Transgene expression system

Abstract

This system provides a mechanism to limit the expression of vector-derived transgenes to a window that mitigates the deletion of disease-causing genes without causing toxicity due to overexpression. [Solution] A construct is provided comprising: a promoter; at least one non-mammalian or synthetic miRNA expressed within an intron, wherein the synthetic miRNA is a sequence not found in nature; a transgene; at least one binding site for the non-mammalian or synthetic miRNA that provides control over the expression of the transgene, wherein the binding site for the synthetic miRNA is a sequence not found in nature; and a polyadenylation signal.

Description

Technical Field

[0001] Gene therapy aims at influencing the correction of genetic diseases by introducing therapeutic transgenes . The present invention provides constructs for generating relatively constant levels of transgene expression among cells receiving different levels of vector-derived transgenes . Also described herein is a method for controlling gene expression, wherein said control is provided using the described gene circuit Background Art

[0002] The concept of gene therapy, which introduces therapeutic genes to influence the correction of genetic diseases, is known However, many genes are highly dose-sensitive, so too little or too much expression of the gene product can cause adverse effects. Virus-mediated gene transfer is a powerful tool for delivering therapeutic transgenes to target tissues and cells, including the nervous system To enable effective systemic transduction to maximize therapeutic effects , high viral titers are generally required. However, such high titers can, in some cells, lead to overexpression toxicity because the achieved transgene expression is at supraphysiological levels An effective system is needed that restricts the expression of vector-derived transgenes within a window that alleviates the genetic defect causing the disease without causing overexpression toxicity

[0003] WO 2016040395 describes the use of synthetic RNA circuits for gene transfer . This circuit is mediated by a first microRNA that is specifically expressed in a cell type ​​​It recognizes at least one sequence and specifically binds to the RNA motif to produce a protein. The first RNA molecule contains a sequence encoding a protein that inhibits the first The microRNA is described as miR-21. Furthermore, in RNA motif cell types... The sequence recognized by the second microRNA that is not expressed, and the sequence encoding the output molecule. A second RNA molecule containing the sequence is also provided. The second microRNA is miR-141 , miR-142 and miR-146 are described. In the same application, different cells ( The expression of output proteins by cancer cells and non-cancer cells provides endogenous It is stated that it depends on the gender miR.

[0004] MicroRNA bases of Strovas TJ, Rosenberg AB, Kuypers BE, Muscat RA, and Seelig G. The single-gene circuit buffers the rate of protein synthesis against perturbations. (ACS Synth Biol.) 2014;3(5):324-331 describes a single-gene microRNA (miRNA)-based feedf This discusses the use of forward loops. It is an introduction that targets its own transcripts. It provides miRNA. Strovas provides engineered gene programmed in mammalian cells. This study examines the difficulty of achieving long-term, stable expression of Gram. The mir-124-3 gene in mice containing the gene was used as a red fluorescent reporter (mCherry). The inserted gene circuit was utilized. This premRNA is doxycycline-inducible promo It is transcribed from and leads to the co-expression of mir-124 and mCherry. miRNA and m The repressive regulatory link between Cherry transcripts is the mir-124 regulation of the Vamp3 gene. This was caused by mRNA with a cleaved 3'UTR.

[0005] International Publication No. 2016040395 provides expression in normal and cancer cells. To that end, the use of differently expressed endogenous miRs is discussed, but the use of this miR is It is used in a limited manner in the treatment of non-cancer diseases. Furthermore, the inventors of the present invention have found that St Existing methods using ovas are not suitable for endogenous miRNAs like miR124, which are used in this paper. Therefore, it was determined that it would have multiple off-target effects on various genes that are known to be controlled by it. In fact, miR124 is known to be associated with several cancers, therefore, genetic It would be inappropriate to use it for this treatment. Thus, providing endogenous microRNAs This presents a problem because, in addition to the introduced gene, an endogenous target may also be provided.

[0006] The inventors have found a more advantageous alternative construct than those provided in the art. The purpose was to provide a tract. [Overview of the initiative] [Problems that the invention aims to solve]

[0007] The inventors have found that the expression of vector-derived transgenes can lead to toxicity due to overexpression. Instead, we determined a system that limits the genetic defect causing the disease to a window that mitigates it, and developed this system. What the researchers call "dose-insensitive" is a type of vector that receives more vector-derived transgenes. Cells or tissues are unbalanced and repressed by an adaptively regulated, innate single-gene circuit. This made it possible to downward control vector-derived transgenes at high vector doses. As a result, the circuit maintains a relatively stable expression level across the range of vector dosages. and maintained, such that the entire cell population expresses the vector-derived transgene at a more uniform and controlled level When the vector dose is increased, more cells in the cell population will express the transgene , but overexpression does not increase simultaneously compared with conventional gene therapy cassettes Sensitive cell types that often experience high vector load, such as heart, liver, and dorsal root ganglia , are less susceptible to the effects of superinfection-mediated overexpression through this mechanism

[0008] The present inventors designed synthetic or non-mammalian miRNA constructs that overcome the drawbacks associated with mammalian-based miRNA constructs that carry the risk of off-target effects The present inventors demonstrated the utility of non-mammalian or fully synthetic (not known to exist in nature) miRNAs to ensure that there are no target sites present in the host (human genome)

[0009] Furthermore, the present inventors found that using such synthetic components enables fine-tuning of the system (number of sites and efficient intron removal) to achieve appropriate dose insensitivity

[0010] Accordingly, a first aspect of the present invention is: - a promoter; - at least one non-mammalian or synthetic miRNA expressed within an intron, wherein said synthetic miRNA is a sequence that does not occur in nature, said at least one non-mammalian or synthetic miRNA; - a transgene; - one or more non-mammalian or synthetic m that provide control of transgene expression ​​iRNA binding site, the binding site of the synthetic miRNA does not exist in nature, Binding sites of one or more non-mammalian or synthetic miRNAs; - Provides a construct that includes a polyadenylation signal.

