vitro, in vivo, or ex vivo. The kits can include a collection of such components (e.g., nucleic acids, recombinant vectors, viral (e.g., AAV) vectors or viral particles) and optionally a second active substance (e.g., another compound, agent, drug, or composition).

[0349] Kit refers to the physical structure that houses one or more components of the kit. The packaging material can sterilely hold the components and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0350] The label or insert may include identification information of one or more components, dosage, clinical pharmacology of the active ingredient, including mechanism of action, pharmacokinetics, and pharmacodynamics. The label or insert may include information identifying the manufacturer, batch number, place of manufacture and date, and expiration date. The label or insert may include information identifying the manufacturer, batch number, manufacturer's address, and date. The label or insert may include information about the disease for which the kit components may be used. The label or insert may include instructions for a clinician or subject to use one or more kit components in a method, use, or treatment plan or therapeutic regimen. The instructions may include dosage, frequency, or duration, as well as instructions for implementing any method, use, treatment plan, or prevention or treatment regimen described herein.

[0351] The label or insert may include information about any benefits (e.g., prophylactic or therapeutic benefits) that the components may provide. The label or insert may include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which a particular composition is not suitable. Adverse side effects or complications may also occur when the subject has, will, or is taking one or more other drugs that may be incompatible with the composition, or when the subject has, will, or is receiving another treatment regimen or treatment plan that may be incompatible with the composition, and therefore, the instructions may include information about such incompatibilities.

[0352] The label or insert includes "printed matter", such as paper or cardboard, either alone or attached to a component, a kit or packaging material (such as a box), or attached to an ampoule, test tube or vial containing a kit component. The label or insert may additionally include a computer-readable medium, such as a barcode printed label, a magnetic disk, an optical disk (such as CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape) or an electronic storage medium, such as RAM and ROM or a hybrid thereof, such as a magnetic / optical storage medium, a FLASH medium or a memory type card.

[0353] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0354] All patents, patent applications, publications, and other references cited herein, including GenBank references and ATCC references, are incorporated herein by reference in their entirety. In the event of a conflict, the specification, including definitions, will control.

[0355]

[00146] Various terms are used above and throughout the specification and claims in connection with the biomolecules of the present invention.

[0356] All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, the disclosed features (e.g., CpG-reduced) nucleic acids, vectors, plasmids, expression / recombinant vector (e.g., rAAV) sequences or recombinant viral particles are examples of classes of equivalent or similar features.

[0357] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids, reference to "a vector" includes a plurality of such vectors, and reference to "a virus" or "a particle" includes a plurality of such viruses / particles.

[0358] As used herein, all values ​​or ranges of values ​​include integers within the range and fractions of the values ​​or integers within the range, unless the context clearly dictates otherwise. Thus, for example, reference to 80% or greater identity 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., and so on.

[0359] Reference to an integer greater than (larger than) or less than includes any number greater than or less than the referenced number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc., up to the number 1 (1); and reference to less than 10 includes 9, 8, 7, etc., up to the number 1 (1).

[0360] As used herein, unless the context clearly indicates otherwise, all values ​​or ranges include values ​​and fractions of integers within the range and fractions of integers within the range. Thus, for example, a reference to a numerical range such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, and so on. Thus, a reference to a range of 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and so on, up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, and so on.

[0361] Reference to a series of ranges includes ranges that combine the boundaries of the different ranges within the series. Thus, examples of reference to a series of ranges are, for example, 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, including 1-20, 1-30, 1-40, 1-50, 1-60, 10-30, 10-40, 10-50, 10-60, , 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, 150-350, 150-400, 150-450, 150-500, etc.

[0362] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments of the present invention. The present invention also specifically includes embodiments in which specific subject matter is excluded in whole or in part, such as substances or materials, method steps and conditions, designs or procedures. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Thus, even though the present invention is not generally described herein as not being included, aspects that are not explicitly excluded from the present invention are disclosed herein.

[0363] A number of embodiments of the present invention have been described. However, without departing from the spirit and scope of the present invention, those skilled in the art may make various changes and modifications to the present invention to adapt it to various uses and conditions. Therefore, the following examples are intended to illustrate and not to limit the scope of the present invention in any way.

[0364] Example

[0365] Example 1 :method

[0366] hFVIII ELISA for NHP plasma: 96-well plates were coated with human-specific FVIII antibodies overnight, washed, and blocked before incubation with diluted NHP study subject plasma samples. The standard curve was generated by adding additional wells of 5% dapoxetine (Pfizer). The plates were washed and then incubated with a biotinylated human-specific FVIII detection antibody. The plates were incubated with horseradish peroxidase (HRP)-conjugated streptavidin, treated with TMB substrate, and read on a microplate reader to determine absorbance at 450 nm.

[0367] hFVIII ELISA for mouse plasma: The hFVIII ELISA for mouse plasma was performed essentially as described above for NHPs; however, the capture and detection antibodies utilized were different.

[0368] Cell-based assay for measuring the potency of AAV vectors encoding the human FVIII transgene: Huh7 cells were plated at 5×10 4 Cells / well were seeded in a 48-well plate overnight. After the study, a 10-fold dose curve (MOI range of 1×10 6 -1×10 3 ) and the diluted vector for administration. The existing culture medium was removed from the Huh7 cells and replaced with the medium containing the viral particles. The cells were maintained at 37°C and 5% CO2 for 72 hours, and the supernatant was collected and stored in a low retention microtiter plate at -80°C until the hFVIII activity was determined. The supernatant was used to generate a recombinant B domain-deleted hFVIII, (Pfizer) diluted into cell growth medium to generate a standard curve SP4 factor VHI (Chromogenix) was used for assay.

[0369] Cell-based assay for measuring protein expression efficiency of plasmid encoding human FVIII transgene: Huh7 cells were cultured in DMEM + 10% FBS + penicillin / streptomycin / L-glutamine at 5×10 4 Cells / well were seeded in a 48-well plate overnight. Plasmids were prepared using the Plasmid Giga Kit (Qiagen) and transfected into cells at 250 ng / well using Polyethylenimine (PEI) Max. The cells were maintained at 37°C and 5% CO2 for 72 hours, and the supernatant was collected and stored in a low-retention microtiter plate at -80°C until the hFVIII activity was assayed. The supernatant was used to generate hFVIII with a recombinant B domain deleted hFVIII, (Pfizer) diluted into cell growth medium to generate a standard curve SP4 Factor VIII (Chromogenix) was used for the assay.

[0370] Example 2

[0371] The FIX structural 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. In total, this unit contains 16 CpGs.

[0372] Design of CpG-reduced promoters

[0373] The cytosine or guanine of the CpG site is changed depending on the consensus sequence. If no potential transcription factor binding site is found, the cytosine (C) of the CpG dinucleotide is replaced by thymine (T). Thus, the pyrimidine-purine structure is maintained. In some cases, C nucleotides or the entire CpG dinucleotide are deleted, and in some cases, the guanine (G) of the CpG dinucleotide is replaced by alanine (A) or C.

[0374] Using this strategy, 22 different sequences were generated based on the ApoE / hAAT regulatory element.Sequences with and without 5' and 3' flanking restriction enzyme sites are illustrated below in SEQ ID NOs: 24-67.

[0375] clone

[0376] Different promoters were synthesized and cloned upstream of the codon-optimized sequence encoding hFIX (SEQ ID NO: 94).

[0377] Mouse studies

[0378] The efficacy of the human alpha-1 antitrypsin (hAAT) gene promoter was assessed by hydrodynamic delivery of plasmid constructs in 8-week-old male wild-type C57BL / 6 mice (Jackson Laboratories). Non-fasting plasma samples were collected with heparin via submandibular blood collection 24 hours after plasmid administration. Plasma was placed on ice and stored at -80°C until analysis. All animal studies were performed according to institutional guidelines and approved protocols.

[0379] Effectiveness Research

[0380] The collected plasma was used to assess hFIX transgene expression.

[0381] The activity level of human FIX was measured by activated partial thromboplastin time (aPTT) assay. The aPTT assay was performed by mixing sample plasma with human FIX-deficient plasma (George King Bio-Medical, Inc.) and aPTT reagent (Trinity Biotech) in a 1:1:1 volume ratio, followed by an 180-second incubation period at 37°C. Coagulation was initiated by adding 25mM calcium chloride. The time for clot formation was measured using a Start 4 coagulation instrument (Diagnostica Stago). A standard curve was generated using pooled normal plasma (George King Bio-Medical, Inc.) with a 1:5 dilution of TBS pH 7.4 (48 μL + 192 μL) followed by a serial 1:2 dilution (120 μL + 120 μL). The human standard curve was used to calculate the activity of each sample at week 17 after AAV vector administration; activity in two untreated mice was also measured. FIX activity in untreated mice was averaged and then subtracted from treated samples to calculate the additional (i.e., human) activity attributable to exogenous FIX protein. Figure 1 middle.

[0382] Example 3

[0383] The FVIII structural regulatory element unit (SEQ ID NO: 2 and SEQ ID NO: 3) consists of a 225 bp TTR promoter. In total, this unit contains 4 CpGs.

