WO2023280323A1 - Recombinant adeno-associated virus vector and method for treating or preventing hemophilia b - Google Patents

Abstract

An expression construct that expresses a blood coagulation factor IX, a recombinant adeno-associated virus (rAAV) vector, a viral particle comprising the rAAV vector, a pharmaceutical composition, and a use thereof for treating or preventing hemophilia B.

Description

Recombinant adeno-associated virus vectors and methods for treating or preventing hemophilia B

This application claims the priority of the Chinese Invention Patent Application No. CN202110778026.9 filed with the State Intellectual Property Office of China on July 9, 2021, and the entire contents of this application including the drawings and sequence listing are incorporated herein by reference.

technical field

The present invention relates to the field of gene therapy. In particular the present invention relates to expression constructs and recombinant adeno-associated viruses (rAAV) expressing (human) coagulation factor IX, viral particles and compositions comprising said rAAV, and their use in the treatment or prevention of human coagulation factor IX deficiency The use of the hemorrhagic symptoms of hemophilia B (referred to as HB) caused by coagulation dysfunction.

Background technique

Hemophilia (hemophilia) is a group of bleeding disorders of hereditary coagulation dysfunction. Gene mutations lead to active thromboplastin production disorders. It is X-chromosomal recessive inheritance. It is divided into hemophilia A (deficiency of coagulation Factor VIII (FVIII) and HB (deficiency of factor IX (FIX). The main clinical manifestations of HB are intra-articular hemorrhage and intra-muscular hemorrhage, and the resulting permanent joint deformation will limit the ability of movement and seriously affect the quality of life.

FIX is a kind of coagulation factor with serine protease activity, mainly produced by the liver and secreted into plasma in the form of inactive zymogen. FIX is converted into activated FIX (FIXa) through the FXIa or FVIIa·TF complex. FIXa can activate FX to FXa together with FVIIIa, phospholipids and Ca ²⁺ , initiate the common coagulation pathway, and play a hemostatic effect.

Currently, the mainstream therapy for HB is still intravenous injection of FIX biological products. However, due to the short half-life of clotting factors, patients need to receive frequent injections throughout their lives. Although this can control the course of the disease to a certain extent, there is still a risk of bleeding. Due to the complex production process, low production capacity and high price of FIX products, there is a large gap between the supply and demand of domestic coagulation factors. So far, the per capita consumption is less than 1/6 of the global average level. In addition, when patients are treated with coagulation factor replacement therapy, they develop complications of inhibitors (anti-drug antibodies) against the injected factor. In patients with these inhibitors, the effectiveness of prophylactic therapy with coagulation factors will be compromised.

Gene therapy in which the gene encoding the coagulation factor is delivered via a viral vector enables stable, long-lasting expression compared to direct injection of the coagulation factor. HB disease is caused by a single gene mutation, and the pathogenic mechanism is clear. The cDNA length of FIX is relatively short, so it is convenient for vector carrying. In addition, mature model animals already exist, including large animal (HB dog) and small animal (HB mouse) models. Therefore, HB has become a research hotspot of gene therapy.

A number of studies on HB gene therapy have made in-depth progress, and clinical data have also proved its effectiveness, sustainability and safety. The clinical data of UniQure's AMT-061 and Spark's SPK-9001 show that delivery of AAV vectors to the liver can maintain the level of FIX activity in subjects exceeding the minimum standard recommended by epidemiological studies (>15%). For more than 3 years, the activity is still maintained at a high level, the annualized bleeding rate, FIX dosage and infusion frequency are all significantly reduced, and no serious adverse events have occurred in existing clinical studies, which proves that AAV vector gene therapy can greatly reduce Improve the quality of life of patients with moderate to severe HB.

UniQure's AMT-061 is an adeno-associated virus 5 (AAV5)-based gene therapy for HB. UniQure provided a method for screening patients who may have anti-AAV antibodies in WO2019011893A1, and also mentioned that AAV5 has lower local neutralizing antibody titers in humans than other serotypes.

CN111647625A describes a method for increasing the expression level of blood coagulation factor IX, including codon optimization and Kozak sequence replacement, and then constructing it into an AAV virus vector, and using a three-plasmid packaging system to transfect cells for expression. This application only proved the successful expression of blood coagulation factor IX in vitro, and did not conduct any in vivo experiments.

There is still a need in the art for efficient and safe gene therapy against HB.

Contents of the invention

The inventors of the present invention have obtained an AAV vector construct capable of efficiently and stably expressing FIX through the screening of expression cassettes and FIX mutants and the optimization of the coding sequence of the target gene, which can mediate stable and efficient FIX in both human liver cells and animal models. The expression of FIX enables exogenous FIX to achieve an effective therapeutic dose without risk of safety, thereby completing the present invention.

Thus, in a first aspect, the present invention provides an expression construct comprising, from 5' to 3', the following elements operably linked:

(1) a transcriptional regulatory element selected from the group consisting of LP1, HLP, TTR, HLP2, enTTR, APOE-hAAT promoters;

(2) an intron selected from the SV40 intron or the FIX gene intron;

(3) Gene coding sequence, which encodes coagulation factor IX with R338L mutation;

(4) Polyadenylation sequence.

In a preferred embodiment, the transcriptional regulatory element is the HLP2 promoter shown in SEQ ID NO: 5 or the enTTR promoter shown in SEQ ID NO: 6. In a more preferred embodiment, the transcriptional regulatory element is the enTTR promoter shown in SEQ ID NO:6.

In a preferred embodiment, the intron is selected from FIX Ti299 shown in SEQ ID NO: 8, the modified SV40 intron (modified SV40intron) shown in SEQ ID NO: 9, the modified SV40 intron shown in SEQ ID NO: 10 FIX intronA shown in, SEQ ID NO: the FIX intronAco intron shown in 11, more preferably the FIX intronAco intron shown in SEQ ID NO: 11.

In a preferred embodiment, the coding sequence of the gene is codon-optimized for expression in human cells.

In some embodiments, the gene coding sequence has the following characteristics:

(1) the sequence identity is less than 80% compared to the FIX wild-type sequence of SEQ ID NO: 13 or the FIX ^PADUA wild-type coding sequence of SEQ ID NO: 12; and

(2) Compared with any of the sequences shown in SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23, the sequence identity is higher than 85%, preferably higher than 90%.

In a further embodiment, the gene coding sequence has the following characteristics:

(1) Compared with the FIX wild-type sequence of SEQ ID NO:13 or the FIX ^PADUA wild-type coding sequence of SEQ ID NO:12, the sequence identity is lower than 80%;

(2) Compared with the sequence shown in any one of SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23, the sequence identity is higher than 85%, preferably higher than 90%;

(3) Compared with the sequences shown in SEQ ID NO:19 and SEQ ID NO:22, the sequence identity is lower than 80%.

In further or additional embodiments, the gene coding sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 16, 17, 18, 20, 21 and 23, or at least 85% thereof Nucleotide sequences that are identical, preferably at least 90% identical, more preferably at least 95% identical, still more preferably at least 98% identical. In further or additional embodiments, the gene coding sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 17, 20 and 23, or has at least 85% identity therewith, preferably at least 90% % identity, more preferably at least 95% identity, still more preferably at least 98% identity of the nucleotide sequences. In a more preferred embodiment, the gene coding sequence is a nucleotide sequence as shown in any one of SEQ ID NOs: 16, 17, 18, 20, 21 and 23. In the most preferred embodiment, the gene coding sequence is the nucleotide sequence shown in SEQ ID NO:23.

In some embodiments, the CpG number of the gene coding sequence is less than 100, preferably less than 10, and most preferably 0. In some embodiments, the gene coding sequence is free of CpG islands.