[0011] The miRNA binding sites discussed herein differ from mammalian sequences found in mammalian cells. It is either synthetically induced to become so, or supplied from another non-mammalian species, such as insects. The miRNA binding site is of insect origin and does not exist in mammalian sequences such as ffluc1. In that case, non-mammalian sources may be used. The miRNA binding site and non-mammalian or synthetic miRNAs. The combination of iRNAs minimizes the off-target regulatory effects of the construct. This regulates the expression of the transgene, thereby providing the desired dosage (expression level) of the transgene. It is possible.

[0012] Preferably, the miRNA binding site that provides control of the expression of the transgene is located at 3' UTR, 5'UTR and / or can be provided within the transgene. Preferably, the transgene If provided within, the miRNA binding site provides a synthetic or non-mammalian binding site. Codon optimization is performed so as not to affect the amino acid sequence of the introduced gene protein. This is also good. This construct allows for feedforward control of expression. It can be used to provide a service.

[0013] Preferably, it may include a stabilizing element to increase the expression of the transgene. Preferably, the stabilizing element may be located at 3'UTR. Preferably, this stabilizing element The ment is a post-transcriptional regulatory element (WPRE) of woodchuck hepatitis virus (WHV) ( SEQ ID NO:74) may also be used. WPRE is gamma, alpha, and β It is a three-element adjustment element including a stabilizer element. Preferably, the stabilizer element is a stable element. A cleaved version of WPRE that retains the qualitative elements but omits the X protein sequence, Alternatively, it may be a ribozyme-stable sequence (WPRE3) (SEQ ID NO: 75). WPRE3 is two of the three adjustment elements of WPRE (minimum gamma and a It is a shortened WPRE sequence containing a rufa element. Preferably, WPRE3 stability The element provides a DNA sequence that forms a tertiary structure in the processed transcript, which leads to It enhances the expression of entered genes.

[0014] Preferably, different promoters can be used with various transgenes. So, by adjusting the strength of the feedforward loop, we can control the expression level of the transgene. This makes it possible to achieve dose sensitivity. Single gene circuit The number of microRNA binding sites in the synthesis is adjusted to enable splicing with different efficiencies. Fine-tuning of the circuit is also possible by using introns.

[0015] The construct may be adapted to express the transgene in mammalian cells. Preferably, the construct is a mammalian cell, preferably, the expression of the transgene is carried out. It may be adapted to provide a specific mammalian cell or cell type.

[0016] Advantageously, the transgenes obtained from different levels of vectors are relatively constant across cells. To generate the expression level of a single gene using intron-derived microRNA We can provide a road.

[0017] As those skilled in the art will understand, the characteristics of the construct (promoter, intron) Synthetic miRNAs, transgenes, and miRs that provide control over the expression of transgenes. The NA binding site (polyadenylation signal) enables the functional expression of the transgene. They need to be provided in relation to each other.

[0018] The construct may be adapted to include a modified Kozak sequence. Preferably, the modified Kozak sequence is any nucleic acid mosaic that functions as a protein translation initiation site. It may be any Kosack sequence including -f. Preferably, the modified Kosack sequence is translated Any modifying sequence that promotes the increase in initiation may be used. Preferably, the Kozak sequence is GC CACCATGG(SEQ ID NO:73) is also acceptable.

[0019] In this embodiment, the construct is (from 5' to 3'): -Promoter and; - At least one non-mammalian or synthetic miRNA expressed within an intron, The aforementioned synthetic miRNA is a sequence that does not exist in nature, containing at least one non-mammalian or synthetic sequence. adult miRNA and; - with introduced genes; - One or more synthetic materials that provide control over the expression of the transgene within the transgene. or a binding site for non-mammalian miRNA, wherein the binding site for synthetic miRNA is naturally It does not contain one or more binding sites for synthetic or non-mammalian miRNAs; -Includes polyadenylation signals.

[0020] In this embodiment, the construct is (from 5' to 3'): -Promoter and; - With at least one non-mammalian or synthetic miRNA expressed within an intron; - An introduced gene in which the synthetic miRNA is a sequence that does not exist in nature. and; One or more synthetic materials that provide control of transgene expression within the -3'UTR or a binding site for non-mammalian miRNA, wherein the binding site for synthetic miRNA is naturally It does not contain one or more binding sites for synthetic or non-mammalian miRNAs; -Includes polyadenylation signals.

[0021] In this embodiment, the construct is (from 5' to 3'): -Promoter and; - At least one non-mammalian or synthetic miRNA expressed within an intron, The aforementioned synthetic miRNA is a sequence that does not exist in nature, at least one non-mammalian or Synthetic miRNA and; - Modified Kozak sequences that can promote the transcription of introduced genes; - with introduced genes; - One that provides control over the expression of a transgene or transgene within the 3'UTR. or a binding site for multiple synthetic or non-mammalian miRNAs, wherein the synthetic miRNA The binding site is not naturally occurring, and involves the binding of one or more synthetic or non-mammalian miRNAs. Body parts and; -Includes polyadenylation signals.

[0022] In this embodiment, the construct is (from 5' to 3'): -Promoter and; - At least one non-mammalian or synthetic miRNA expressed within an intron, The aforementioned synthetic miRNA is a sequence that does not exist in nature, at least one non-mammalian or Synthetic miRNA and; - with introduced genes; - One that provides control over the expression of a transgene or transgene within the 3'UTR. or a binding site for multiple synthetic or non-mammalian miRNAs, wherein the synthetic miRNA The binding site does not exist in nature, and the binding site of one or more synthetic or non-mammalian miRNAs The binding site is designed to partially reduce miRNA binding, and consists of one or more Binding sites for synthetic or non-mammalian miRNAs; -Includes polyadenylation signals.