[0384] Design of CpG-reduced TTR promoter

[0385] The cytosine or guanine residues at CpG sites were altered based on the consensus sequence. If no potential binding site was found, the cytosine residues of the CpG dinucleotides were replaced with thymine residues. This maintained the pyrimidine-purine structure. Using this strategy, five novel sequences were generated based on the TTRm (SEQ ID NO: 3) regulatory element. CpG-reduced TTR sequences with and without restriction enzyme sites are described in SEQ ID NOs: 4-13.

[0386] Another set of four different shorter TTR hybrid promoters was designed. In silico simulations were performed to assess the presence of putative transcription factor binding sites within 1000 nucleotides of the transcription start site (TSS) of the native genes of five different liver-specific promoters. Subsequently, TTR hybrid promoters were assembled by selecting specific regions from the original native promoters and assembling these specific regions in tandem. TTR hybrid sequences with and without restriction enzyme sites are illustrated below in SEQ ID NOs: 14-21.

[0387] clone

[0388] Different promoters were synthesized and cloned upstream of the codon-optimized nucleotide sequence encoding hFVIII-BDD (SEQ ID NO: 77).

[0389] Mouse studies

[0390] The efficacy of the TTR promoter was initially assessed by hydrodynamic delivery in 8-week-old male wild-type C57BL / 6 mice (Jackson Laboratories). Non-fasting plasma samples were collected with heparin via submandibular blood collection 24 hours after plasmid administration. 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 performed according to institutional guidelines and approved protocols.

[0391] hFVIII antigen content in mouse plasma

[0392] The level of hFVIII transgene product in mouse plasma was quantified using a sandwich ELISA as follows: First, each well of a microtiter plate was coated with anti-hFVIII capture antibody (Green Mountain Antibodies, diluted to 2 μg / mL). The next day, the plate was washed four times and blocked (6% BSA, 0.2% Tween 20 in PBS) for 30 minutes at room temperature. Pooled mouse plasma was spiked with known concentrations of recombinant B-domain deleted hFVIII (XYNTHA). ) and serially diluted (1:2) to generate an 8-point standard curve ranging from 300 ng / mL to 2.34 ng / mL. The limit of quantification of the assay was 4.8 ng / mL. Three levels of quality control samples were prepared and included on each plate to assess assay performance. After adding samples to each well, the plate was incubated at 37°C for 1 hour and then washed four times. A 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 bind to the captured hFVIII protein. After washing, a peroxidase-conjugated streptavidin reagent (Thermo Fisher Scientific) at a 1:5000 dilution was added to the plate at room temperature for 30 minutes to bind to the biotinylated anti-hFVIII antibody. After washing the plate to remove unbound conjugated antibody, peroxidase activity was revealed following a 15-minute incubation with 3,3',5,5'-tetramethylbenzidine substrate (TMB) at room temperature. The reaction was stopped with TMB stop solution and the plate was read for optical density (OD) by an absorbance plate reader. The absorbance value obtained is proportional to the concentration of hFVIII present in the plasma sample. The data show that Figure 2-5 middle.

[0393] RNA isolation and qPCR

[0394] Brain, testis, kidney, spleen and liver mouse tissues were collected, rinsed with DPBS, cut / minced into multiple small fragments, and about 30 mg was used for RNA isolation as described in the kit protocol (RNeasy plus universal mini kit, Qiagen). RNA concentration was measured using a Nanodrop 2000 instrument, and the sample was diluted to 150 ng / gL in a nuclease-free state. DNase treatment was performed using a Turbo DNA kit (Invitrogen) according to the manufacturer's instructions. For cDNA reactions, 200 ng RNA was used as directed in the High Capacity cDNA Reverse Transcription Kit (ABI). The cDNA sample was diluted 5 times, and 20 ng cDNA was used for PCR reactions. Quantitative real-time PCR was performed using the following: 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). The murine actB (Integrated DNA Technologies) gene served as a housekeeping gene for normalization. Data are shown in Figure 6 middle.

[0395] Example 4

[0396] Factor VIII expression was increased by altering components within the expression cassette that facilitate transgene expression. An intron-less version of hFVIII expressing the B domain deleted from an AAV vector (AAV-INTL) was generated and compared to an intron-containing version (AAV-WINT). In addition, the AAV-WINT hFVIII had a synthetic intron (SEQ ID NO: 93) positioned between the TTRm promoter and the transgene encoding the B domain deleted human Factor VIII ( Figure 7 ), the AAV-INTL hFVIII expression cassette (SEQ ID NO: 1) contains the same components as the AAV-WINT hFVIII expression cassette.

[0397] Example 5: Efficacy in mice

[0398] To evaluate the efficacy of an intronless cassette (intronless TTRm hFVIII; SEQ ID NO: 1) relative to an intron-containing cassette (TTRm hFVIII) in mammals, approximately 8-week-old male C57BL / 6 mice (Jackson Laboratories) were injected intravenously in the lateral tail vein with the AAV encapsidation cassette at a dose of 6.4e9 or 1.6e10 vg / mouse. Plasma was collected at several time points, as indicated ( Figure 8 and 9 ), and circulating hFVIII levels were assessed by hFVIII ELISA.

[0399] Determination of hFVIII content showed that the potency of TTRm hFVIII without introns was significantly increased compared to TTRm hFVIII (with introns) ( Figure 8 and 9 ), and this effect was seen at all doses and time points tested (Study #1). These results were replicated in a subsequent study (Study #2) with 10 mice in each group using a dose of 1.6e10 vg / mouse ( Figure 10 Study #2 confirmed that intronless TTRm hFVIII (AAV-INTL) was more effective than a vector containing a synthetic intron (AAV-WINT), and these differences persisted for at least 8 weeks.

[0400] Example 6: Efficacy in NHPs, Study 1

[0401] Comparison of AAV vector efficacy of AAV-INTL and AAV-WINT in NHPs (Study #1). Twelve male cynomolgus macaques (Macaca fascicularis) aged between 24 and 50 months, weighing between 2-6 kg, and negative for AAV neutralizing antibodies were divided into four randomized groups and injected intravenously with a single dose of AAV-WINT or AAV-INTL based on the dose groups shown in Table 1. Subsequently, plasma samples were obtained weekly to determine circulating hFVIII levels.

[0402] Table 1. Group names and dose levels from the NHP study

[0403] Groups# Number of animals (male) Dose level (vg / kg) Dose concentration (vg / kg) 1(AAV-WINT low) 3 <![CDATA[2.0×10 12 ]]> <![CDATA[2.0×10 11 ]]> 2 (AAV-WINT High) 3 <![CDATA[6.0×10 12 ]]> <![CDATA[6.0×10 11 <!-- 43 -->]]> 3 (AAV-INTL low) 3 <![CDATA[2.0×10 12 ]]> <![CDATA[2.0×10 11 ]]> 4 (AAV-INTL high) 3 <![CDATA[6.0×10 12 ]]> <![CDATA[6.0×10 11 ]]>

[0404] The hFVIII levels in the plasma of monkeys dosed with AAV-WINT or AAV-INTL were determined by ELISA at weekly intervals throughout the 8-week study. At any dose tested in this study, hFVIII levels of 2e12 vg / kg ( Figure 11 ) or 6e12vg / kg( Figure 12), regardless of time point, circulating hFVIII levels were observed to increase 2- to 4-fold based on peak circulating values. As expected, we observed decreased expression 2-3 weeks after treatment, indicating the emergence of inhibitory antibodies against BDD hFVIII. The results of this first study demonstrate that AAV-INTL exhibits increased efficacy relative to AAV-WINT in NHPs.

[0405] Example 7 : Efficacy in NHPs, Study 2

[0406] A second study (Study #2) was conducted in NHPs to confirm the increased vector efficacy of AAV-INTL relative to AAV-WINT. Ten male cynomolgus macaques (Macaca fascicularis) aged between 24 and 50 months, weighing between 2-6 kg, and negative for AAV neutralizing antibodies were divided into two randomized groups and injected intravenously with a single dose (2e12 vg / kg) of AAV-WINT or AAV-INTL. Subsequently, plasma samples were obtained weekly to determine circulating hFVIII levels.

[0407] The hFVIII levels in the plasma of macaques dosed with AAV-WINT or AAV-INTL were determined by ELISA at weekly intervals throughout the 8-week study. At the dose tested in this study, 2e12 vg / kg, a 4- to 7-fold increase in circulating hFVIII levels based on peak circulating values ​​was observed regardless of time point ( Figure 13 As previously seen in NHPs, decreased transgene expression was observed 2-3 weeks after treatment due to the development of inhibitory antibodies against BDDhFVIII. The results of Study #2 confirmed that AAV-INTL showed increased efficacy relative to AAV-WINT in NHPs.

[0408] Example 8: Determination of carrier efficacy

[0409] To confirm that the appropriate vector was administered to each group of NHPs at the appropriate concentration, the dose formulation titer was determined by qPCR, and the presence or absence of the synthetic intron in the stock vector was determined by a genotyping PCR assay that allows for the distinction between AAV-WINT and AAV-INTL. To directly assess the vector potency of both the undiluted stock vector and the 2e12 vg / kg dose formulation, a cell-based potency assay was utilized.