In a preferred embodiment, the polyadenylation sequence is selected from SV40 polyA shown in SEQ ID NO:14 or bGH polyA shown in SEQ ID NO:15, preferably SV40 polyA shown in SEQ ID NO:14.

In a specific embodiment, the expression construct described in the first aspect of the present invention is used for treating or preventing the hemorrhagic symptoms of HB caused by the coagulation dysfunction caused by the deficiency of human coagulation factor IX.

In a second aspect, the present invention provides a recombinant adeno-associated virus (rAAV) vector, which comprises the nucleic acid construct of the first aspect and at least one AAV inverted terminal repeat (ITR). Preferably, the rAAV vector comprises two AAV ITRs. Preferably, the AAV ITR is derived from an AAV2 ITR, such as an AAV2 ITR or a variant thereof, such as an AAV2 ITR lacking a C region or a C' region. In a specific embodiment, the rAAV vector comprises an AAV2 ITR and an AAV2 ITR variant lacking a C or C' region. In a specific embodiment, the rAAV vector comprises an AAV2 ITR upstream of the coding sequence and an AAV2 ITR variant lacking a C region downstream of the coding sequence.

In a specific embodiment, the rAAV vector according to the second aspect of the present invention is used to treat or prevent the hemorrhagic symptoms of HB caused by the coagulation dysfunction caused by the deficiency of human coagulation factor IX.

In a third aspect, the present invention provides rAAV virus particles comprising the rAAV vector of the second aspect and a capsid. Preferably, the capsid is selected from AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVLK03, AAVS3, AAVKP1, AAVrh10, AAVNP40, AAVNP59, AAV-DJ, AAVAnc80L65, AAVsL65, AAVHSC15, AAVC102, AAV204 , AAV214 serotype capsids and variants thereof. Preferably, the rAAV virus particle comprises an AAV8 serotype capsid.

In a specific embodiment, the rAAV virus particles described in the third aspect of the present invention are used for treating or preventing the hemorrhagic symptoms of HB caused by coagulation dysfunction caused by the deficiency of human coagulation factor IX.

In the fourth aspect, the present invention provides a pharmaceutical composition, which comprises the nucleic acid construct of the first aspect, the rAAV vector of the second aspect or the rAAV virus particle of the third aspect, and a pharmaceutically acceptable carrier.

In a specific embodiment, the pharmaceutical composition of the fourth aspect of the present invention is used to treat or prevent the bleeding symptoms of HB caused by the coagulation dysfunction caused by the deficiency of human coagulation factor IX.

In a fifth aspect, the present invention relates to the use of the nucleic acid construct of the first aspect, the rAAV vector of the second aspect or the rAAV particle of the third aspect in the preparation of a medicament for treating hemophilia B in a subject. Preferably, the medicament is for intravenous administration.

In a preferred embodiment, the medicament is administered at 4×10 ¹¹ to 2×10 ¹² viral genomes per kilogram of body weight (vg/kg).

In specific embodiments, said subject is a mammal, preferably a human.

In preferred embodiments, the subject produces an insufficient amount of Factor IX protein, or produces a defective or abnormal Factor IX protein.

Description of drawings

Figure 1 is a histogram showing the results of scAAV vector-mediated expression level and activity detection of FIX mutants in HepG2 cells.

Figure 2 is a histogram showing the results of ssAAV vector-mediated expression level and activity detection of FIX mutants in HepG2 cells.

Fig. 3 shows the structural representations of nine scAAV vector expression cassettes (HB1-HB9), eight ssAAV vector expression cassettes (HB10-HB17) and a control construct (HB0) constructed in the present invention. The white arrow is the promoter, the black box or black dotted line is the intron, the middle arrow is the FIX-PADUA coding sequence, and the gray box on the right is the poly(A) signal.

The histogram in Figure 4 shows the detection results of FIX expression levels in HepG2 cells mediated by HB1-HB7 and HB0 as a control.

The histogram in Figure 5 shows the detection results of FIX expression levels in HepG2 cells mediated by HB1, HB8, HB9, and HB0 (left and right are two repeated experiments).

The histogram in Figure 6 shows the detection results of FIX expression levels in Huh7 cells mediated by HB1, HB8, HB9, and HB0.

The histogram in Figure 7 shows the detection results of FIX expression level in HepG2 cells mediated by HB10-HB14 and HB0 as a control.

The histogram in Figure 8 shows the detection results of FIX expression levels in HepG2 cells mediated by HB11, HB15-HB17, and HB0.

The histogram in Figure 9 shows the detection results of FIX expression levels in Huh7 cells mediated by HB11, HB15-HB17, and HB0.

The histogram in Figure 10 shows the detection results of the expression levels and activities of different codon-optimized FIX ^PADUA coding sequences delivered by the HB9 vector in HepG2 cells.

Figure 11 is a histogram showing the expression levels and activity detection results of different codon-optimized FIX ^PADUA coding sequences delivered by the HB17 vector in HepG2 and Huh7 cells.

Figure 12 shows the data distribution graph (median) of tail docking bleeding time 30 weeks after administration of VGB-R04.

Fig. 13 shows the data distribution graph (median) of the amount of tail docking bleeding 30 weeks after administration of VGB-R04.

Detailed description of the invention

The practice of some of the methods disclosed herein employs, unless otherwise indicated, conventional techniques of botany, biochemistry, chemistry, molecular biology, cell biology, genetics and recombinant DNA, which are within the skill of the art. See, eg, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) et al.

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, ie, limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly for biological systems or processes, the term may mean an order of magnitude of a value, ie 10 times, preferably within 5 times, more preferably within 2 times. Where specific values are described in the application and claims, unless otherwise stated, the term "about" should be understood as within a range of error acceptable in the context.

The term "gene" as used herein refers to a nucleic acid (eg, DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence encoding an RNA transcript. As used herein, the term with respect to genomic DNA includes intervening non-coding regions as well as regulatory regions, and may include 5' and 3' ends. In some uses, the term includes transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" encompasses only the coding sequence necessary to encode a polypeptide (eg, an "open reading frame" or "coding region"). In some cases, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. A gene may refer to a "foreign gene" or a non-native gene. A non-native gene may refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions and/or deletions (eg, a non-native sequence).

The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotides may comprise synthetic nucleotides. Nucleotides may comprise synthetic nucleotide analogs. A nucleotide may be the monomeric unit of a nucleic acid sequence (eg, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphate such as dATP, dCTP , dITP, dUTP, dGTP, dTTP or a derivative thereof. These derivatives can include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well known techniques. Labeling can also be done with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length that can in single-, double-, or multi-stranded form. Polynucleotides can be exogenous or endogenous to the cell. Polynucleotides can be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide may comprise one or more analogs (eg, altered backbones, sugars or nucleobases).

The terms "hemophilia B" and "hemophilia B" have the same meaning in the context of the present invention and may be abbreviated as "HB". HB is a genetic disease caused by the defect of coagulation factor IX (FIX), which is a chromosomal recessive genetic disease. Due to the lack of coagulation factors, patients will have coagulation disorders, with bleeding as the main clinical manifestation.

The term "treating" includes curing a disease, or at least partially alleviating, alleviating one or more symptoms of a disease. For example, in the context of the present invention, treatment may reduce the frequency of bleeding, shorten the duration of bleeding, or reduce the total amount of bleeding, among other things.

The term "subject" in the present invention refers to animals, preferably vertebrates, more preferably mammals, such as rodents, such as mice, rats; primates, such as monkeys; most preferably humans.

FIX expression construct

In the context of the present invention, "expression construct" is synonymous with "expression cassette", which is usually part of or will be introduced into a vector or construct, which usually contains one or more The coding sequence and the regulatory sequences that regulate the expression of the coding sequence operably linked thereto, such as promoters, enhancers, translation termination signals such as polyadenylation sequences, etc.