[0023] In this embodiment, the construct is (from 5' to 3'): -Promoter and; - At least one non-mammalian or synthetic miRNA expressed within an intron, The aforementioned synthetic miRNA is a sequence that does not exist in nature, at least one non-mammalian or Synthetic miRNA and; - with introduced genes; - One that provides control over the expression of a transgene or transgene within the 3'UTR. or multiple miRNA binding sites, wherein the binding sites of the synthetic miRNAs are naturally One or more miRNA binding sites that are not present; -3'UTR with stability elements; -Includes polyadenylation signals.

[0024] In some embodiments, the construct is expressed within the promoter and intron. At least one non-mammalian or synthetic miRNA, a transgene, and a transgene or 3 'One or more binding sites within the UTR that provide control over the expression of the transgene and , polyadenylation signal and optionally any one of the embodiments described above It may include multiple features. In some embodiments, one or more of the features mentioned above may be included. The characteristics may be included in the order in which they are mentioned.

[0025] Preferably, the construct is modified to provide enhanced expression, control, and stability. It may be decorated. Preferably, the construct may include a reporter transgene. Preferably, the construct may include a Kozak sequence that promotes potent expression. The construct may include a stabilizing element in the 3'UTR. Preferably, Instruct reduces the effectiveness of miRNA binding (but does not completely eliminate it). (i) It may contain one or more binding sites that have been manipulated to include mutations.

[0026] Preferably, the target gene may be MECP2. Alternatively, the target gene may be the following: Any one of the target genes may be selected: FMR1, UBE3A, CDKL5, FXN , SMN1, or INS. The target gene is used for the treatment of genetic disorders or developmental disabilities. This could be any gene required to be supplied using genetic therapy. Target gene When delivered to a target to treat a genetic condition or developmental disorder, the expression of the drug is controlled. It may be any gene that is required to be filtered.

[0027] Transgene Preferably, the introduced gene is a protein-coding gene that is artificially introduced into the target cell. Yes, it exists. For example, gene therapy cassettes, under the control of a selected promoter. Provided as part of the construct of the first aspect of the present invention. Introduced gene The DNA sequence of a child may represent a specific isoform of a particular gene. (Transgene DNA) The sequence may be codon-optimized. Codon optimization is a specific and unique DNA sequence. While it can provide a column, the changes in DNA and subsequent mRNA are related to the protein. It does not affect the wild-type amino acid sequence; that is, the wild-type amino acid sequence is maintained.

[0028] Preferably, the introduced gene can be selected from the following: [Array 1] TIFF2026143582000002.tif107160

[0029] [Array 2-1] TIFF2026143582000003.tif107160[Array 2-2] TIFF2026143582000004.tif121150

[0030] [Array 3-1] TIFF2026143582000005.tif106150[Array 3-2] TIFF2026143582000006.tif63151

[0031] [Array 4-1] TIFF2026143582000007.tif158151[Array 4-2] TIFF2026143582000008.tif187159

[0032] Preferably, functional variants of these transgenes may be provided, where the functional variant is It retains the function provided by the introduced gene, with at least 60% sequence identity, and at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity , at least 95% sequence identity, at least 97% sequence identity, at least 99% It has sequence identity. Preferably, the functional mutant provides the function of the introduced gene. It may be a gene fragment. Preferably, it is a mi that provides control over the expression of the transgene. If the RNA binding site is located within the transgene, the miRNA binding site is mi The RNA is incorporated into the functional mutant so that it can bind to the transgene and control its expression. It can be done.

[0033] The identity of the sequences can be determined by any method known in the art. Preferably, sequence identity can be determined over the entire length of the transgene.

[0034] For appropriate transgenes, it is desirable to control the expression of any transgene. This includes those based on single-gene disorders. Suitable transgenes are those in which the expression of the transgene is controlled. This includes any monogenic disorder that is desired to be controlled. Examples of transgenes include single genes whose expression is desired to be controlled. This includes those based on genetic CNS disorders. When the nervous system is overexpressed, it impairs nervous system function. It expresses many genes that are known to be harmful. However, this invention is non-CNS. Applicable to all situations where overexpression of a transgene is harmful, including gene therapy for disorders. It is possible. For example, in the substitution of the dystrophin gene in muscle cells, moderate overproduction While the current level does not cause harmful side effects, very high levels of overexpression can lead to severe cardiotoxicity. Wake up.

[0035] miRNA expression from within introns MicroRNAs (miRNAs) are small, single-stranded, non-coding molecules, approximately 22 nucleotides in length. It is a type of RNA. Many miRNAs are independently processed by RNA polymerase II. It is transcribed either as a transcript or as RNA incorporated into the introns of mRNA. The primary transcript of miRNA is converted by two RNase III enzymes into a hair-sized tubular It is processed into a precursor miRNA, and finally into a mature miRNA of ~22nt (Dros (ha and Dicer). miRNAs are involved in protein-level regulation, translational repression, and / or mRNA. It functions by targeting messenger RNA (mRNA) for the degradation of A. ru.

[0036] The inventors have found that it is possible to knock down the expression of transcripts containing each binding region. In some examples of the present invention, non-mammalian or synthetic miRNAs were developed. These are insect-derived proteins originally designed to target firefly luciferase protein. These are miRNA sequences. In other examples, these are synthetic miRNA sequences, which are naturally occurring. There is no homology with the miRNA. In some cases, the synthetic miRNA sequence is codon-first. It is designed to target optimized coding sequences, and the coding sequences are the same amino acid sequence. It is modified at the DNA level while retaining its original properties. In the context of gene therapy, this means that the external While genetically introduced transgenes are exclusively targeted by synthetic miRNAs, endogenous It becomes possible to ensure that sex genes are not affected. In the final example of the present invention, complete Novel synthetic miRNA sequences were created by in silico generation of large DNA sequences, This is used in conjunction with existing miRNA design tools to identify sequences suitable for miRNA targeting. Preferably, since all of these miRNAs are non-mammalian or synthetic, mammalian It does not have a predicted endogenous target within the transcriptome.