[0410] Human liver cells were transduced with serial dilutions of vector, and potency was determined by assessing secreted BDD hFVIII in the supernatant using a hFVIII activity assay (Chromogenix Coatest SP4). AAV-INTL exhibited increased potency relative to AAV-WINT at all MOIs ( Figure 14Notably, the stock virus and diluted formulations showed similar potency within the vector group, further confirming that the titer of the dose formulation in the 2e12 vg / kg group was properly prepared. Additionally, AAV-INTL showed a roughly 4-fold increase in potency when compared to AAV-WINT at each MOI ( Figure 15 These values ​​are consistent with the increased potency of AAV-INTL observed in NHP studies 1 and 2.

[0411] Example 9 : Batch comparison

[0412] The first and second NHP studies used different batches of AAV-WINT and AAV-INTL vectors. To assess whether vector potency was comparable across batches, potency was measured using an in vitro assay ( Figure 16 The results of these comparisons showed that the variation between batches was minimal, and AAV-INTL was still similarly about 4- to 5-fold more potent than AAV-WINT.

[0413] Example 10 : Determination of expression cassette efficiency

[0414] To explore the mechanism of increased potency in vivo, transcription efficiency was determined in the absence of viral transduction. Human liver cell lines were transfected with plasmids containing expression cassettes constituting AAV-WINT and AAV-INTL, TTRm-intron-BDD-hFVIII, and intronless TTRm-BDD-hFVIII (SEQ ID NO: 1), respectively. Supernatants from these cells were assayed for hFVIII content by a human FVIII activity assay (Chromogenix Coatest SP4).

[0415] Comparison of three independent DNA preparations of TTRm-intron-BDD-hFVIII with two independent DNA preparations of intronless TTRm-BDD-hFVIII (SEQ ID NO: 1) showed similar hFVIII content, and a trend towards decreased expression upon intron removal ( Figure 17 While not wishing to be bound by any theory, the data suggest that non-transcriptional mechanisms are driving the increased potency of AAV-INTL over AAV-WINT.

[0416] Example 11 :Data discussion

[0417] At equivalent doses, AAV-INTL demonstrated increased potency and expression of BDD-hFVIII compared to AAV-WINT in cell culture, mouse, and NHP models. Mechanistically, the increased potency was not clearly due to increased transcription of the FVIII transgene from the expression cassette, but rather may be due to increased viral packaging efficiency or alternative mechanisms. These results indicate that the intronless expression cassette may have increased potency in human clinical trials and provide benefits to patient safety and efficacy.

[0418] Example 12: Human clinical trial results

[0419] A single dose study was conducted in four males with hemophilia A (N=4), as summarized in Table 4. All four participants received 5×10 11 Vaccines were dosed at 400 vg / kg with a single infusion of the AAV-INTL hFVIII-BDD expression cassette (SEQ ID NO: 1) encapsidated in the LK03 AAV vector (SEQ ID NO: 91), referred to herein as "LK03-INTL hFVIII-BDD."

[0420] Table 4.

[0421]

[0422] The LK03-INTL hFVIII-BDD vector was found to drive FVIII expression in all four participants ( Figure 18-19 ).

[0423] Example 13: Sequence

[0424] Table 2. SEQ ID NO and description

[0425]

[0426]

[0427]

[0428]

[0429] The entire nucleic acid sequence of the AAV-INTL expression cassette (5' ITR, TTRm, hFVIII-BDD, PolyA, and 3' ITR) (SEQ ID NO: 1) and the legend (Table 3):

[0430]

[0431] Table 3. Characteristics of SEQ ID NO: 1

[0432]

[0433] Wild-type TTR promoter (SEQ ID NO: 2). The following four underlined nucleotides are changed in the mutant TTR promoter (SEQ ID NO: 3).

[0434] gtgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatt tgtg taggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctg

[0435] Mutated TTR promoter (4 nucleotide changes; underlined) "TTRm" (SEQ ID NO: 3):

[0436] gtgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatt gact taggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctg

[0437] Nucleic acid sequence of CpG1-TTRm (SEQ ID NO: 4). In CpG1, for all four CpGs, each C was changed to a T (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 5 is CpG1-TTRm with these restriction enzyme sites (underlined).

[0438] SEQ ID NO:4:

[0439]

[0440] SEQ ID NO:5:

[0441]

[0442] The nucleic acid sequence of CpG2-TTRm (SEQ ID NO: 6). In CpG2, the Cs in the second, third, and fourth CpGs are changed to Ts (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 7 is CpG2-TTRm with these restriction enzyme sites (underlined).

[0443] SEQ ID NO:6:

[0444]

[0445] SEQ ID NO:7:

[0446]

[0447] The nucleic acid sequence of CpG3-TTRm (SEQ ID NO: 8). In CpG3, the Cs in the first, third, and fourth CpGs are changed to Ts (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 9 is CpG3-TTRm with these restriction enzyme sites (underlined).

[0448] SEQ ID NO:8:

[0449]

[0450] SEQ ID NO:9:

[0451]

[0452] The nucleic acid sequence of CpG4-TTRm (SEQ ID NO: 10). In CpG4, the Cs in the first, second, and fourth CpGs are changed to Ts (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 11 is CpG4-TTRm with these restriction enzyme sites (underlined).

[0453] SEQ ID NO: 10:

[0454]

[0455] SEQ ID NO:11:

[0456]

[0457] The nucleic acid sequence of CpG5-TTRm (SEQ ID NO: 12). In CpG5, the Cs in the first, second, and third CpGs are changed to Ts (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. SEQ ID NO: 13 is CpG5-TTRm with these restriction enzyme sites (underlined).

[0458] SEQ ID NO:12:

[0459]

[0460] SEQ ID NO:13:

[0461]

[0462] Nucleic acid sequence of the Hybrid6 promoter (TTR / hAAT / albumin hybrid) (SEQ ID NO: 14). In a single CpG dinucleotide, the G was changed to an A (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcg) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. (Italics = TTR, underlined = albumin, bold = hAAT.) SEQ ID NO: 15 is the Hybrid6 promoter with these restriction enzyme sites.

[0463]

[0464] Nucleic acid sequence of the Hybrid7 promoter (TTR / hAAT hybrid) (SEQ ID NO: 16). In two CpG dinucleotides, the Cs were changed to Ts (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcg) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. (Italics = TTR, bold = hAAT.) SEQ ID NO: 17 is the Hybrid7 promoter with these restriction enzyme sites.

[0465]

[0466] Nucleic acid sequence of the Hybrid8 promoter (TTR / FGG (fibrinogen gamma chain gene promoter) / albumin promoter hybrid) (SEQ ID NO: 18). In a single CpG dinucleotide, the G is changed to an A (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and PmeI (gtttaaac) restriction sites were located at the 5' and 3' ends, respectively. (Underline = albumin, underlined italics = FGG, italics = TTR.) SEQ ID NO: 19 is the Hybrid8 promoter with these restriction enzyme sites.

[0467]

[0468] Nucleic acid sequence of the Hybrid9 promoter (TTR / FGG / hAAT / SAAl hybrid) (SEQ ID NO: 20). In all three CpG dinucleotides, the Cs were changed to Ts (double underlined). When the sequence was cloned into the FVIII expression cassette, MluI (acgcgt) and Pmel (tttaaac) restriction sites were located at the 5' and 3' ends, respectively. (Italic = TTR, bold = hAAT, underlined italic = FGG, underlined bold = SAA1.) SEQ ID NO: 21 is the Hybrid9 promoter with these restriction enzyme sites.

[0469]

[0470] Nucleic acid sequence of the non-CpG reduced ApoE / hAAT regulatory element (SEQ ID NO: 22). The total number of CpGs contained in the sequence is double underlined. The C / EBP (CCAAT / enhancer-binding protein) site is underlined.

[0471]

[0472] Nucleic acid sequence of the non-CpG reduced ApoE / hAAT regulatory unit flanked by Apal restriction sites at the 5' and 3' ends (SEQ ID NO: 23). The sequence contains a total of 16 CpGs (double underlined). The Apal restriction site was used when cloning the sequence into the FIX expression cassette.

[0473]

[0474] The nucleic acid sequence of CpG1-ApoE / hAAT (SEQ ID NO: 24) is shown. Each C was changed to a T, except for the seventh CpG where a G was changed to an A. When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 25 is CpG1-ApoE / hAAT with these restriction enzyme sites (underlined).

[0475]

[0476] The nucleic acid sequence of CpG2-ApoE / hAAT (SEQ ID NO: 26) was unchanged, with the first CpG remaining unchanged and the G changed to an A in the seventh CpG. The unchanged "c"s are in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 27 is CpG2-ApoE / hAAT with these restriction enzyme sites (underlined).

[0477]

[0478] The nucleic acid sequence of CpG3-ApoE / hAAT (SEQ ID NO: 28) was unchanged, with the second CpG remaining unchanged and the G in the seventh CpG changed to an A. Each C was changed to a T. Unchanged "c"s are in bold. When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 29 is CpG3-ApoE / hAAT with these restriction enzyme sites (underlined).