The term "operably connected" means that various elements are connected in a manner capable of performing their respective functions.

The expression cassette of the present invention may also contain non-coding sequences such as introns, untranslated region sequences and the like.

Regulatory sequences can be selected for a particular purpose. For example, a tissue-specific promoter can be selected for expression of a gene of interest in a particular tissue. However, the effects of various elements, including coding sequences and control sequences, combined in different ways, and their effects on the coding sequence of a specific gene of interest, are generally difficult to predict.

The expression construct of the present invention is designed for high-efficiency and stable expression of the FIX gene, especially in the liver, so it at least comprises transcriptional regulatory elements (such as promoter, enhancer-promoter), intron, FIX gene coding sequence and translation termination signals.

transcriptional regulatory element

In the context of the present invention, "transcriptional regulatory element" refers to a non-coding DNA sequence that regulates the transcription of a gene of interest. Transcriptional regulatory elements can be divided into cis-acting elements and trans-acting elements. The cis-acting element and the gene being regulated are located on the same nucleic acid molecule, usually in close proximity. Cis-acting elements contain transcription factor binding sites, which initiate gene transcription and regulate transcription efficiency by binding to transcription factors. Cis-acting elements generally include promoters, enhancers, and silencers. Trans-acting elements are located on different chromosomes or nucleic acid molecules, which encode trans-acting factors, ie transcription factors, and regulate gene expression by interacting with cis-acting elements.

Transcriptional regulatory elements in the constructs and recombinant vectors of the invention comprise promoter or enhancer-promoter elements.

A "promoter" is a 5' cis-acting DNA sequence that initiates transcription of a gene. According to the characteristics of promoters, they can be divided into constitutive promoters, tissue or cell-specific promoters, and developmental stage-specific promoters. The promoter may be a native promoter of a naturally occurring gene, or an artificially modified promoter.

"Enhancer" is also a cis-acting element, which acts on the promoter to activate and enhance the transcription level of the gene.

In order to achieve the purpose of the present invention, it is particularly hoped that the construct or vector of the present invention or the enhancer-promoter can mediate high expression of FIX in the liver. Strong constitutive promoters, more preferably liver-specific promoters, can therefore be used.

A variety of liver-specific promoters or enhancer-promoter elements are known in the art, including but not limited to Alb (albumin) promoter, Cyp3a4 (cytochrome P450 3A4) promoter, ET (transthyretin) promoter, hAAT (human α1-antitrypsin; human α-1-antitrypsin) promoter, HLP (hybrid liver-specific promoter; hybrid liver-specific promoter), HLP2, apolipoprotein 2 promoter, LP1 (liver-specific promoter; Liver-specific promoter 1), miR-122 (miRNA-122) promoter, hemopexin promoter, TTR (transthyretin) promoter, enTTR, APOE-hAAT.

In a preferred embodiment, the promoter or enhancer-promoter element is selected from LP1, HLP, TTR, HLP2, enTTR, APOE-hAAT, most preferably enTTR.

"LP1 promoter" is a highly specific liver-specific promoter (Amit C. Nathwani et al., Blood. 2006, 107(7):2653-2661). The LP1 promoter contains liver-specific elements from the liver regulatory region (HCR) of the human apolipoprotein E/C-I locus and the hAAT promoter encoded by SERPINA1 (Serpin family A member 1). The LP1 promoter of the present invention has the sequence shown in SEQ ID NO:2. In several studies including the aforementioned study by Amit C. Nathwani et al. (Amit C. Nathwani et al., 2006, supra; Wei Lu et al., Front Med. 2016 Jun;10(2):212-8), Both used the LP1 promoter to construct recombinant AAV vectors expressing human coagulation factor IX.

"HLP" and "HLP2" both mean a hybrid liver-specific promoter. In the present invention, the sequences of HLP and HLP2 are shown in SEQ ID NO:3 and SEQ ID NO:5 respectively.

"TTR" includes the enhancer and promoter of transthyretin, and the commonly used TTR sequences are different. The TTR sequence of the present invention is shown in SEQ ID NO:4. Although it contains an enhancer region, it is also sometimes referred to as a "TTR promoter" in the context of the present invention.

"enTTR" further includes a liver-specific cis-regulatory element—3xSERP (see EP3270944B1 for its detailed sequence) on the basis of TTR, and its sequence is shown in SEQ ID NO:6 in the present invention. Although it contains an enhancer region and cis-regulatory elements, it is also sometimes referred to as the "enTTR promoter" in the context of the present invention.

"APOE-hAAT" consists of the hepatic locus regulatory element of the apolipoprotein E (APOE) gene and the AAT promoter. LP1, HLP, HLP2 are all truncated versions of APOE-hAAT. APOE-hAAT of the present invention is shown in SEQ ID NO:7.

intron

The expression construct of the present invention also comprises an intron sequence, which is generally located downstream of the promoter sequence and upstream of the FIX gene coding sequence. In some cases, introns can enhance expression of a gene in an expression construct or recombinant vector in eukaryotic cells.

Natural introns of other genes or modified introns can be used, such as modified SV40 introns, such as the SV40 intron shown in SEQ ID NO:9. Introns of the human FIX gene itself can also be used. In a preferred embodiment, the FIX intronAco intron shown in FIX Ti299 shown in SEQ ID NO: 8, FIX intronA shown in SEQ ID NO: 10, and SEQ ID NO: 11 can be used, preferably SEQ ID NO : FIX intronAco intron shown in 11.

polyadenylation sequence

The expression constructs of the invention also comprise a polyadenylation sequence, also known as polyA or poly(A). The polyadenylation sequence is the untranslated region 3' of the coding sequence. A variety of polyadenylation sequences commonly used in expression constructs are known in the art. In an embodiment of the present invention, the polyA may be SV40 polyA or bGH polyA, such as SV40 polyA of SEQ ID NO: 14 or bGH polyA of SEQ ID NO: 15. In a preferred embodiment, the polyadenylation sequence is SV40 polyA as shown in SEQ ID NO: 14.

coding sequence

As the target gene coding sequence delivered by a viral vector, the sequence encoding the FIX gene can encode wild-type FIX, and can also encode mutant FIX, such as encoding mutant FIX with higher activity than wild-type, such as encoding blood coagulation factor IX with R338L mutation FIX ^PADUA .

In some embodiments, the coding sequence of the target gene is a codon-optimized coding sequence to achieve better expression in a subject, such as safer and more efficient expression. Although some principles of codon optimization are known in the art, for FIX and FIX ^PADUA coding sequences, it is difficult to know in advance which sequence will have better expression efficiency or activity level without further experimental verification , especially the expression efficiency or activity level in vivo.

In a preferred embodiment, the codon-optimized sequence may have a higher expression level and/or higher protein activity, for example at least 10%, at least 20%, at least 30% higher than the non-optimized sequence , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even at least 100% expression level and/or protein activity. The measurement of expression level and protein activity can be carried out according to methods known to those skilled in the art, such as the methods described in the Examples of the present invention.

On the other hand, the present invention also contemplates the CpG content and distribution of coding sequences. "CpG content" refers to the content of cytosine (C) guanine (G) dinucleotides linked by phosphate (p) in a DNA sequence. "CpG islands" are regions of the genome where CpG dinucleotides occur with a high probability. For example, the presence of CpG islands is determined in the present invention using the algorithm described by Gardiner-Garden and Frommer (1987). Specifically, a region containing at least 200 bp, in which the proportion of GC exceeds 50%, and the ratio of observed value/predicted value of CpG is higher than 0.6, is called "CpG island". The predicted value of CpG is: the number of C in the window multiplied by the number of G in the window, divided by the window length. In mammals, the unmethylated CpG of exogenous genes will be recognized by TLR9, which activates CD8 ⁺ T cells to clear infected cells, which is not conducive to the long-term expression of exogenous genes. Therefore, in order to express the gene encoding FIX more safely and efficiently in the human body, it is most preferable to reduce the number of CpG islands to zero in the present invention. The coding sequences of the invention also preferably contain a low CpG content. When the CpG content and the number of CpG islands are also considered as conditions for sequence optimization, it further increases the difficulty of sequence design.