[0037] Preferably, the miRNA may be incorporated into different introns. Examples of introns are provided below. The human EF1a intron is commonly used. It is an intron present in the F1a promoter and is known to be able to be spliced ​​efficiently. MINIX introns are also known to splice efficiently. Furthermore, due to its short sequence, it is useful in the context of gene therapy. The inventors of this invention have developed EF1a Pro We demonstrated that the motor and the MINIX intron can function in combination. It also demonstrates that the JeT Promotor and MINIX Intron work together. .

[0038] Preferably, the intron can be selected from the following: [Array 5] TIFF2026143582000009.tif94155[Array 6] TIFF2026143582000010.tif31155

[0039] Preferably, non-mammalian miRs that target firefly luciferase (ffluc1) are preferred. NA may be provided.

[0040] [Array 7] TIFF2026143582000011.tif41151

[0041] BLAST search results showed that the identical (21 bp) RNA matched this one was found in human cells. It is not present in any RNA transcript produced by (therefore, this is a "non-mammalian" sequence) It was found that... Research shows that if miRNA is perfectly complementary to the seed sequence, It has been shown to tolerate mismatches in target sites. Seed sequences are typically miRNAs. Located at positions 2-7 in the 5' region, it is essential for miRNA binding. However, off-target None of the candidate seed RNAs contained a sequence that perfectly matched the seed sequence.

[0042] miRNAs are incorporated in a hairpin loop structure to enable correct recognition and processing. Preferably, the embedded non-mammalian miRNA may be selected from the following: [Arrays 9-12] TIFF2026143582000012.tif158159

[0043] Preferably, the miRNA is homologous to mammalian, insect, or plant miRNAs, and This can be provided by novel synthetic miRNAs that originally target the sequences generated in the dam.

[0044] Preferably, the embedded synthetic miRNA can be selected from the following: [Array 13-20] TIFF2026143582000013.tif159157TIFF2026143582000014.tif164161

[0045] Preferably, the integrated synthetic miRNA is a target gene (i.e., a therapeutic transgene) The code array can be targeted.

[0046] The target gene is codon-optimized and has no homology to mammalian, insect, or plant miRNAs. Synthetic miRNAs are codon-optimized introduction genes that do not target endogenous transcripts of the same gene. The ability to target genes may be screened.

[0047] Preferably, the embedded synthetic miRNA targeting the codon-optimized sequence is as follows: Possible options: [Arrays 21-32] TIFF2026143582000015.tif38159TIFF2026143582000016.tif220157TIFF2026143582000017.tif157163

[0048] miRNAs function by binding to specific sequences that are complementary to the mature miRNA sequence. These binding sites may be located in the 3' untranslated region (3'UTR) of endogenous mRNA. The binding site may also be located in the 5'UTR, exons, and introns. Yes. In further alternative embodiments, the binding site is a codon-optimized transgene sequence. It may be located inside. Preferably, miR that provides control of the expression of the transgene. The NA binding site may be provided within the 3'UTR, 5'UTR, or the transgene.

[0049] Preferably, the "seed" sequence of the binding site is located at the 2nd to 7 / 8th position of the 5' end of the miRNA. It forms a Watson-Crick pair with the base. However, those skilled in the art can, for example, preserve the sequence. Strong base pairing at the 3' end of miRNA, local AU content, and miRNA within the 3'UTR The goal is to understand methods that can alter the binding specificity and strength based on the location of the binding site A. cormorant.

[0050] Preferably, by using a different number of binding sites, the strength of control over the introduced gene can be varied. This can be done. Furthermore, by using the mismatch introduced at the binding site, control of the introduced gene can be achieved. The roll level can be lowered. Such changes can reduce the level of dose insensitivity. It can be configured.

[0051] Preferably, the binding site reduces, rather than completely inhibits, the binding of miRNA to its target. These mutations can be induced to target several miRNs. Preferably, these mutations are induced to target several miRNs. A still binds to the binding site, thereby regulating the expression of the transgene. It can be used to enhance the expression of a transgene while maintaining [the original state].

[0052] When miRNA target binding is successful, translational repression or mRNA degradation mechanisms usually occur. Knockdown occurs at the protein level.

[0053] Preferably, the binding site for non-mammalian or synthetic miRNA can be selected from the following: [Arrays 33-47] TIFF2026143582000018.tif160155TIFF2026143582000019.tif167147

[0054] The following composite sequences are designed to optimize code sequences and target them miRNAs are designed to target sequence regions different from those of the mammalian endogenous sequence. . [Arrays 48-67] TIFF2026143582000020.tif41152TIFF2026143582000021.tif211151 TIFF2026143582000022.tif54150

[0055] Promoter Use any suitable promoter, constitutive or conditional, to drive the expression of the transgene. This is possible. Preferably, the promoter is an Ef1a promoter, a CAG promoter, Jet Promotor, CMV Promotor, CBA Promotor, CBH Promotor, Synapse N1 Promotor, Mecp2 Promotor, U1a Promotor, U6 Promotor, Ubikichi C promoter, neuron-specific enolase promoter, oligodendrocyte transcription factor Child 1 or GFAP promoter may be included.

[0056] In this embodiment, the feedforward miRNA is suitable, for example, the promoter described above. It can be incorporated into an intron sequence to which it is bound.

[0057] The exact promoter used depends on the required expression strength and the size of the gene. In this case, for example, it depends on the available packaging capacity in the AAV delivery vector. This will result in the following suitable promoters being provided: [Arrays 68-69] TIFF2026143582000023.tif62142[array 76] TIFF2026143582000024.tif91154

[0058] Polyadenylation signal This approach can be used with synthetic poly(A) sequences or cleaved fragments of natural poly(A) sequences. This is possible. In this embodiment, the binding site of the feedforward miRNA is within the 3'UTR. It can be incorporated into the gene. Preferably, the miRNA binding site is embedded within the transgene sequence. Unless otherwise specified, it can be incorporated into 3'UTR.