[0479]

[0480] The nucleic acid sequence of CpG4-ApoE / hAAT (SEQ ID NO: 30) was unchanged, with the third CpG remaining unchanged and the G changed to an A in the seventh CpG. The unchanged "c"s are in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 31 is CpG4-ApoE / hAAT with these restriction enzyme sites (underlined).

[0481]

[0482] The nucleic acid sequence of CpG5-ApoE / hAAT (SEQ ID NO: 32) was obtained. Each C was changed to a T, except that the fourth CpG remained unchanged and the G was changed to an A in the seventh CpG. The unchanged CpG (the fourth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 33 is CpG5-ApoE / hAAT with these restriction enzyme sites (underlined).

[0483]

[0484] The nucleic acid sequence of CpG6-ApoE / hAAT (SEQ ID NO: 34) was obtained. Each C was changed to a T, except that the fifth CpG remained unchanged and the G was changed to an A in the seventh CpG. The unchanged CpG (the fifth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 35 is CpG6-ApoE / hAAT with these restriction enzyme sites (underlined).

[0485]

[0486] The nucleic acid sequence of CpG7-ApoE / hAAT (SEQ ID NO: 36) is shown. The sixth CpG remains unchanged, and the G in the seventh CpG is changed to an A. Each C is changed to a T. The unchanged CpG (the sixth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 37 is CpG7-ApoE / hAAT with these restriction enzyme sites (underlined).

[0487]

[0488] The nucleic acid sequence of CpG8-ApoE / hAAT (SEQ ID NO: 38) is shown. Each C was changed to a T, except for the seventh position, which remained unchanged. The unchanged CpG (the seventh) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 39 is CpG8-ApoE / hAAT with these restriction enzyme sites (underlined).

[0489]

[0490] The nucleic acid sequence of CpG9-ApoE / hAAT (SEQ ID NO:40) was obtained. The eighth CpG remained unchanged and the G in the seventh CpG was changed to an A, with each C changed to a T. The unchanged CpG (the eighth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO:41 is CpG9-ApoE / hAAT with these restriction enzyme sites (underlined).

[0491]

[0492] The nucleic acid sequence of CpG10-ApoE / hAAT (SEQ ID NO:42) was unchanged, with the ninth CpG remaining unchanged and the seventh CpG being changed from G to A. Each C was changed to a T. The unchanged CpG (ninth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO:43 is CpG10-ApoE / hAAT with these restriction enzyme sites (underlined).

[0493]

[0494] The nucleic acid sequence of CpG11-ApoE / hAAT (SEQ ID NO: 44) is shown. The tenth CpG remains unchanged, and the G in the seventh CpG is changed to an A. Each C is changed to a T. The unchanged CpG (the tenth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 45 is CpG11-ApoE / hAAT with these restriction enzyme sites (underlined).

[0495]

[0496] The nucleic acid sequence of CpG12-ApoE / hAAT (SEQ ID NO: 46) was obtained. The eleventh CpG was unchanged and the G in the seventh CpG was changed to an A, with each C changed to a T. The unchanged CpG (the eleventh) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 47 is CpG12-ApoE / hAAT with these restriction enzyme sites (underlined).

[0497]

[0498] The nucleic acid sequence of CpG13-ApoE / hAAT (SEQ ID NO: 48) was unchanged, with the exception of the twelfth CpG, which was left unchanged and the seventh CpG, where G was changed to A. The unchanged CpG (the twelfth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 49 is CpG13-ApoE / hAAT with these restriction enzyme sites (underlined).

[0499]

[0500] The nucleic acid sequence of CpG14-ApoE / hAAT (SEQ ID NO: 50) was unchanged, with the thirteenth CpG remaining unchanged and the G changed to an A in the seventh CpG. Each C was changed to a T. The unchanged CpG (the thirteenth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 51 is CpG14-ApoE / hAAT with these restriction enzyme sites (underlined).

[0501]

[0502] The nucleic acid sequence of CpG15-ApoE / hAAT (SEQ ID NO: 52) is shown. The 14th CpG remains unchanged, and the G in the 7th CpG is changed to an A, with each C changed to a T. The unchanged CpG (the 14th) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 53 is CpG15-ApoE / hAAT with these restriction enzyme sites (underlined).

[0503]

[0504] The nucleic acid sequence of CpG16-ApoE / hAAT (SEQ ID NO: 54) is shown. The fifteenth CpG remains unchanged, and in the seventh CpG, each C is changed to a T. The unchanged CpG (the fifteenth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 55 is CpG16-ApoE / hAAT with these restriction enzyme sites (underlined).

[0505]

[0506] The nucleic acid sequence of CpG17-ApoE / hAAT (SEQ ID NO: 56) is shown. The sixteenth CpG remains unchanged, and the G in the seventh CpG is changed to an A. Each C is changed to a T. The unchanged CpG (the sixteenth) is in bold. When cloning the sequence into the FIX expression cassette, ApaI restriction sites are located at the 5' and 3' ends. SEQ ID NO: 57 is CpG17-ApoE / hAAT with these restriction enzyme sites (underlined).

[0507]

[0508] The nucleic acid sequence of CpG18-ApoE / hAAT (SEQ ID NO: 58) was removed from the fifth, seventh, eighth, tenth, and eleventh CpGs, and the remaining Cs were changed to Ts. When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 59 is CpG18-ApoE / hAAT with these restriction enzyme sites (underlined).

[0509]

[0510] The nucleic acid sequence of CpG19-ApoE / hAAT (SEQ ID NO: 60) is shown. At the first through fifth and eighth through eleventh CpGs, the Cs were changed to Ts, and the C of the seventh CpG was removed. All remaining CpGs (sixth and twelfth through sixteenth) remained unchanged (in bold). When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 61 is CpG19-ApoE / hAAT with these restriction enzyme sites (underlined).

[0511]

[0512] The nucleic acid sequence of CpG20-ApoE / hAAT (SEQ ID NO:62) is the result of multiple deletions of a region that does not contain putative transcription factor binding sites. The G was changed to an A in the only remaining CpG (the seventh CpG in SEQ ID NO:22). When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO:63 is CpG20-ApoE / hAAT with these restriction enzyme sites (underlined).

[0513]

[0514] The nucleic acid sequence of CpG21-ApoE / hAAT (SEQ ID NO: 64) was modified from C to T in all CpGs except for the fifth and sixth CpGs, where G was modified to C and A, respectively. When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 65 is CpG21-ApoE / hAAT with these restriction enzyme sites (underlined).

[0515]

[0516] The nucleic acid sequence of CpG22-ApoE / hAAT (SEQ ID NO: 66) was modified from C to T in all CpGs except for the fifth CpG, which was left unchanged (in bold) and the sixth CpG, where G was changed to A. When the sequence was cloned into the FIX expression cassette, ApaI restriction sites were located at the 5' and 3' ends. SEQ ID NO: 67 is CpG22-ApoE / hAAT with these restriction enzyme sites (underlined).

[0517]

[0518] Amino acid sequence of FVIII-BDD (SQ sequence bold / underlined) (SEQ ID NO: 68).

[0519]

[0520] SQ sequence (SEQ ID NO:69).

[0521] SFSQNPPVLKRHQR

[0522] Wild-type FVIII-BDD cDNA (SEQ ID NO:70).

[0523]

[0524] CpG-reduced nucleic acid variant encoding FVIII-BDD (SEQ ID NO:71)