The inventors of the present invention developed a variety of optimized FIX ^PADUA coding sequences and surprisingly found that some of them had significantly improved expression levels and increased protein activity relative to the wild-type FIX ^PADUA coding sequence. On this basis, it is also found that these effect-improved sequences have high sequence identity, and when compared with other sequences (such as optimized sequences with insignificant or unimproved effects, or wild-type sequences), The sequence identity is lower.

"Sequence identity" is used in the present invention to describe the percentage of the degree of similarity between two nucleotide sequences, which is calculated as follows: After aligning the two nucleotide sequences, the two nucleotide sequences have the same nucleoside The total number of acid positions is divided by the total number of nucleotides and multiplied by 100%. The way of aligning nucleotide sequences and the method of calculating sequence identity are known to those skilled in the art, for example, the tool blastn provided by NCBI can be used in "highly similar sequences (megablast)" mode.

rAAV vectors and viral particles

In healthy people, FIX factor is produced in the liver. In order to enhance the expression efficiency of the liver and avoid unnecessary expression, the AAV gene therapy of the present invention specifically delivers the FIX factor gene to the liver through the AAV vector, and mediates the spatiotemporal specific expression of the FIX factor through the liver-specific expression regulatory sequence.

A "vector" generally refers in the art to a recombinant plasmid or virus used to deliver nucleic acid. The recombinant AAV (rAAV) vector of the present invention refers to an AAV viral vector comprising a heterologous nucleic acid to be delivered, and the two ends or at least one end of the heterologous nucleic acid are AAV inverted terminal repeat (inverted terminal repeat; ITR) sequences.

The heterologous nucleic acid may be or comprise a recombinant construct or expression cassette of the invention. In one embodiment, rAAV vectors of the invention may comprise one or more recombinant constructs or expression cassettes.

The AAV ITR sequence refers to the sequence at both ends of the natural single-stranded AAV genome, which is about 145 nucleotides long. The outermost 125 nucleotides contain multiple self-complementary regions, including a large palindromic sequence A-A' and two small palindromic sequences B-B' and C-C'. A 20-nucleotide D region is also included in the outermost 125 nucleotides. The D region contains a terminal recognition sequence TRS, which is cleaved by the AAV rep protein during viral DNA replication. The ITR sequence of the present invention may be an ITR sequence of any AAV serotype or a variant thereof, preferably an AAV2 ITR or a variant thereof. The ITR sequence can be from the same or a different AAV serotype as the capsid.

The design of self-complementary AAV (scAAV) connects the two complementary strands together by deleting the ITR terminal recognition sequence TRS on one side, which is similar to double-stranded DNA molecules. After infection, it can self-fold into double strands with transcriptional activity and improve The replication efficiency of the virus greatly improves the infection efficiency and achieves high expression of FIX. Although the self-complementary structure also reduces the packaging size allowed by the expression cassette (the single-stranded form allows a maximum packaging of 5kb, and each single-stranded self-complementary can only allow a maximum of 3.3kb), but the FIX cDNA is only 1.4kb, so it can meet the requirements of scAAV. packaging design.

The present invention also provides a viral particle comprising a recombinant AAV vector, the viral particle further comprising a capsid. The capsid used in the present invention can be selected from capsids of any serotype, including but not limited to capsids selected from AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVLK03, AAVS3, AAVKP1, AAVrh10, AAVNP40 , AAVNP59, AAV-DJ, AAVNc80L65, AAVsL65, AAVHSC15, AAVC102, AAV204, AAV214 serotype capsids and their variants. In a preferred embodiment, the viral particle comprises a capsid with tissue tropism for the liver, especially when the viral particle is delivered systemically by intravenous administration. For example, a viral particle of the invention may have a capsid of the AAV8 serotype.

pharmaceutical composition

The pharmaceutical composition of the present invention may comprise the expression construct of the present invention, rAAV vector, rAAV virus particle and pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" means an excipient suitable for delivery into a subject and consistent with the therapeutic purposes of the invention, which does not cause unacceptable toxicity, allergic reactions or other problems in the subject . The pharmaceutically acceptable carrier can be liquid, such as water, ethanol, or solid, such as starch.

In addition to the active ingredient and pharmaceutically acceptable carrier for delivering the gene encoding FIX, the pharmaceutical composition may also contain other additives, such as stabilizers, diluents, coloring agents and the like.

apply

HB can be treated by delivering a transgene encoding FIX or a FIX variant by administering the expression constructs, rAAV vectors, rAAV virions and pharmaceutical compositions of the invention to animals.

Administration can be by systemic administration, or locally into a desired organ, tissue or cell, such as the liver.

In some instances, rAAV vectors or rAAV virions of the invention, or pharmaceutical compositions comprising them, are administered intravenously, such as by intravenous infusion.

The administered dose may be in the range of 1×10 ¹¹ -1×10 ¹³ vg/kg, preferably 4×10 ¹¹ -2×10 ¹² vg/kg body weight.

The invention also relates to the following:

Item 1. A nucleic acid construct comprising the following elements operably linked from 5' to 3':

(1) a transcriptional regulatory element selected from the group consisting of LP1, HLP, TTR, HLP2, enTTR, APOE-hAAT;

(2) an intron selected from the SV40 intron or the FIX gene intron;

(3) Gene coding sequence, which encodes coagulation factor IX with R338L mutation;

(4) A polyadenylation sequence selected from SV40 polyA or bGH polyA.

Item 2. the nucleic acid construct of item 1, wherein said transcription control element is the HLP2 promoter shown in SEQ ID NO:5 or the enTTR promoter shown in SEQ ID NO:6, preferably as SEQ ID NO:6 The indicated enTTR promoters.

Item 3. the nucleic acid construct of item 1 or 2, wherein said intron is selected from the modified SV40 intron (mSV40) shown in FIX Ti299 shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:9. The FIX intronA shown in ID NO:10, the FIX intronAco intron shown in SEQ ID NO:11, preferably the FIX intronAco intron shown in SEQ ID NO:11.

Item 4. The nucleic acid construct according to any one of Items 1-3, wherein the gene coding sequence is codon-optimized for expression in human cells.

Item 5. The nucleic acid construct of item 4, the gene coding sequence has the following characteristics:

(1) the sequence identity is less than 80% compared to the FIX wild-type sequence of SEQ ID NO: 13 or the FIX ^PADUA wild-type coding sequence of SEQ ID NO: 12; and

(2) Compared with any of the sequences shown in SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23, the sequence identity is higher than 85%, preferably higher than 90%.

Item 6. The nucleic acid construct of item 5, the gene coding sequence further has the following characteristics:

(3) Compared with the sequences shown in SEQ ID NO:19 and SEQ ID NO:22, the sequence identity is lower than 80%.

Item 7. The nucleic acid construct of any one of items 4-6, the gene coding sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 16, 17, 18, 20, 21 and 23, Or a nucleotide sequence having at least 85% identity thereto, preferably at least 90% identity, more preferably at least 95% identity, still more preferably at least 98% identity thereto.

Item 8. The nucleic acid construct of any one of Item 4-7, the gene coding sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 17, 20 and 23, or at least 85% thereof Nucleotide sequences that are identical, preferably at least 90% identical, more preferably at least 95% identical, still more preferably at least 98% identical.