[0059] By utilizing any suitable polyadenylation signal known in the art This is possible. Preferably, the polyA signal may be one of the following: [Arrays 70-72] TIFF2026143582000025.tif81156

[0060] Stability element Preferably, it may include a stabilizing element to increase the expression of the transgene. Preferably, the stabilizing element may be located at 3'UTR. The following may also be used.

[0061] [Arrays 74-75] TIFF2026143582000026.tif137164

[0062] vector The miRNA feedforward construct of the present invention functions in vivo. It is designed to deliver these constructs to the necessary tissues / organs. Any suitable viral vector can be used. In this embodiment, Kutar is an adeno-associated virus (AAV) delivery system, or lentivirus, adenovirus Herpes simplex virus, retrovirus, alphavirus, flavivirus, rad Other therapeutic viruses such as zovirus, measles virus, picornavirus and poxvirus It could be an ilus vector system. In the case of AAV, the entire construct (promoter, miR) Cloned NA, transgene, binding site, and polyA into an AAV-compatible plasmid. It is possible to sandwich the AAV with terminal inversion (ITR). A strict size requirement is required for AAV manufacturing. Due to limitations, the entire construct must be 4.4kb or less (excluding ITR). ). This size limitation means that certain transgenes would take up a large portion of the available space. The use of offspring may be restricted. Or, in order to accommodate a larger transgene, Smaller promoters and polyA can also be used. Preferably, construct In the process, the 3'UTR region is removed and a codon-optimized sequence of the transgene is synthesized using miRN. A can be targeted. Codon-optimized transgenes have different DNA / mRNA Because it has a sequence, endogenous mRNA derived from the target gene (GOI) is not targeted.

[0063] According to a second aspect of the present invention, a vector comprising the construct of the first aspect of the present invention It will be provided.

[0064] Preferably, the construct allows for the delivery of the construct to target cells. It can be delivered within a viral vector. Target cells are neurons, neuronal subtypes. Oligodendrocytes, astrocytes, Schwann cells, and other cells of the central nervous system and peripheral nerves It can be a transcellular cell. Advantageously, the viral vector is adeno-associated virus (AAV). , especially AAV9, AAV1, 2, 4, 5, 6, 6.2, 8, 9, rh10, PHP.B, You can choose between PHP.S and PHP.eB vectors.

[0065] According to a third aspect of the present invention, in order to express the introduced gene, the construct of the first aspect A method for using the ct is provided. Preferably, a second embodiment provides cells that can be provided to the subject. This includes a method for expressing the introduced gene. Preferably, the required dose adjustment level is evaluated. To evaluate, it is necessary to effectively screen constructs in vitro. Yes, it is possible. In vitro, the transgene is contained within the plasmid, and lipid-mediated gene transduction is performed. It can be introduced into cell lines via an inlet. Strong expression of the transgene is confirmed after 24 hours. This can be confirmed. Subsequently, the feedforward transgene cassette preferably expresses rAAV. By inserting into a vector, it can be vectorized, and this can be used to produce AAV particles. It is possible to achieve this.

[0066] According to a fourth aspect of the present invention, the insufficient expression of genes in the subject is caused A method for treating disorders caused by insufficient gene expression in the subject To treat the condition, the wild-type or codon-specific transgene expressed in the target is used. A construct of the first aspect of the present invention having an optimized or modified copy or a second A method is provided which includes the step of providing a vector of a particular form. Preferably, the introduced gene is packaged The cased AAV virus vector can be administered by various methods, including systemic intravenous injection, or Administration via intrathecal lumbar spine, intraventricular, and intracisional injections within the CSF, or by injection into neurites. It will be introduced to that target group.

[0067] Preferably, the introduced gene is expressed at a low level in subjects with Rett syndrome, a neurological disorder. It can be the gene causing the condition. Typically, Rett syndrome is caused by the X-linked gene MECP2. This is caused by loss-of-function mutations in MECP2. Preferably, the transgene is MECP2 It may be a functional copy of a gene. Preferably, the construct is an adeno-associated virus. This invention provides a method for delivering transgenes to the nervous system using (AAV) vectors.

[0068] The construct provides transgene expression within a narrow / desired range in target cells. For example, the introduced gene is the wild-type protein-coding sequence of the MECP2 gene. If it is a type or codon-optimized copy, the construct provides the appropriate therapeutic effect. However, it is possible to provide the transgene at an expression level lower than the level at which side effects are observed. It is thought that this is the case for MECP2, FMR1, and UBE3A, which are genes that undergo overexpression. It is known to be harmful.

[0069] For example, in Rett syndrome, low levels of expression improve the disease phenotype in mice. As previously shown by the inventors, overexpression in patients and experimental animals (gene locus) Doubling (more than twice) leads to adverse neurological outcomes. This is why gene therapy While the treatment area is narrow and limited, feedforward technology is well-suited for this purpose. Furthermore, the FMR1, UBE3A, and SYNGAP1 genes are thought to be dose-sensitive. In this situation, transgenes are being introduced to improve the disease and minimize side effects. The present invention aims to determine the expression level of [the substance] and to suitably provide that expression level to patients. It is possible.

[0070] Many other genes associated with isolated disorders are dose-sensitive, and such external Benefit from the use of the constructs and systems of the present invention that regulate the expression of intrinsically modified genes. It is likely that human copy number variations (CNVs) can serve as indicators of dose-sensitive genes, and multiple The research suggests that dose sensitivity of individual genes is a common cause of CNV pathogenicity. . Gu W & Lupski JR.CNV and nervous system diseases - What's new? Cytogenet Genom e Res. 2008; 123:54-64 discusses the association between dose-sensitive genes and neurodevelopmental disorders. He gives several examples. For instance, MECP2 duplication syndrome (involving the MECP2 gene), Adult-onset autosomal dominant leukodystrophy (ADLD, involving the LMNB1 gene), isolated Orthostatic anencephaly syndrome (ILS, involving the PAFAH1B1 / LIS1 gene), Miller-Di Examples include scare syndrome (MDS, involving the YWHAE gene).