[0525] atgcagattgagctgtctacctgcttcttcctgtgcctgctgaggttctgcttctctgct

[0526] accaggaggtactacctgggggctgtggagctgagctgggattacatgcagtctgacctg

[0527] ggggagctgcctgtggatgccaggtttccccccagggtgcccaagagcttccccttcaat

[0528] acctctgtggtgtataagaagaccctgtttgtggagttcactgatcatctgttcaacatt

[0529] gctaaacccaggcccccctggatggggctgctgggccctaccatccaggctgaggtgtat

[0530] gacactgtggtgatcactctgaagaacatggctagccatcctgtgtctctgcatgctgtg

[0531] ggggtgagctactggaaggcttctgagggggctgagtatgatgatcagactagccagagg

[0532] gagaaggaggatgacaaggtgttccctgggggctctcacacctatgtctggcaggtgctg

[0533] aaggagaatggccccatggcctctgatcctctgtgtctgacctatagctacctgagccat

[0534] gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgtagggag

[0535] gggagcctggccaaggagaagacccagaccctgcacaagttcattctgctgtttgctgtg

[0536] tttgatgagggcaagagctggcattctgaaaccaagaacagcctgatgcaggacagggat

[0537] gctgcctctgctagggcctggcccaagatgcacactgtgaatgggtatgtcaataggtct

[0538] ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggc

[0539] accacccctgaggtgcacagcatctttctggagggccacaccttcctggtgaggaatcac

[0540] agacaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg

[0541] gacctgggccagtttctgctgttctgccacatctctagccaccagcatgatggcatggag

[0542] gcctatgtgaaggtggactcctgccctgaggagccccagctgaggatgaagaataatgag

[0543] gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgagatttgat

[0544] gatgacaattctcccagcttcattcagatcaggtctgtggccaagaagcatcccaagacc

[0545] tgggtgcactacattgctgctgaggaggaggactgggactatgcccccctggtgctggcc

[0546] cctgatgacaggagctataagagccagtacctgaataatggcccccagaggattgggagg

[0547] aagtataagaaggtgaggttcatggcctatactgatgaaaccttcaagaccagagaggcc

[0548] atccagcatgagtctgggatcctggggcccctgctgtatggggaggtgggggacaccctg

[0549] ctgatcatcttcaagaaccaggccagcaggccctacaacatctaccctcatggcatcact

[0550] gatgtgaggcctctgtacagcagaaggctgcccaagggggtgaagcatctgaaggacttc

[0551] cccattctgcctggggagattttcaagtacaagtggactgtgactgtggaggatggccca

[0552] accaagtctgaccctaggtgcctgactaggtactacagcagctttgtgaatatggagagg

[0553] gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag

[0554] aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag

[0555] aacaggagctggtacctgactgagaacattcagaggtttctgcccaaccctgctggggtg

[0556] cagctggaggaccctgaattccaggcctctaacatcatgcacagcattaatggctatgtg

[0557] tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacattctgagc

[0558] attggggcccagactgacttcctgtctgtgttcttctctggctacacctttaagcacaag

[0559] atggtgtatgaggataccctgaccctgtttcctttctctggggagactgtgttcatgagc

[0560] atggagaaccctggcctgtggatcctgggctgccacaactctgacttcaggaacaggggg

[0561] atgactgctctgctgaaggtgagcagctgtgataagaacactggggactactatgaggac

[0562] agctatgaggacatctctgcctatctgctgagcaagaataatgctattgagcccaggagc

[0563] ttctctcagaacccccctgtgctgaagaggcaccagagggagatcaccagaactactctg

[0564] cagtctgaccaggaggagattgactatgatgacaccatctctgtggagatgaagaaggag

[0565] gattttgatatttatgatgaggatgaaaaccagagccccaggagctttcagaagaagact

[0566] aggcactatttcattgctgctgtggagaggctgtgggactatggcatgtcttctagcccc

[0567] catgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttc

[0568] caggagttcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcac

[0569] ctggggctgctgggcccttatatcagggctgaggtggaggataacatcatggtgaccttc

[0570] aggaaccaggccagcaggccctacagcttctactctagcctgatcagctatgaggaggac

[0571] cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacttat

[0572] ttctggaaggtgcagcaccatatggcccccaccaaggatgagtttgattgcaaagcctgg

[0573] gcctacttctctgatgtggacctggagaaggatgtgcactctgggctgattggccccctg

[0574] ctggtgtgccacaccaacactctgaaccctgcccatggcaggcaggtgactgtgcaggag

[0575] tttgccctgttcttcaccatctttgatgagactaagagctggtacttcactgagaacatg

[0576] gagaggaactgcagggccccctgcaatatccagatggaggaccccacctttaaggaaaat

[0577] tataggtttcatgccattaatggctacatcatggacaccctgcctggcctggtgatggcc

[0578] caggaccagaggatcaggtggtacctgctgagcatgggcagcaatgagaacattcacagc

[0579] atccacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg

[0580] tataatctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc

[0581] tggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctg

[0582] gtgtattctaacaagtgtcagacccccctgggcatggcctctggccatatcagggacttc

[0583] cagatcactgcctctggccagtatgggcagtgggcccccaagctggccaggctgcattac

[0584] tctggcagcatcaatgcctggagcaccaaggagccattcagctggattaaggtggacctg

[0585] ctggctccaatgattatccatggcatcaagacccagggggccaggcagaagtttagcagc

[0586] ctgtacatctctcagtttatcatcatgtactctctggatggcaaaaagtggcagacctac

[0587] aggggcaattctactggcactctgatggtgttctttggcaatgtggacagctctgggatc

[0588] aagcacaacatctttaacccccctatcattgccaggtacattaggctgcaccccacccat

[0589] tacagcatcaggagcaccctgaggatggagctgatgggctgtgatctgaacagctgcagc

[0590] atgcccctgggcatggagagcaaggctatctctgatgcccagattactgccagcagctac

[0591] ttcaccaatatgtttgccacctggagccccagcaaggccaggctgcacctgcagggcagg

[0592] tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag

[0593] aagaccatgaaggtgactggggtgaccacccagggggtgaagagcctgctgactagcatg

[0594] tatgtgaaggagttcctgatcagcagcagccaggatggccatcagtggaccctgttcttc

[0595] cagaatggcaaggtgaaggtgttccagggcaatcaggacagcttcacccctgtggtgaac

[0596] agcctggacccccccctgctgaccagatacctgaggatccacccccagagctgggtgcat

[0597] cagattgccctgaggatggaggtgctggggtgtgaggcccaggacctgtactga

[0598] Nucleic acid variant with reduced CpG encoding FVIII - BDD (SEQ ID NO:72)