Item 9. The nucleic acid construct according to any one of items 4-8, wherein the gene coding sequence is a nucleotide sequence shown in any one of SEQ ID NOs: 16, 17, 18, 20, 21 and 23.

Item 10. The nucleic acid construct according to any one of items 4-9, wherein the gene coding sequence has less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, CpG content below 30, below 20, below 10, below 5 or zero.

Item 11. The nucleic acid construct according to any one of Items 4-10, wherein the gene coding sequence does not contain a CpG island.

Item 12. The nucleic acid construct described in any one of items 1-11, wherein the polyadenylation sequence is SV40 polyA shown in SEQ ID NO: 14 or bGH polyA shown in SEQ ID NO: 15, preferably SEQ ID NO: SV40 polyA shown in ID NO:14.

Item 13. A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid construct of any one of Items 1-12 and at least one AAV inverted terminal repeat (ITR).

Item 14. The rAAV vector of Item 13, comprising two AAV ITRs.

Item 15. The rAAV vector of Item 13 or 14, wherein the AAV ITR is derived from the AAV2 ITR.

Item 16. The rAAV vector of item 15, said rAAV vector comprising an AAV2 ITR and an AAV2 ITR variant.

Item 17. The rAAV vector of Item 16, wherein the AAV2 ITR variant is an AAV2 ITR variant lacking a C region or a C' region.

Item 18. The rAAV vector of item 16 or 17, said AAV2 ITR is located upstream of a coding sequence, and said AAV2 ITR variant is located downstream of a coding sequence.

Item 19. An rAAV virus particle comprising the rAAV vector of any one of Items 13-18 and a capsid.

Item 20. The rAAV virus particle of Item 19, comprising an AAV8 serotype capsid.

Item 21. A pharmaceutical composition comprising the nucleic acid construct described in any one of Items 1-12, the rAAV construct described in any one of Items 13-18, or the rAAV described in Item 19 or 20 Virus particles, and a pharmaceutically acceptable carrier.

Item 22. The nucleic acid construct described in any one of Items 1-12, the rAAV construct described in any one of Items 13-18, or the rAAV virus particle described in Item 19 or 20 in preparation for treatment or Use of a medicament for preventing hemophilia B or bleeding symptoms associated therewith in a subject.

Item 23. The use of Item 22, the medicament is for intravenous administration.

Item 24. The use of Item 23, wherein the drug is administered at a dose of 4×10 ¹¹ to 2×10 ¹² viral genomes per kilogram of body weight (vg/kg).

Item 25. The use of any one of Items 22-24, wherein the subject is a mammal, preferably a human.

Item 26. The use of any one of Items 22-25, wherein the subject produces insufficient or defective or abnormal Factor IX protein.

Example

In order to understand and apply the present invention more fully, the present invention will be described in detail below with reference to the examples and accompanying drawings, which are only intended to illustrate the present invention and not intended to limit the scope of the present invention. The scope of the invention is specifically defined by the appended claims.

Experimental materials and methods

Experimental materials and methods used in the examples are described below.

Unless otherwise specified, the recombinant adeno-associated virus vector of the present invention is delivered using the AAV8 serotype.

1. Detection method of blood coagulation factor IX concentration based on ELISA method

1.1 Method principle

Coagulation factor IX/FIX/F9 antibodies and rabbit monoclonal antibodies (Rabbit Mab) were used as capture reagents and coated onto 96-well microtiter plates. After binding the analyte, add the detection antibody Biotin-F2645 (biotin anti-factor IV antibody, mouse monoclonal-clone HIX-1, purified hybridoma cell culture) to combine with the analyte, add enzyme-labeled streptavidin Heparin (SA-HRP), followed by the addition of the substrate TMB, produces a color reaction. The depth of the color is directly proportional to the concentration of the analyte. Add stop solution to terminate the reaction, detect at a wavelength of 450nm (reference wavelength is 630nm) with a microplate reader, and read the OD value by shaking for 5s. The theoretical concentration is taken as the abscissa, and the difference between the measured corrected OD value and the average OD value of the blank duplicate wells is taken as the ordinate, and the relevant parameters of the standard curve are fitted by a four-parameter regression model, so that the sample concentration can be calculated.

1.2 Experimental steps

a) Add coating solution to the 96-well plate, wash the plate with washing buffer after incubation;

b) Add blocking solution, wash the plate with washing buffer after incubation;

c) Add standard music, quality control and samples to be tested respectively, and wash the plate with washing buffer after incubation;

d) adding the detection antibody, and washing the plate with washing buffer after incubation;

e) adding SA-HRP, washing the plate with washing buffer after incubation;

f) adding TMB for color development;

g) adding a stop solution to terminate the reaction;

h) Use a microplate reader to read the absorbance value under the condition of 450-630nm.

2. Activity detection method of blood coagulation factor FIX

2.1 Method principle

BIOPHEN FIX activity assay kit was used to detect hFIX activity. The principle of the method is as follows: under the action of phospholipids and calcium, factor XIa activates the fixed factor IX in the sample to be tested, and converts it into activated factor IX. Factor VIII:C is activated by thrombin and forms an enzyme complex with factor IXa to activate factor X. The resulting factor Xa hydrolyzes the chromogenic substrate, resulting in the release of p-aminoaniline, which is proportional to the concentration of factor IX in the sample, adding 2% citric acid to terminate the reaction, and detecting it with a microplate reader at a wavelength of 405nm Read the OD value. Take the theoretical FⅨ activity (IU/mL) as the abscissa, measure the difference between the OD mean value of the standard curve duplicate hole and the blank duplicate hole OD mean value as the ordinate, and use the four-parameter regression model to fit the relevant parameters of the standard curve, The FIX activity in the sample can thus be calculated.

2.2 Experimental steps

a) Add the diluted standard curve, quality control and samples to the 96-well plate respectively;

b) Add R1 (Reagent 1; Reagent 1): Human Factor X and lyophilized FVIII:C (Human Factor X and lyophilized FVIII:C), mix well, and incubate;

c) Add R2 (Reagent 2; Reagent 2): Activator reagent, mix well, and incubate;

d) Add R3 (Reagent 3; Reagent 3): Substrate-Lyophilized chromogenic substrate specific to Factor Xa (Substrate-Lyophilized chromogenic substrate specific to Factor Xa), mix well, and incubate;

e) adding 2% citric acid to terminate the reaction;

f) Detect the absorbance value at 405 nm with a microplate reader.

3. AAV8 neutralizing antibody (NAB) detection method

3.1 Method principle

HEK293 cells can express luciferase after infection with recombinant adeno-associated virus 8 (AAV8) carrying the luciferase gene. After adding the corresponding substrate, the luciferase can catalyze the substrate to generate a luminescent product, and the more the product, the higher the fluorescence value. If there is an anti-AAV8 neutralizing antibody, AAV8 carrying the luciferase gene cannot infect HEK293 cells and express luciferase, and the fluorescence value decreases accordingly. Therefore, the content of anti-AAV8 neutralizing antibody in plasma can be evaluated by detecting the fluorescence value.

3.2 Experimental steps

a) HEK293 cells are recovered and resuspended;

b) Plating: add the cell suspension to the cell culture plate and incubate;

c) Sealing of the sample dilution plate: take another cell culture plate and add 1% BSA solution, incubate, and wash the plate with 1xPBS;

d) Sample incubation: add the diluted test sample and AAV8-Luciferase Work Solution to the sealed cell culture plate, mix and incubate;

e) Sample addition: add the incubated sample to the culture plate where the cells are placed, shake gently, and wash the plate with 1xPBS after incubation;

f) Cell lysis: add cell lysate to the cell culture plate, shake at room temperature;

g) Detection: absorb the supernatant after cell lysis, transfer to 96F NONTREATED WHITE MICROWELL SH, add luciferase detection reagent, and shake in the dark;

h) Reading: RLU was determined using a microplate reader.