[0071] Rice AM & McLysaght. Dosage sensitivity is a major determinant of human copy n umber variant pathogenicity. Nature Communications. 2017; 8:14366 | DOI: 10.1038 This indicates that isolated pathogenic genes involved in disease-related CNVs play a rich role in neurodevelopment. This indicates that many dose-sensitive genes, such as PRKCZ, TTC34, and P, are involved. RDM16, ARHGEF16, PARK7, PRDM2, IGSF21, PTCH2, NFIA, ST6GALNAC3, DPYD, COL11A1, PDZK1, GPR89 A, NBPF11, GPR89B, KCNT2, CFHR2, ASPM, PTPRC, G PATCH2, DUSP10, GPR137B, RYR2, CHRM3, RGS7, AK T3, KIF26B, SMYD3, LPIN1, EPCAM, MSH2, NRXN1, X PO1, LRP1B, ZEB2, ACVR2A, MBD5, KIF5C, SCN1A, C OL3A1, PMS1, PLCL1, SATB2, PARD3B, EPHA4, SPHK AP, CHL1, GRM7, TRANK1, DOCK3, FAM19A1, FOXP1, ROBO1, CADM2, FOXL2, SOX2, LPP, RASGEF1B, GRID 2, FAT4, NR3C2, LRBA, FGA, GALNTL6, WWC2, TLR3, IRX2、IRX1、CDH12、CDH9、NIPBL、HEXB、MEF2C、GR AMD3、FBN2、PRELID2、TCOF1、GABRG2、MSX2、NSD1 、FOXC1、CDYL、TBC1D7、RUNX2、MUT、RIMS1、NKAIN 2, LAMA2, ARID1B, PARK2, PACRG, QKI, TNRC18, FB XL18、SUGCT、GLI3、AUTS2、MLXIPL、COL1A2、PPP1 R9A、CFTR、TSPAN12、GRM8、CNTNAP2、MNX1、CSMD1 、MCPH1、LPL、ANK1、IMPAD1、CHD7、VCPIP1、TRPS1 、PARP10、DOCK8、KANK1、GLIS3、PTPRD、MLLT3、RO R2、PTCH1、AL162389.1、ARRDC1、EHMT1、PCDH15、 CTNNA3、ADK、BMPR1A、PAX2、BTRC、INPP5A、MRPL2 3、ELP4、PAX6、CPT1A、DYNC2H1、KIRREL3、WNK1、C ACNA1C、PPFIBP1、TBX5、MED13L、NALCN、CHD8、MY H7, TTC6, DAAM1, NRXN3, MTA1, SNRPN, UBE3A, OCA 2、HERC2、CHRFAM7A、ARHGAP11B、OTUD7A、FBN1、H EXA、SNUPN、NRG4、AC112693.2、IGF1R、LRRC28、H BA2, HBQ1, CREBBP, RBFOX1, CDR2, CDH13, CYBA, N XN、YWHAE、SMG6、METTL16、PAFAH1B1、ADORA2B、N T5M、RAI1、NF1、C17orf67、PITPNC1、ACOX1、TCF4 、DOCK6、CACNA1A、LPHN1、ZSCAN5A、BMP2、MYT1、P EX26, USP18, DGCR6L, USP41, UBE2L3, NF2, LARGE BRD1 and SHANK3 have been identified.

[0072] The inventors have identified any suitable gene, in particular, any dose sensitivity as described above. We believe that genes can be suitably utilized in the present invention as needed. For example, the present technology As understood in the field, the constructs and systems of the present invention are for the treatment of diseases or conditions. The expression of any appropriate protein, especially the expression level of the protein being offered, It can be used when the rule is important.

[0073] The inventors of this invention have a concept and a construct that has an appropriate introduced gene inside, and introduced The method of expressing the gene is suitable for any other clinically relevant and dose-sensitive gene. I believe it is usable.

[0074] Preferably, this construct is used for fragile X syndrome (using the FMR1 transgene), and German syndrome (e.g., using the UBE3A transgene), or Syngap-related intellectual disability. It can be used in other gene therapy programs, including those for disorders (using SYNGAP1). .

[0075] Using specific vectors, we can provide vectors to specific cell types determined by the disease. This can be assumed.

[0076] For example, SYNGAP1 is a neural gene and is expressed only in nerve cells, but UB E3A, MECP2, and FMR1 are ubiquitously expressed across multiple tissues. However, The manifest characteristics of the disease arise from the disappearance of expression in the nervous system, therefore therapeutic feed The target of forward genes is predominantly the nervous system.

[0077] The inventors have found that in order to achieve appropriate dose insensitivity, the synthetic component is a micro-component of the system. We developed a construct that is expected to perform adjustments (number of sites and efficient intron elimination). In the context of this invention, dose insensitivity refers to the situation where the expression level of the therapeutic transgene is too high. Undesirable effects observed in combination (for example, if an individual has two copies of the MECP2 gene, Severe M syndrome presents with severe symptoms similar to Rett syndrome, characterized by a drastic decrease or complete absence of MeCP2 levels. Protein expression that does not lead to ECP2 duplication syndrome (which is known to occur) The intention is to estimate the range.