[0599] atgcagattgagctgtctacctgctttttcctgtgtctgctgaggttctgcttctctgcc

[0600] actaggaggtactacctgggggctgtggagctgtcttgggattacatgcagtctgatctg

[0601] ggggagctgcctgtggatgccaggtttcctcccagggtgcccaagtctttccccttcaat

[0602] acctctgtggtgttaagaagaccctgtttgtggagtttactgatcacctgttcaacatt

[0603] gccaagcccaggcccccttggatgggcctgctggggcccaccatccaggctgaggtgtat

[0604] gacactgtggtgatcaccctgaagaacatggcctctcaccctgtgagcctgcatgctgtg

[0605] ggggtgagctactggaaggcctctgaggggctgagtatgatgaccagaccagccagagg

[0606] gagaaggaggatgataaggtgttccctgggggagccaacacttatgtgtggcaggtgctg

[0607] aaggaatggcccaatggcctctgatcccctgtgcctgacctattcttacctgagccat

[0608] gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag

[0609] ggctctctggctaaggaagagacccagaccctgcacaagttcatcctgctgtttgctgtg

[0610] tttgatgagggaagagctggcactctgagaccaagaacagcctgatgcaggacagggat

[0611] gctgcccttgccagggcctggcccaaaatgcacactgtgaatggctatgtgaataggagc

[0612] ctgcctggcctgattggctgccacaggaagtctgtgtattggcatgtgattggcatgggc

[0613] accacccctgaggtgcactctatcttcctggagggccatactttcctggtgaggaatcat

[0614] aggcaggccagcctggagattagccccattacctttctgactgcccagaccctgctgatg

[0615] gacctgggccagttcctgctgttttgccacatcagctctcaccagcatgatggcatggag

[0616] gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaagaacaatgag

[0617] gaggctgaggattatgatgatgatctgactgattctgaaatggatgtggtgaggtttgat

[0618] gatgacaatagcccctctttcatccagatcaggtctgtggccaagaagcatcctaagacc

[0619] tgggtgcactacattgctgctgaggaggaggactgggactatgctcccctggtgctggcc

[0620] cctgatgacaggtcttacaagagccagtacctgaacaatggcccccagagaattgggagg

[0621] aagtataagaaggtgagattcatggcttacactgatgagaccttcaagactagggaggcc

[0622] atccagcatgagtctggcattctgggccccctgctgtatggggaggtgggggacaccctg

[0623] ctgatcatcttcaagaaccaggcctctaggccctacaatatttacccccatgggatcact

[0624] gatgtgaggcccctgtacagcaggaggctgcctaagggggtgaagcatctgaaggacttc

[0625] cccatcctgcctggggagatcttcaagtataagtggactgtgactgtggaagatggcccc

[0626] accaagtctgaccctaggtgcctgaccaggtactactcttcttttgtgaacatggagagg

[0627] gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag

[0628] agggggaaccagattatgtctgacaagaggaatgtgattctgttctctgtgtttgatgag

[0629] aacaggagctggtatctgactgagaacatccagaggttcctgcccaatcctgctggggtg

[0630] cagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatgggtatgtg

[0631] tttgattctctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc

[0632] attggggctcagactgatttcctgtctgtgttcttttctggctacacctttaagcataag

[0633] atggtgtatgaggacactctgaccctgtttcccttctctggggagactgtgtttatgagc

[0634] atggagaaccctggcctgtggatcctgggctgccacaactctgatttcaggaacaggggc

[0635] atgactgctctgctgaaggtgtcttcttgtgacaagaacactggggactattatgaggac

[0636] agctatgaggacatctctgcctacctgctgagcaagaacaatgctattgagcccagatct

[0637] ttcagccagaacccccctgtgctgaagaggcaccagagggagatcactaggaccaccctg

[0638] cagtctgaccaggaggagattgactatgatgacactatctctgtggagatgaagaaggag

[0639] gactttgatatctatgatgaggatgagaaccagtctcccaggagcttccagaaaaagacc

[0640] aggcactacttcattgctgctgtggagaggctgtgggactatggcatgtcttctagcccc

[0641] catgtgctgaggaacagggcccagtctgggtctgtgccccagttcaagaaggtggtgttc

[0642] caggagttcactgatgggagcttcacccagcctctgtacaggggggagctgaatgagcac

[0643] ctggggctgctgggcccttatattagggctgaggtggaggacaacatcatggtgactttc

[0644] aggaatcaggcctctaggccctatagcttctacagctctctgatcagctatgaggaggat

[0645] cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacctac

[0646] ttctggaaggtgcagcaccacatggctcctaccaaggatgagtttgactgcaaggcctgg

[0647] gcctacttttctgatgtggacctggagaaggatgtgcactctggcctgattggccccctg

[0648] ctggtgtgtcataccaacaccctgaaccctgcccatggcaggcaggtgactgtgcaggag

[0649] tttgccctgttcttcaccatctttgatgagaccaagagctggtactttactgagaacatg

[0650] gagaggaattgcagagccccttgcaacatccagatggaggacccaaccttcaaagagaac

[0651] tacaggttccatgccatcaatgggtacatcatggacaccctgcctggcctggtgatggct

[0652] caggaccagaggatcaggtggtatctgctgagcatgggcagcaatgagaatatccatagc

[0653] attcacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg

[0654] tataacctgtaccctggggtgtttgagactgtggagatgctgccaagcaaggctgggatt

[0655] tggagggtggagtgcctgattggggagcacctgcatgctggcatgtctaccctgttcctg

[0656] gtgtactccaataagtgccagacccccctgggcatggcctctggccacatcagggacttc

[0657] cagatcactgcctctggccagtatgggcagtgggccccaaagctggccaggctgcactat

[0658] tctgggagcatcaatgcttggagcaccaaggagcctttcagctggattaaggtggatctg

[0659] ctggcccccatgatcattcatggcatcaaaacccagggggctagacagaagttttctagc

[0660] ctgtacatcagccagttcatcatcatgtacagcctggatggcaagaagtggcagacttac

[0661] aggggcaatagcactggcaccctgatggtgttttttggcaatgtggacagctctggcatc

[0662] aagcacaacatctttaacccccccattattgccaggtatatcaggctgcatcccacccac

[0663] tattctattaggtctactctgagaatggagctgatgggctgtgacctgaacagctgtagc

[0664] atgcccctggggatggagagcaaggctatctctgatgcccagatcactgccagctcttat

[0665] ttcaccaatatgtttgccacctggtctccctctaaggccaggctgcacctgcagggcagg

[0666] agcaatgcttggaggccccaggtgaataaccccaaggagtggctgcaggtggacttccag

[0667] aagaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgactagcatg

[0668] tatgtgaaggagttcctgatcagcagcagccaggatgggcatcagtggactctgttcttc

[0669] cagaatggcaaggtgaaggtcttccaggggaaccaggatagcttcactcctgtggtgaac

[0670] tctctggacccccccctgctgactaggtatctgaggatccacccccagagctgggtgcac

[0671] cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtattga

[0672] CpG-reduced nucleic acid variant encoding FVIII-BDD (SEQ ID NO:73)

[0673] atgcagattgaactgtctacttgtttcttcctgtgcctgctgaggttttgcttctctgct

[0674] actaggaggtactatctgggggctgtggagctgtcttgggactatatgcagtctgacctg

[0675] ggggagctgcctgtggatgctaggtttccccccagggtgcccaagagcttcccctttaac

[0676] acctctgtggtgtataagaagactctgtttgtggagttcactgaccatctgttcaacatt

[0677] gccaagccaaggcccccctggatgggcctgctgggccccaccatccaggctgaggtgtat

[0678] gacactgtggtgattactctgaagaacatggccagccatcctgtgagcctgcatgctgtg

[0679] ggggtgtcttactggaaggcctctgagggggctgagtatgatgaccagacctctcagagg

[0680] gagaaggaggatgacaaggtgttccctggggctctcatacctatgtgtggcaggtcctg

[0681] areaatgggcccatggcctctgaccccctgtgcctgacctactcttatctgtctcat

[0682] gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag

[0683] ggcagcctggctaaggaagacccagactctgcacaagttcatcctgctgtttgctgtg

[0684] tttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagggat

[0685] gctgccctctgctagggcctggcccaagatgcacactgtgaatgggtatgtgaacaggagc

[0686] ctgccaggcctgattggctgccataggaagtctgtgtattggcatgtgattgggatgggg

[0687] actacccctgaggtccacagcattttcctggaggggcatacctttctggtgaggaaccac

[0688] aggcaggcctctctggagatctctcccattactttcctgactgcccagaccctgctgatg

[0689] gacctgggccagttcctgctgttctgccacatcagcagccaccagcatgatggcatggag

[0690] gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaaaaacaatgag

[0691] gaggctgaggattatgatgatgacctgactgattctgagatggatgtggtgaggtttgat

[0692] gatgataacagccccagcttcatccagattaggtctgtggccaagaagcatcccaagacc

[0693] tgggtgcactacattgctgctgaggagaggattgggactatgctccctctggtgctggcc

[0694] cctgatgacaggagctacaagagccagtacctgaataatggcccccagaggattggcagg

[0695] aagtataagaaggtgaggttcatggcctacactgatgagacctttaagaccagggaggcc

[0696] atccagcatgaatctgggatcctgggccccctgctgtatgggaggtggggcaccctg

[0697] ctgattatctttaagaaccaggctagcaggccctacaacatttacccccatggcattact

[0698] gatgtgaggcccctgtacagcaggaggctcccaagggggtgaagcacctgaaggatttc

[0699] cccattctgcctggggagatctttaagtacaaatggactgtgactgtggaggatggccct

[0700] actaagtctgatcccaggtgtctgaccagatatactacagcagctttgtgaatatggagagg

[0701] gacctggcttctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag

[0702] aggggcaatcagattatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag

[0703] aacagaagctggtacctgactgagaacatccagaggttcctgcccaaccctgctggggtg

[0704] cagctggaggaccctgagttccaggctagcaatatcatgcacagcattaatggctatgtg

[0705] tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctattggtacattctgagc

[0706] attggggcccagactgatttcctgtctgtgttcttttctggctacaccttcaagcacaag

[0707] atggtgtatgaggatactctgaccctgtttcccttctctggggagactgtgttcatgagc

[0708] atggagaaccctggcctgtggatcctgggctgtcacaactctgacttcaggaacaggggc

[0709] atgactgccctgctgaaggtgagctcttgtgataagaacactggggactactatgaggac

[0710] tcttatgaggacatctctgcctacctgctgagcaagaacaatgctattgagcccaggagc

[0711] ttctctcagaatccccctgtgctgaagaggcatcagagggagatcactaggactaccctg

[0712] cagtctgaccaggaagagattgactatgatgacaccatctctgtggaaatgaagaaggag

[0713] gactttgatatctatgatgaggatgaaaaccagagccccaggagcttccagaagaagacc

[0714] aggcattacttcattgctgctgtggagaggctgtgggactatgggatgagctcttctccc

[0715] catgtgctgaggaatagggctcagtctggctctgtcccacagttcaagaaggtggtgttt

[0716] caggagttcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcat

[0717] ctgggcctgctggggccctacatcagggctgaggtggaggataacattatggtgactttc

[0718] aggaaccaggcctctaggccctacagcttctacagcagcctgatcagctatgaggaggac

[0719] cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagactaagacctat

[0720] ttctggaaggtgcagcatcacatggctcccactaaagatgagtttgactgcaaggcctgg

[0721] gcctacttctctgatgtggatctggagaaggatgtgcattctgggctgattggccctctg

[0722] ctggtctgccatactaacaccctgaatcctgcccatggcaggcaggtgactgtgcaggag

[0723] tttgccctgttctttaccatctttgatgagaccaagtcttggtacttcactgagaacatg

[0724] gagaggaactgcagggccccctgtaacatccagatggaggaccccacctttaaggagaac

[0725] tacaggttccatgccatcaatggctacatcatggacactctgcctggcctggtgatggcc

[0726] caggaccagaggatcaggtggtacctgctgtctatgggctctaatgagaacattcattct

[0727] atccacttctctggccatgtgtttactgtgaggaagaaggaggagtacaagatggccctg

[0728] tacaatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatc

[0729] tggagggtggagtgcctgattggggaacacctgcatgctggcatgagcaccctgttcctg

[0730] gtctatagcaataagtgccagacccccctggggatggcctctgggcatatcagagacttc

[0731] cagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactac

[0732] tctggcagcattaatgcctggagcaccaaggagcccttctcttggatcaaggtggacctg

[0733] ctggctcccatgatcatccatgggatcaagacccagggggccaggcagaagttcagcagc

[0734] ctgtacatctctcagttcatcatcatgtactctctggatggcaagaagtggcagacctac

[0735] aggggcaatagcactgggaccctgatggtgttctttgggaatgtggacagctctggcatc

[0736] aagcacaatatcttcaacccccccatcattgccaggtacatcagactgcaccccactcat

[0737] tacagcatcaggagcactctgaggatggagctgatgggctgtgacctgaatagctgctct

[0738] atgcccctgggcatggagagcaaggccatttctgatgcccagattactgcctcttcttac

[0739] ttcactaatatgtttgccacctggagccccagcaaggccaggctgcatctgcaggggagg

[0740] agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag

[0741] aagactatgaaggtgactggggtgaccactcagggggtgaagagcctgctgaccagcatg

[0742] tatgtgaaggagttcctgatctcttctagccaggatgggcaccagtggaccctgtttttc

[0743] cagaatgggaaggtgaaggtgtttcagggcaatcaggacagctttactcctgtggtgaac

[0744] agcctggacccccccctgctgactaggtacctgaggattcacccccagagctgggtgcac

[0745] cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga

[0746] CpG-reduced nucleic acid variant encoding FVIII-BDD (SEQ ID NO:74)