4. Blood coagulation factor FIX-padua neutralizing antibody detection method (FⅨ-padua NAB assay)

4.1 Method principle

Human hFⅨ-deficient plasma was used to serially dilute the test samples according to a certain dilution factor, and then mixed with standard human plasma to detect residual hFⅨ activity using BIOPHEN FⅨ activity assay kit. The amount of anti-hFIX-Padua neutralizing antibody is represented by Bethesda Unit (Bu), when the residual activity ratio (the ratio of residual activity to blank control sample activity) is 50%, it is 1BU.

The principle of using the BIOPHEN FIX activity assay kit to detect hFIX activity is as follows: under the action of phospholipids and calcium, factor XIa activates the immobilized factor IX in the test sample and converts it into activated factor IX. Factor VIII:C is activated by thrombin and forms an enzyme complex with factor IXa to activate factor X. The resulting factor Xa hydrolyzes the chromogenic substrate, resulting in the release of p-aminoaniline, which is proportional to the concentration of factor IX in the sample, adding 2% citric acid to terminate the reaction, and detecting it with a microplate reader at a wavelength of 405nm Read the OD value. hFIX residual activity (100%, %RA) = hFIX activity in test sample/hFIX activity in negative control sample*100.

BU=(2-log%RA)/0.301*dilution factor. ≥0.5BU was judged as anti-hFIX neutralizing antibody positive.

4.2 Experimental steps

a) Add standard music, quality control, samples to be tested and negative control samples to the 96-well plate;

b) Add R1, mix and incubate;

c) Add R2, mix well, and incubate;

d) Add R3, mix well, and incubate;

e) adding 2% citric acid to terminate the reaction;

f) Detect the absorbance value at 405 nm with a microplate reader.

Embodiment 1. Selection of highly active FIX mutants

In the early clinical trials of FIX gene therapy, the wild-type FIX protein sequence was selected, and the codon optimization of the FIX coding sequence was not performed, and the steady-state maintenance level of FIX activity was low and the time was short.

FIX ^PADUA is a naturally occurring FIX mutant with a single amino acid mutation (R338L) found in an Italian adolescent patient with venous thrombosis. Compared with wild-type FIX, the coagulation activity (FIX:C) can be increased by 5-10 times under the same expression level (FIX:Ag). Spark's SPK-9001, UniQure's AMT-061 and Freeline's FLT180a all selected highly active FIX ^PADUA variants as transduction genes.

To confirm whether FIX ^PADUA variants indeed have improved expression levels and protein activity relative to wild-type FIX, FIX ^WT , FIX ^{PADUA were} ligated into scAAV vectors and ssAAV vectors and used to transfect human liver tumor cell lines HepG2 and Huh7.

Specifically, HepG2 and Huh7 cells were cultured in high-glucose DMEM+10% FBS, and digested with TrypLE when passaged. ¹ day before transfection, 2.2×105 cells/well were seeded in a 12-well plate. Transfection was performed using Lipofectamine 3000 kit (Invitrogen, L3000008) and following the HepG2 transfection protocol recommended by the official website. 1 μg of plasmid was transfected per well, and each plasmid was transfected in triplicate wells. After transfection, the medium was replaced with DMEM+1% GlutaMax+10 μg/mL vitamin K. Cell culture supernatant was collected 72 hours after transfection for FIX ELISA detection (abcam, ab168546).

Experimental results using HepG2 cells showed that the expression level of FIX ^WT was high but the specific activity of the protein was low (Figure 1); the expression level of FIX ^PADUA was slightly lower than that of FIX ^WT but the specific activity of the protein was increased by 10.01-12.85 times (Figure 2). Therefore, FIX ^{PADUA was chosen} for further development.

Screening of Regulatory Elements in Example 2.FIX Gene Expression Cassette

The inventors selected different regulatory elements to design and prepare 9 scAAV vector expression cassettes (HB1-HB9) and 8 ssAAV vector expression cassettes (HB10-HB17) (Figure 3 and Table 1), and compared their expression in human liver tumor cells HepG2 and the expression efficiency in Huh7, and CMV-FIX ^PADUA as a control (HB0). Considering the difference in the DNA loading capacity of scAAV and ssAAV, the FIX expression efficiency of scAAV and ssAAV vectors were compared separately.

Table 1. Combinations of regulatory elements in the constructs

The transfection conditions were the same as in Example 1. Cell culture supernatants were collected 72 hours after transfection for FIX ELISA detection (abcam, ab168546) and Biophen FIX activity detection (HYPHEN BioMed, 221802).

The HepG2 cell experiment results of the first batch of 7 scAAV vectors (HB1-HB7) constructed showed that the expression efficiency mediated by the intron FIX Ti299 (Ti299) was higher than that of the SV40 intron (mSV40, the modified SV40 intron ) (HB1 is higher than other constructs); among the five promoters of LP1, HLP, TTR, HLP2, and enTTR, the highest expression efficiency is enTTR and HLP2 (HB5, HB6, HB7, and the difference between HB5 and HB6 is only in the promoter ); the contribution of translation termination signal SV40 polyA and bGH polyA to expression efficiency is similar (HB6 and HB7 have similar performance, the difference is only in polyA) (Figure 4). Based on these results, FIX Ti299 with better effect was selected as the intron in the second round of screening. Regarding the translation termination signal, considering the capacity of the AAV vector, a shorter SV40 polyA was selected for the second round of scAAV expression cassette screening.

After inserting FIX Ti299 into the two promoters with the highest expression efficiency in the first round of screening—HLP2 and enTTR, and using SV40 polyA, the second batch of two constructs, HB8 and HB9, were constructed. These two constructs were subjected to a second round of scAAV vector selection together with HB1, which performed best in the previous round, and still using HB0 as a control, the same experiment was carried out in both HepG2 and Huh7 cells. The results showed that the construct with the highest expression efficiency in both cells was HB9, which adopted the enTTR promoter and Ti299 intron (Fig. 5, Fig. 6).

CMV-FIX ^PADUA was selected as a control to compare the expression efficiency of the ssAAV expression cassette. The HepG2 cell experiment results of the first batch of 5 constructed ssAAV vectors (HB10-HB14) showed that the expression efficiency mediated by the intron FIX intronAco was higher than that of FIX intronA (HB11 was higher than HB10, the difference was only in the intron); APOE - Among the four promoters of hAAT, LP1, HLP2 and enTTR, enTTR (HB14) had the highest expression efficiency (Fig. 7). Therefore, after inserting FIX intronAco into APOE-hAAT, LP1, HLP2, and enTTR, a second round of ssAAV vector selection was carried out, and experiments were carried out in HepG2 and Huh7 cells. The results showed that the construct with the highest expression efficiency in both cells was HB17, which contained enTTR promoter and FIX intronAco intron (Fig. 8, Fig. 9). This result further verified that enTTR is the most efficient promoter.

Example 3. Optimization of Coding Sequences

The inventors designed and synthesized eight codon-optimized FIX ^PADUA coding sequences, named FIX-PADUA-co1 to FIX-PADUA-co8, respectively, and their nucleotide sequences are shown in SEQ ID NOs: 16-23. Using these codon-optimized coding sequences, the corresponding recombinant vectors were constructed and the transduction experiments as described in previous examples were repeated in HepG2 and Huh7 cells.