[0078] In this embodiment, the construct allows control over the transgene level. It may include two elements. Preferably, the first element is a promoter This could be a microRNA sequence located within an intron situated between the introduced gene and the other gene. MicroRNAs containing introns are spliced ​​during premRNA processing. Afterward, the miRNAs are processed to produce mature miRNAs that can degrade target transcripts. To achieve this, a key element of the design is that miRNAs are mammalian gland markers to prevent off-target effects. The goal is to be designed not to target insects. In some cases, miRNAs are designed not to target insects. It could be of origin (for example, from the firefly family, but from any suitable insect or other suitable (A non-mammalian miRNA may be optimized for this use.) In an alternative example, the sequence is complete. It is synthetic (designed not to bind to the mammalian genome and is not a naturally occurring sequence) Therefore, it does not have known off-target effects within the mammalian genome. The ment corresponds to the miRNA produced from the intron, at the 3'UT of the construct. These could be binding sites for several non-mammalian or synthetic miRNAs in R. The presence of a matching site makes the transgene a target of the delivered microRNA. This reduces the level of the transgene, prevents overexpression, and results in the desired dose-insensitivity of the system. It produces an effect.

[0079] In another embodiment of the feedforward principle, synthetic microRNAs are used in gene therapy synthesis. It is delivered within the set intron, but targets the miRNA binding site contained within the 3'UTR. Instead of targeting the gene, the target is the coding sequence of the introduced gene itself. In embodiments such as this, the sequence of the introduced gene remains the same at the amino acid level, but the DNA sequence The codons are optimized so that the sequence is modified by the bell. This is intrinsic A novel D2 that allows synthetic miRNAs to uniquely target transgenes without targeting mammalian sequences. An NA sequence is created. This version of the feedforward system is more compact. Therefore, large genes (for example, Syng) that are close to the packaging capacity of a viral vector This is advantageous for ap1). Overall, a single-gene loop maintains a certain level of expression. This makes it possible, and as a result, the circuit remains at a relatively fixed level across a wide range of gene dosages. This makes it possible to maintain the expression of (i.e., this relatively fixed or constant expression) The level is such that it results in the desired dose insensitivity. This experimental system allows the gene A regimen was developed in which the relative change in gene expression was very small even when the dosage was changed. For gene therapy aimed at broadly and evenly expressing genes throughout the entire population of cells into which they have been introduced. This is an important characteristic when applying the virus, as it increases the dose of the viral vector and affects the effects of overexpression. A high adoption rate can be achieved without the need for [unspecified action].

[0080] In one embodiment, the construct is a cell that is sensitive to AAV gene therapy. Suitable for expression in and / or tissues. In embodiments, this construct This involves cells that typically overexpress the transgene delivered using an AAV vector. This enables control of the expression of the introduced gene. In one embodiment, construct The ct prevents cytotoxicity in these cells and / or tissues. Therefore, the construct can prevent cytotoxicity in the dorsal root ganglia. In an embodiment, The construct can prevent cytotoxicity in hepatocytes. In one embodiment, the construct The lacto can prevent cytotoxicity in cardiomyocytes. In this embodiment, the lacto in virions The packaging of the construct does not affect the quality of the construct, or does it have any effect on the quality of the construct. Even if it is minimal, it will be to the greatest extent possible.

[0081] In one embodiment, the construct is drawn by a specific genetic condition or developmental disorder. It can be used to reduce the severity of the clinical symptoms that occur. A construct is a clinical symptom caused by a specific genetic condition or developmental disorder. It can be used to completely reverse it. In an embodiment, the construct is It can be used to treat specific genetic conditions or developmental disorders. In this context, the construct can be used to treat Rett syndrome. In terms of morphology, the construct is designed to alleviate the clinical symptoms of Rett syndrome. It can be administered via VO.

[0082] In one embodiment, the construct is used to reduce the toxicity of gene therapy. This is possible. In the embodiment, the feedforward mechanism regulates the expression of the introduced gene. Reduces toxicity to cells. In embodiments, the construct reduces adverse health effects. It can be administered in vivo without causing any adverse effects.

[0083] Hereinafter, embodiments of the present invention will be described only illustratively with reference to the attached figures. [Brief explanation of the drawing]