[0747] atgcagattgagctgtctacctgcttctttctgtgcctgctgaggttctgtttctctgcc

[0748] actaggaggtattatctggggctgtggagctgtcctgggactacatgcagtctgatctg

[0749] ggggagctgcctgtggatgccaggttccctcccagggtgcccaagtctttccctttcaat

[0750] acctctgtggtgtacaagaagaactctgtttgtggagtttactgatcacctgtttaacatt

[0751] gccaagcccaggcccccctggatggggctgctgggccccaccatccaggctgaggtgtat

[0752] gacactgtggtgattactctgaagaatatggcttctcaccctgtgagcctgcatgctgtg

[0753] ggggtgagctactggaaggcctctgaggggctgagtatgatgaccagaccagccagagg

[0754] gagaaggaggatgacaaggtgttccctggggcagccacacttatgtgtggcaggtgctg

[0755] aaggaatggcccaatggcctctgaccccctgtgcctgacctacagctatctgagccat

[0756] gtggatctggtgaaggatctgaactctggcctgattggggccctgctggtgtgcagggag

[0757] ggctctctggccaaggaagagaactcagactctgcacaagttcatcctgctgtttgctgtg

[0758] tttgatgagggcaagagctggcactctgagaccaagaactctctgatgcaggatagggat

[0759] gctgcttctgccagggcctggcccaagatgcacactgtgaatgggtatgtgaataggagc

[0760] ctgcctgggctgattgggtgtcacaggaagtctgtgtactggcatgtgattggcatgggc

[0761] accactcctgaggtgcacagcatctttctggagggccacacttttctggtgaggaatcac

[0762] aggcaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg

[0763] gatctgggccagttcctgctgttttgccatatcagcagccatcagcatgatgggatggag

[0764] gcttatgtgaaggtggactcttgccctgaggagcctcagctgaggatgaagaataatgaa

[0765] gaggctgaggactatgatgatgatctgactgactctgagatggatgtggtgaggtttgat

[0766] gatgacaacagccccagctttatccagattaggtctgtggccaagaagcaccccaagacc

[0767] tgggtgcattacattgctgctgaggaagaggattgggactatgcccccctggtgctggcc

[0768] cctgatgacaggagctacaagtctcagtacctgaacaatggccctcagaggattggcagg

[0769] aagtacaagaaggtgaggttcatggcttacactgatgagaccttcaagaccagggaggcc

[0770] attcagcatgaatctgggatcctgggccccctgctgtatggggaggtgggggacaccctg

[0771] ctgattattttcaagaaccaggccagcaggccctacaacatttatcctcatggcattact

[0772] gatgtgagacccctgtacagcaggaggctgcctaagggggtgaagcacctgaaggacttc

[0773] cccatcctgcctggggagatcttcaagtacaagtggactgtgactgtggaggatggcccc

[0774] actaagtctgaccccaggtgcctgactaggtactactccagctttgtgaacatggagagg

[0775] gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag

[0776] aggggcaaccagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgag

[0777] aataggtcttggtacctgactgagaacatccagaggtttctgcctaatcctgctggggtg

[0778] cagctggaggatcctgagttccaggcctctaacattatgcacagcatcaatgggtatgtg

[0779] tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc

[0780] attggggcccagactgactttctgtctgtgttcttctctggctacacctttaagcataag

[0781] atggtgtatgaggacaccctgactctgttccccttctctggggagactgtgttcatgagc

[0782] atggagaacccaggcctgtggatcctgggctgccacaactctgatttcaggaataggggc

[0783] atgactgccctgctgaaggtgagcagctgtgataagaacactggggactattatgaggat

[0784] agctatgaggacatctctgcctacctgctgagcaagaacaatgccattgagcccaggagc

[0785] ttcagccagaatcctcctgtgctgaagaggcaccagagggagatcaccaggaccaccctg

[0786] cagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggag

[0787] gactttgacatctatgatgaggatgagaatcagagccccaggagcttccagaagaagact

[0788] agacactactttattgctgctgtggagaggctgtgggactatggcatgagctcttctccc

[0789] catgtgctgagaaacagggcccagtctggctctgtgccccagttcaagaaggtggtcttc

[0790] caggagttcactgatggctctttcacccagcctctgtatagaggggagctgaatgagcac

[0791] ctgggcctgctgggcccttacatcagggctgaggtggaggacaatatcatggtgaccttc

[0792] aggaaccaggctagcaggccctactctttctacagcagcctgatcagctatgaggaggac

[0793] cagaggcagggggctgagcctaggaagaattttgtgaagcccaatgagaccaagacctac

[0794] ttctggaaggtgcagcaccacatggctcccactaaggatgagtttgactgcaaggcctgg

[0795] gcctacttttctgatgtggacctggagaaggatgtgcattctggcctgattggccccctg

[0796] ctggtctgccacaccaatactctgaaccctgctcatgggagacaggtgactgtgcaggag

[0797] tttgccctgttcttcaccatctttgatgagaccaagtcctggtactttactgagaacatg

[0798] gagaggaattgcagggccccttgcaacatccagatggaggaccccaccttcaaggaaaat

[0799] tataggttccatgccatcaatggctacatcatggacaccctgcctggcctggtgatggcc

[0800] caggaccagaggatcaggtggtatctgctgtctatgggctctaatgagaacatccacagc

[0801] atccatttctctggccatgtgttcactgtgaggaagaaggaggagtataagatggctctg

[0802] tacaacctgtaccctggggtctttgagactgtggagatgctgcccagcaaggctggcatt

[0803] tggagggtggagtgcctgattggggaacacctgcatgctgggatgagcaccctgttcctg

[0804] gtgtactctaacaagtgccagaccccactgggcatggcttctggccacatcagggatttc

[0805] cagattactgcctctggccagtatggccagtgggctcccaagctggctaggctgcactac

[0806] tctgggagcatcaatgcctggtctactaaggagcctttctcttggatcaaagtggacctg

[0807] ctggcccctatgatcatccatgggatcaagactcagggggccaggcagaagttcagcagc

[0808] ctgtacatctctcagttcatcattatgtacagcctggatggcaagaagtggcagacctac

[0809] aggggcaacagcactggcaccctgatggtgttctttgggaatgtggacagctctgggatt

[0810] aagcacaacatctttaacccccccatcattgccaggtatatcaggctgcaccctacccac

[0811] tacagcattaggagcaccctgaggatggagctgatgggctgtgacctgaacagctgcagc

[0812] atgcccctggggatggagagcaaggccatttctgatgctcagatcactgcttctagctac

[0813] ttcactaacatgtttgccacctggtctcccagcaaggctagactgcacctgcaggggagg

[0814] agcaatgcctggaggccccaggtgaataatcccaaggagtggctgcaggtggatttccag

[0815] aaaaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgaccagcatg

[0816] tatgtgaaggagttcctgatcagcagcagccaggatgggcatcagtggaccctgttcttt

[0817] cagaatgggaaggtgaaggtgtttcagggcaatcaggacagcttcacccctgtggtgaac

[0818] agcctggacccccccctgctgaccaggtacctgaggatccacccccagagctgggtgcat

[0819] cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga

[0820] Nucleic acid variant with reduced CpG encoding FVIII - BDD (SEQ ID NO:75)