Figure 10 shows the detection results of the expression level and activity of the above 8 different codon optimized sequences delivered by the HB9 vector in HepG2 cells, and compared with the FIX ^PADUA coding sequence of SEQ ID NO:12 without codon optimization compared. As can be seen from the results in Figure 10, FIX-PADUA-co8, FIX-PADUA-co5 and FIX-PADUA-co2 are the three codon-optimized sequences with the best performance, especially those of FIX-PADUA-co8 and FIX-PADUA-co5 The performance is more than 2 times that of the control. In addition, the expression levels of FIX-PADUA-co3, FIX-PADUA-co6 and FIX-PADUA-co1 were also significantly increased relative to the control.

The applicant analyzed the sequence similarity between these optimized sequences and unoptimized sequences, as well as the CpG content and the number of CpG islands in the sequences through sequence comparison, and the results are shown in Table 2 below.

Table 2. Comparison of 8 optimized FIX ^PADUA coding sequences and non-optimized FIX ^PADUA coding sequences

the	CpG number	CpG island	Identity%
PADUA_co1	78	Have	76.63
PADUA_co2	87	Have	76.12
PADUA_co3	123	Have	73.52
PADUA_co4	63	Have	77.27
PADUA_co5	94	Have	74.31
PADUA_co6	113	Have	73.73
PADUA_co7	75	Have	75.18
PADUA_co8	0	none	78.51
PADUA_wt	19	none	100

It can be seen from the above Table 2 that the identity of the optimized sequence and the unoptimized sequence is lower than 80%, indicating that their similarity in structure is low. Applicants also analyzed the sequence similarity between each optimized sequence by alignment. The comparison was performed using NCBI's blastn in the "highly similar sequences (megablast)" mode, and the results are shown in Table 3 below.

Table 3. Nucleotide sequence identity between 8 optimized FIX ^PADUA coding sequences

N.S.S. = No significant similarity

Combining the sequence similarity data in Table 3 with the results in Figure 10, it was unexpectedly found that there is a certain correspondence between the sequence similarity and the expression level of the optimized sequence. For example, according to the expression level results in Figure 10, only FIX-PADUA-co4 and FIX-PADUA-co7 are not as effective as the unoptimized sequences, while these two sequences in Table 3 have no significant similarity to the other six optimized sequences , or less than 80% identity (FIX-PADUA-co7 has only 78.27% identity with FIX-PADUA-co1). On the other hand, the six optimized sequences in Figure 10 that outperformed the unoptimized sequences had a sequence identity of more than 85% between each pair, such as FIX-PADUA-co5 and FIX-PADUA-co4 and FIX-PADUA Other optimized sequences except -co7 have more than 90% similarity. These results show that the optimized sequence of the present invention with better effect, such as FIX-PADUA-co2 of SEQ ID NO:17, FIX-PADUA-co5 of SEQ ID NO:20 or FIX-PADUA-co5 of SEQ ID NO:23 Any optimized sequence of co8 having a higher sequence identity, such as at least 85% identity, or even at least 90% identity, is more likely to have improved expression levels relative to a non-optimized sequence.

Figure 11 shows the expression results of the three optimized sequences (FIX-PADUA-co2, FIX-PADUA-co5 and FIX-PADUA-co8) with the best expression efficiency when delivered by ssAAV HB17 in HepG2 and Huh7 cells . Under either scAAV or ssAAV delivery conditions, the expression efficiency of the three optimized sequences was significantly improved compared with the unoptimized sequence.

In subsequent experiments, considering the GC content of the coding sequence (Table 2), PADUA_co8 (SEQ ID NO: 23) was finally selected for the construction of gene therapy vectors for subsequent experiments.

Considering that after AAV infects the body, the mutated ITR sequence of scAAV will be recognized by TLR9 and cause an immune response, so scAAV has a greater risk of immunogenicity than ssAAV, so ssAAV-enTTR-FIXintronAco-FIX ^PADUA _co8-SV40 polyA was selected as the The final AAV vector was used for subsequent experiments and named VGB-R04.

Embodiment 4. Drug efficacy test in vivo

This example describes the efficacy test in mice using ssAAV-enTTR-FIXintronAco-FIX ^PADUA_co8 -SV40 polyA (VGB-R04).

1. Pharmacodynamics pre-test of single intravenous injection of VGB-R04 in C57BL/6 mice

C57BL/6 mice are highly sensitive to AAV8, and hemophilia B is an X-linked recessive genetic disease, and the affected population is male. Therefore, only male mice were used for evaluation in this study.

All the animals in this experiment were given a single intravenous injection of the corresponding dose of VGB-R04; a total of 2 dose groups (Group 1-3) were established, as shown in Table 4 below.

Table 4. Dose Grouping

Plasma was collected from all animals at three time points before administration, the first week after administration (W1), and the second week after administration (W2) for FIX ^PADUA activity analysis. The results are shown in Table 5 below.

Table 5. Test results

From the results in Table 5, it can be seen that VGB-R04 of the present invention can increase the activity of plasma coagulation factor FIX to a supraphysiological level in mice, and shows an obvious dose-effect relationship.

2. Pharmacodynamic long-acting test of VGB-R04 given to mice with hemophilia B (HB) by single intravenous injection test

The pharmacodynamic characteristics of VGB-R04 in hemophilia B (HB) pathological model animals, namely HB mice, were further studied, especially the long-term effect.

The experiment was carried out with HB mice (FIX KO mice), and normal C57 mice were used as controls. The FIX gene knockout mouse model has clear HB phenotypes such as coagulation disorders. The HB mice used in this experiment were constructed by the Institute of Zoology, Chinese Academy of Sciences. CRISPR/Cas technology was used to cut the genome at the second exon and the second intron of the C57 mouse FIX gene, resulting in the second exon. Partial deletion of the exon, frameshift mutation of the mutant gene sequence, leading to premature termination of transcription, and degradation of the wrong protein by the body.

HB is an X-chromosome-linked recessive genetic disease, and the onset population is male, so the test uses male animals.

All animals in this experiment were given the corresponding dose of VGB-R04 by single intravenous injection. A total of 5 dosage groups (groups 1-5) were established, and a vehicle control group (group 6) and a group of wild C57 control group (group 7, wild-type control group for acute tail-docking hemorrhage study) were designed in addition. See Table 6 for amounts and dose level information.

Table 6. Dose Grouping

Plasma was cross-collected from all animals in Group 1-Group 6 for FIX ^PADUA activity and protein concentration analysis. At least 6 individuals were ensured at each sampling point, and the blood collection time points were as follows (W=week):

Group 1-Group 5: before administration (D-3～D-1), W1, W2, W4, W8, W12, W16, W20, W24, W28, W30;

Group 6: before administration (D-3 to D-1), W12, W30.

All surviving mice in Group 1-Group 7 were subjected to tail-docking drug efficacy evaluation at W30, and the bleeding volume and bleeding time after tail-docking were observed. The results of the FIX activity measured by the test are shown in Table 7 below, where the FIX activity of the human standard plasma is set as 100%, and the detected human FIX activity in the mouse plasma is expressed as a percentage with reference to it.

Table 7. FIX activity in mouse plasma before and after administration

From the results in Table 7, it can be known that VGB-R04 of the present invention can significantly increase the plasma FIX activity of HB mice to physiological or supraphysiological levels in HB mice, and exhibits an obvious dose-effect relationship and long-term effect. The results of the tail-docking bleeding model showed that after administration of VGB-R04 of the present invention, the median of the bleeding volume and bleeding time of HB mice (as shown in Figure 12 and Table 8) showed a dose-dependent decline, indicating that VGB-R04 can significantly improve HB mice bleed.

Table 8. Tail docking bleeding time and bleeding volume data (median) 30 weeks after administration of VGB-R04

3. Pharmacodynamic test of single intravenous injection of VGB-R04 in cynomolgus monkeys

The pharmacodynamic characteristics of VGB-R04 in healthy cynomolgus monkeys were further studied.

Cynomolgus monkeys are sensitive to AAV. In the reported studies, cynomolgus monkeys have a good correlation with human clinical dose-effect relationship, and are ideal animal species for evaluating the pharmacodynamic behavior of AAV gene therapy products in vivo. The patients with hemophilia B are males. Therefore, male animals were used in this experiment.

Before administration, it is necessary to screen the anti-AAV8 neutralizing antibody in animals and select animals with a neutralizing antibody titer <1:5 for the test. All the animals in this experiment were given a single intravenous injection of the corresponding dose of VGB-R04; a total of 3 dose groups (groups 1-3) were established, as shown in Table 9 below.

Table 9. Dose Grouping

All animals were subjected to hematology and blood biochemical tests before administration and after administration on D8, D15, D22, D29, and D36 (D stands for day, indicating the number of days).

Plasma was collected from all animals for analysis of FIX ^PADUA activity, FIX ^PADUA protein concentration and anti-hFIX inhibitors. The time points of blood collection were as follows:

Group 1-Group 3: Before administration (D-3 to D-1, D1), after administration, once a week from D8 for 18 weeks in total.

FIX activity was detected by a one-phase method based on partial thrombin time (activated partial thromboplastin time, APTT), and the results are shown in Table 10 below, wherein the FIX activity of the cynomolgus monkey before administration was deducted and expressed as a percentage (%) Detected human FIX activity in cynomolgus monkey plasma. The results show that after the VGB-R04 of the present invention is given to cynomolgus monkeys, it can significantly improve the plasma FIX activity of cynomolgus monkeys, and show an obvious dose-effect relationship; the fluctuation of plasma FIX activity that occurs over time may be affected by the corresponding time Effect of anti-hFIX inhibitors detected in plasma.

Table 10. FIX activity and concentration in cynomolgus monkey plasma after administration

The sequence numbers and corresponding sequences of the present invention are shown in the table below.

SEQ ID NO	code name	describe
1	CMV	Promoter
2	LP1	Promoter
3	HLP		Promoter
4	TTR	Promoter
5	HLP2	Promoter
6	TTTR	Promoter
7	APOE-hAAT		Promoter
8	Ti299	intron
9	mSV40 intron	intron

10	FIX intron A	intron
11	FIX intronAco	intron
12	FIX-PADUA	CDS
13	FIX-WT	CDS
14	SV40 polyA		polyA
15	bGH polyA	polyA
16	FIX-PADUA-co1	codon-optimized CDS
17	FIX-PADUA-co2	codon-optimized CDS
18	FIX-PADUA-co3	codon-optimized CDS
19	FIX-PADUA-co4	codon-optimized CDS
20	FIX-PADUA-co5	codon-optimized CDS
twenty one	FIX-PADUA-co6	codon-optimized CDS
twenty two	FIX-PADUA-co7	codon-optimized CDS
twenty three	FIX-PADUA-co8	codon-optimized CDS
twenty four	FIX-WT	PRT
25	FIX-PADUA	PRT

Claims (26)

1. A nucleic acid construct comprising operably linked elements from 5' to 3':

   (1) a transcriptional regulatory element selected from the group consisting of LP1, HLP, TTR, HLP2, enTTR, APOE-hAAT;

   (2) an intron selected from the SV40 intron or the FIX gene intron;

   (3) Gene coding sequence, which encodes coagulation factor IX with R338L mutation;

   (4) A polyadenylation sequence selected from SV40 polyA or bGH polyA.
2. The nucleic acid construct of claim 1, wherein said transcriptional regulatory element is the HLP2 promotor as shown in SEQ ID NO:5 or the enTTR promotor as shown in SEQ ID NO:6, preferably as shown in SEQ ID NO:6 The enTTR promoter.
3. The nucleic acid construct of claim 1 or 2, wherein said intron is selected from the modified SV40 intron (mSV40) shown in FIX Ti299 shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO FIX intronA shown in: 10, the FIX intronAco intron shown in SEQ ID NO: 11, preferably the FIX intronAco intron shown in SEQ ID NO: 11.
4. The nucleic acid construct according to any one of claims 1-3, wherein the gene coding sequence is codon-optimized for expression in human cells.
5. The nucleic acid construct of claim 4, said gene coding sequence has the following characteristics:

   (1) the sequence identity is less than 80% compared to the FIX wild-type sequence of SEQ ID NO: 13 or the FIX ^PADUA wild-type coding sequence of SEQ ID NO: 12; and

   (2) Compared with any of the sequences shown in SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23, the sequence identity is higher than 85%, preferably higher than 90%.
6. The nucleic acid construct of claim 5, said gene coding sequence further has the following characteristics:

   (3) Compared with the sequences shown in SEQ ID NO:19 and SEQ ID NO:22, the sequence identity is lower than 80%.
7. The nucleic acid construct of any one of claims 4-6, the gene coding sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 16, 17, 18, 20, 21 and 23, or with it Nucleotide sequences having at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, still more preferably at least 98% identity.
8. The nucleic acid construct of any one of claims 4-7, said gene coding sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 17, 20 and 23, or has at least 85% identity therewith , preferably at least 90% identical, more preferably at least 95% identical, still more preferably at least 98% identical nucleotide sequences.
9. The nucleic acid construct of any one of claims 4-8, the gene coding sequence is the nucleotide sequence shown in any one of SEQ ID NOs:16,17,18,20,21 and 23.
10. The nucleic acid construct according to any one of claims 4-9, wherein the gene coding sequence has an 30. CpG content below 20, below 10, below 5 or zero.
11. The nucleic acid construct according to any one of claims 4-10, wherein the gene coding sequence does not contain CpG islands.
12. The nucleic acid construct according to any one of claims 1-11, wherein the polyadenylation sequence is SV40 polyA shown in SEQ ID NO:14 or bGH polyA shown in SEQ ID NO:15, preferably SEQ ID NO : SV40 polyA shown in 14.
13. A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid construct according to any one of claims 1-12 and at least one AAV inverted terminal repeat (ITR).
14. The rAAV vector of claim 13 comprising two AAV ITRs.
15. The rAAV vector of claim 13 or 14, wherein the AAV ITR is derived from the AAV2 ITR.
16. The rAAV vector of claim 15, said rAAV vector comprising an AAV2 ITR and an AAV2 ITR variant.
17. The rAAV vector of claim 16, wherein the AAV2 ITR variant is an AAV2 ITR variant lacking a C region or a C' region.
18. The rAAV vector of claim 16 or 17, the AAV2 ITR is located upstream of the coding sequence, and the AAV2 ITR variant is located downstream of the coding sequence.
19. A rAAV virus particle comprising the rAAV vector of any one of claims 13-18 and a capsid.
20. The rAAV virion of claim 19 comprising an AAV8 serotype capsid.
21. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 1-12, the rAAV construct of any one of claims 13-18, or the rAAV of claim 19 or 20 Virus particles, and a pharmaceutically acceptable carrier.
22. The nucleic acid construct described in any one of claims 1-12, the rAAV construct described in any one of claims 13-18, or the rAAV virus particle described in claim 19 or 20 are prepared for treatment or Use of a medicament for preventing hemophilia B or bleeding symptoms associated therewith in a subject.
23. The use of claim 22, said medicament is for intravenous administration.
24. The use of claim 23, wherein the drug is administered at a dose of 4×10 ¹¹ -2×10 ¹² viral genomes per kilogram of body weight (vg/kg).
25. The use according to any one of claims 22-24, wherein the subject is a mammal, preferably a human.
26. The use of any one of claims 22-25, wherein the subject produces insufficient or defective or abnormal Factor IX protein.

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