[0821] atgcagattgagctgtctacttgcttcttcctgtgcctgctgaggttctgcttctctgcc

[0822] actaggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctg

[0823] ggggagctgcctgtggatgccaggtttcctcccagggtgcctaagagcttccccttcaac

[0824] acctctgtggtgtacaagaagactctgtttgtggagtttactgatcatctgttcaacatt

[0825] gccaagcccaggcctccttggatggggctgctgggccccaccatccaggctgaggtgtat

[0826] gacactgtggtgattaccctgaagaatatggccagccatcctgtgagcctgcatgctgtg

[0827] ggggtgagctattggaaggcctctgaggggctgagtatgatgatcagactagccagagg

[0828] gagaaggaggatgacaaggtgttccctgggggagccatacctatgtgtggcaggtgctg

[0829] aaggaatggccccatggcctctgaccctctgtgcctgactatagatctacctgagccat

[0830] gtggatctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag

[0831] ggcagcctggccaaggaagagaactcagaccctgcacaagttcatcctgctgtttgctgtg

[0832] tttgatgaggggaagtcctggcactctgagactaagaacagcctgatgcagtagtagggat

[0833] gctgcttctgccagggcctggcctaagatgcacactgtgaatggctatgtgaataggagc

[0834] ctgcctggcctgattggctgccataggaagtctgtgtactggcatgtgattgggatgggc

[0835] accacccctgaggtgcactctattttcctggagggccatactttcctggtgaggaaccat

[0836] aggcaggccagcctggagatcagccccatcactttcctgactgcccagactctgctgatg

[0837] gacctgggccagttcctgctgttctgccacatcagcagccatcagcatgatggcatggag

[0838] gcttatgtgaaggtggacagctgccctgaggagcctcagctgaggatgaagaataatgag

[0839] gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgaggtttgat

[0840] gatgacaactctccctctttcatccagatcaggtctgtggccaagaagcaccctaagacc

[0841] tgggtgcactacattgctgctgaggaggaggattgggactatgcccccctggtgctggcc

[0842] ccagatgacaggagctacaagtcccagtacctgaacaatggcccccagaggattggcagg

[0843] aagtacaagaaggtgaggttcatggcttatactgatgagactttcaagaccagggaggcc

[0844] atccagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctg

[0845] ctgattatcttcaagaaccaggcttctaggccctacaatatctaccctcatggcatcact

[0846] gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcatctgaaggatttc

[0847] cccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggcccc

[0848] actaagtctgaccccaggtgcctgaccaggtattacagcagctttgtgaacatggagagg

[0849] gatctggcttctgggctgattggccccctgctgatctgctacaaggagtctgtggaccag

[0850] aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag

[0851] aataggagctggtacctgactgagaacatccagaggtttctgcccaatcctgctggggtg

[0852] cagctggaggatcctgagtttcaggcctctaatatcatgcacagcatcaatggctatgtg

[0853] tttgactctctgcagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagc

[0854] attggggcccagactgactttctgtctgtgtttttttctggctacaccttcaagcacaag

[0855] atggtgtatgaggatactctgactctgttccctttttctggggagactgtgttcatgtct

[0856] atggagaaccctgggctgtggattctgggctgccacaattctgacttcaggaacagaggc

[0857] atgactgctctgctgaaggtgagcagctgtgacaagaacactggggactactatgaggac

[0858] tcttatgaggacatttctgcctacctgctgagcaagaacaatgccattgagcccagaagc

[0859] ttttctcagaacccccctgtgctgaagaggcaccagagggagatcaccaggaccaccctg

[0860] cagtctgaccaggaggagattgactatgatgatactatttctgtggagatgaagaaggag

[0861] gactttgacatctatgatgaggatgagaaccagagccccaggtctttccagaagaagact

[0862] aggcactactttattgctgctgtggagaggctgtgggactatgggatgtctagctctcct

[0863] catgtgctgaggaacagggcccagtctggctctgtgccccagtttaaaaaggtggtgttc

[0864] caggaattcactgatggcagctttacccagcctctgtacaggggggagctgaatgagcac

[0865] ctggggctgctggggccttacattagggctgaggtggaggacaacatcatggtgaccttc

[0866] aggaatcaggccagcaggccctactctttctacagcagcctgatctcttatgaggaggac

[0867] cagaggcagggggctgaacccaggaagaactttgtgaagcccaatgagaccaagacctac

[0868] ttctggaaggtgcagcaccacatggctcccaccaaggatgagtttgattgcaaggcctgg

[0869] gcttacttctctgatgtggatctggagaaggatgtgcactctgggctgattggccccctg

[0870] ctggtgtgccacaccaacactctgaaccctgcccatggcagacaggtgactgtgcaggag

[0871] tttgccctgttcttcactatctttgatgagactaagagctggtacttcactgagaacatg

[0872] gagaggaattgcagggccccttgcaacatccagatggaggaccccacctttaaggagaac

[0873] tacaggtttcatgccattaatggctacatcatggacaccctgcctggcctggtgatggcc

[0874] caggaccagaggatcaggtggtacctgctgtctatggggagcaatgagaacatccacagc

[0875] attcacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg

[0876] tacaacctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctgggatc

[0877] tggagggtggagtgcctgattggggagcacctgcatgctgggatgagcaccctgttcctg

[0878] gtgtatagcaacaagtgccagacccccctgggcatggcctctggccacatcagagacttt

[0879] cagattactgcctctggccagtatgggcagtgggcccccaagctggccaggctgcactat

[0880] tctggctctattaatgcctggagcactaaggagcccttcagctggattaaggtggacctg

[0881] ctggctcccatgatcatccatggcatcaagactcagggggccaggcagaagttctcttct

[0882] ctgtacatcagccagttcattatcatgtactccctggatggcaagaagtggcagacctat

[0883] aggggcaacagcactggcaccctgatggtgttctttgggaatgtggacagctctggcatc

[0884] aagcataatatcttcaatccccccatcattgctaggtacatcaggctgcaccccacccac

[0885] tactctattaggtctaccctgaggatggagctgatgggctgtgacctgaacagctgcagc

[0886] atgcctctgggcatggagagcaaagccatctctgatgcccagatcactgccagcagctac

[0887] tttaccaacatgtttgctacttggagccccagcaaggccaggctgcacctgcaggggagg

[0888] tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag

[0889] aagactatgaaggtgactggggtgaccacccagggggtgaagagcctgctgacctctatg

[0890] tatgtgaaggagttcctgattagcagcagccaggatggccaccagtggaccctgtttttc

[0891] cagaatgggaaggtgaaggtgtttcaggggaaccaggacagcttcactcctgtggtgaac

[0892] tctctggacccccccctgctgaccaggtatctgaggatccaccctcagagctgggtgcac

[0893] cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga

[0894] CpG-reduced nucleic acid variant encoding FVIII-BDD (SEQ ID NO:76)

[0895]

[0896] A nucleic acid variant encoding FVIII-BDD with reduced CpG (SEQ ID NO: 77).

[0897]

[0898] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 78)

[0899]

[0900] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 79)

[0901]

[0902] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 80)

[0903]

[0904] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 81)

[0905]

[0906] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 82)

[0907]

[0908] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 83)

[0909]

[0910] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 84)

[0911]

[0912] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 85)

[0913]

[0914] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 86)

[0915]

[0916] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 87)

[0917]

[0918] Reduced CpG nucleic acid variant encoding FVIII-BDD (SEQ ID NO: 88)

[0919]

[0920] FVIII V3 cDNA(SEQ ID NO:89)

[0921]

[0922] FVIII CO3 cDNA(SEQ ID NO:90)

[0923]

[0924] AAV-LK03 VP1 Capsid (SEQ ID NO:91)

[0925] MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0926] AAV-SPK VP1 Capsid (SEQ ID NO:92)

[0927] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0928] Nucleic acid sequence of the intron in AAV-WINT (SEQ ID NO:93)

[0929] AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTGACACTGACATCCACTTTTTCTTTTTCTCCACAG

[0930] FIX (SEQ ID NO:94)

[0931]

Claims

1. A polynucleotide comprising the regulatory element nucleic acid sequence of SEQ ID NO:

58.

2. The polynucleotide according to claim 1, comprising the nucleotide sequence of SEQ ID NO:

59.

3. An expression cassette comprising the polynucleotide according to any one of claims 1 to 2 and a transgene, wherein the regulatory element nucleic acid sequence is operably linked to the transgene.

4. The expression cassette of claim 3, wherein the regulatory element nucleic acid sequence is located 5' to the transgene.

5. The expression cassette of claim 4, wherein the transgene encodes a protein.

6. The expression cassette of claim 5, wherein the protein is a therapeutic protein.

7. The expression cassette of claim 6, wherein the therapeutic protein is a blood coagulation or clotting factor protein.

8. The expression cassette of claim 6, wherein the therapeutic protein is Factor IX (FIX).

9. The expression cassette of claim 6, wherein the therapeutic protein is Factor VIII (FVIII), Factor VII (FVII), or Protein C.

10. The expression cassette of claim 6, wherein the therapeutic protein is a lysosomal storage enzyme.

11. The expression cassette of claim 10, wherein the lysosomal storage enzyme is acid α-glucosidase (GAA) or α-galactosidase (GLA).

12. The expression cassette of claim 6, wherein the therapeutic protein is C1 esterase inhibitor (C1EI).

13. The expression cassette of claim 3, wherein the untranslated (non-coding) nucleic acid located between the regulatory element nucleic acid sequence and the transgene is not an intron.

14. An adeno-associated virus (AAV) vector comprising the expression cassette of any one of claims 3-13.

15. The AAV vector of claim 14, wherein the AAV vector comprises: a) AAV capsid; and b) a 5' AAV inverted terminal repeat (ITR) flanking the 5' end of the expression cassette and a 3' ITR flanking the 3' end of the expression cassette.

16. The AAV vector of claim 15, wherein the capsid comprises the amino acid sequence of SEQ ID NOs: 91 or 92.

17. The AAV vector of claim 15, wherein the capsid is an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 capsid.

18. The AAV vector of any one of claims 15-17, wherein the 5' AAV inverted terminal repeat (ITR) and the 3' ITR are selected from the ITRs of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rhl0, or Rh74 AAV.

19. The AAV vector of claim 15, wherein there is no more than 107 nucleotides of untranslated nucleic acid sequence between the regulatory element nucleic acid sequence and the 5' end of the transgene.

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