[0084] [Figure 1] This figure shows the challenges of dose sensitivity in gene therapy. [Figure 2] Figure 2A illustrates how gene dosage is a challenge in gene therapy, resulting in a very narrow safety margin. The dosage of genes is a challenge in gene therapy, and the safety margin can be very narrow. For example, the median survival time for a Rett syndrome model mouse is ~11 weeks. Treatment with a therapeutic gene therapy vector can normalize body weight and increase the 40-week survival rate to 100% (left box). However, doubling this therapeutic dose is lethal (right), highlighting dose sensitivity and a narrow safety margin. Figure 2B illustrates how gene dosage is a challenge in gene therapy, resulting in a very narrow safety margin. [Figure 3] This figure shows that a single-gene feedforward gene therapy circuit can reduce dose sensitivity, as demonstrated by quantitative evaluation at the transgene level using flow cytometry. [Figure 4]This diagram illustrates the feedback related to transgene expression, mediated by the viral level of a transgene delivered to any given cell, in cases where different cells would otherwise express very different levels of the transgene due to different infections. MECP2 is an example of a dose-sensitive gene, where too little or too much can cause disease. In gene therapy, cells receiving different levels of transduction have different levels of feedforward control (indicated by line thickness). In a single-gene circuit, the expression of a therapeutic transgene and its negative regulator (synthetic miRNA) is driven by the same input (the level of therapeutic vector entering the cell). As the input level (vector level) increases, the circuit achieves a higher level of miRNA-mediated downregulation. As a result, this circuit can maintain a more fixed level of transgene expression across the entire cell population. In the absence of such regulation (unregulated gene therapy cassette), cells express vector-derived proteins at more diverse levels, as shown in the shading. [Figure 5]Figure 5A shows a method for optimizing a construct (cassette) to enable the treatment of different conditions using different transgenes, or the expression of different therapeutic levels of transgenes. It shows key components of a feedforward construct. Figure 5B shows a method for optimizing a construct (cassette) to enable the treatment of different conditions using different transgenes, or the expression of different therapeutic levels of transgenes. The transgene component is replaced, but the remaining components of the cassette are maintained. Figure 5C shows a method for optimizing a construct (cassette) to enable the treatment of different conditions using different transgenes, or the expression of different therapeutic levels of transgenes. A new intron / miRNA and 3'UTR / miRNA binding site (dashed line) is introduced, but the remaining components of the cassette are maintained. Figure 5D shows a method for optimizing a construct (cassette) to enable the treatment of different conditions using different transgenes, or the expression of different therapeutic levels of transgenes. Two copies of non-mammalian or synthetic miRNA can be expressed from the same intron or from two different introns. Introns may be located within the 5'UTR of the transgene and / or within the open reading frame. Figure 5E shows how constructs (cassettes) can be optimized to enable the treatment of different conditions using different transgenes, or the expression of different therapeutic levels of transgenes. The 3'UTR may contain one, three, six, or any number in between copies of non-mammalian or synthetic miRNA binding sites. [Figure 6] This figure shows a construct in which the synthetic miRNA targets the codon-optimized sequence of the transgene, rather than the UTR. [Figure 7]Figure 7A shows the effect of non-mammalian miRNA expression on MeCP2-NeonGreen protein levels as assessed by FACS. This demonstrates feedforward using a native miRNA lacking a predicted binding site within the mammalian genome. The feedforward construct (lower line) was compared to a control construct (upper line) that lacks miRNA regulation due to the inclusion of a scrambled miRNA binding site (all subsequent experiments follow this same structure). The feedforward construct included three non-mammalian miRNA binding sites in the 3'UTR. The graph shows mRuby (x-axis - a measure of plasmid quantity relative to cells, unaffected by miRNA regulation) versus MeCP2-NeonGreen (y-axis - miRNA-regulated protein) levels. The upper graph shows results for miR124-3, an endogenous mammalian miRNA used in the feedforward circuit described in a Strovas publication in the art. The results demonstrate that miRNA is effective in regulating MeCP2 expression in the feedforward sample compared to the control, as indicated by the difference in the slope of the linear regression lines. Figure 7B shows the effect of non-mammalian miRNA expression on MeCP2-NeonGreen protein levels as assessed by FACS. This demonstrates feedforward using a native miRNA lacking a predicted binding site within the mammalian genome. The feedforward construct (lower line) was compared to a control construct (upper line) that lacks miRNA regulation because it contains a scrambled miRNA binding site (all subsequent experiments follow this same structure). The feedforward construct contained three non-mammalian miRNA binding sites in the 3'UTR. The graph shows mRuby (x-axis - a measure of plasmid quantity relative to cells, unaffected by miRNA regulation) versus MeCP2-NeonGreen (y-axis - a protein regulated by miRNA) levels. The lower graph shows the results for ffluc1, a non-mammalian miRNA originally designed to knock down the firefly luciferase fluorescent protein.The results, as indicated by the difference in the slope of the linear regression lines, show that miRNAs are effective in controlling MeCP2 expression in feedforward samples compared to controls. [Figure 8]Figure 8A shows a non-mammalian miRNA. An example of a compact intron used to hold non-mammalian or synthetic miRNA (in this experiment, the miRNA is synthetic firefly luciferase (ffluc1) as shown in the previous figure) can be incorporated into a gene therapy cassette and expressed from an intron located between the promoter and the MECP2 coding sequence to achieve feedforward control. Robust expression of non-mammalian miRNA depends on the efficient splicing of this intron, and different introns may enable different levels of protein regulation. A feedforward molecule was constructed to express a non-mammalian miRNA from intron 1 of the human EF1a gene. The construct included three non-mammalian miRNA binding sites in the 3'UTR. This intron exhibits robust regulation at the MeCP2 level. Figure 8B shows a non-mammalian miRNA. An example of a compact intron used to hold non-mammalian or synthetic miRNA (in this experiment, the miRNA is synthetic firefly luciferase (ffluc1) as shown in the previous figure) for integration into a gene therapy cassette and to achieve feedforward control is expressed from an intron located between the promoter and the MECP2 coding sequence. Robust expression of non-mammalian miRNA depends on efficient splicing of this intron, and different levels of protein regulation can be enabled by using different introns. A feedforward molecule expressing non-mammalian miRNA was constructed from a small synthetic intron (MINIX). The construct included three non-mammalian miRNA binding sites in the 3'UTR. This intron shows robust regulation at the MeCP2 level, but as can be seen from the reduced slope of the linear regression line, the MINIX intron shows the same level of MeCP2 expression as the control even at low plasmid expression levels. This is considered therapeutically beneficial because it can deliver therapeutic levels of protein at low plasmid levels and prevent protein toxicity at high plasmid delivery levels. [Figure 9]Figure 9A shows the results of changing the number of non-mammalian miRNA binding sites in the 3'UTR. Constructs with one non-mammalian miRNA binding site in the 3'UTR were constructed and evaluated by FACS. Figure 9B shows the results of changing the number of non-mammalian miRNA binding sites in the 3'UTR. Constructs with three non-mammalian miRNA binding sites in the 3'UTR were constructed and evaluated by FACS. The construct with three binding sites showed a more pronounced suppression of MeCP2 levels, as indicated by the decrease in the slope of the linear regression line. The strength of feedforward control, i.e., dose sensitivity, can be fine-tuned by changing the number of non-mammalian or synthetic miRNA binding sites. Figure 9C shows the results of changing the number of non-mammalian miRNA binding sites in the 3'UTR. Constructs with six non-mammalian miRNA binding sites in the 3'UTR were constructed and evaluated by FACS. The construct with six binding sites showed a more pronounced suppression of MeCP2 levels, as indicated by the decrease in the slope of the linear regression line. The strength of the feedforward control, i.e., dose sensitivity, can be fine-tuned by changing the number of binding sites for non-mammalian or synthetic miRNAs. [Figure 10]Figure 10A shows the construct with an unmodified binding site. Figure 10B shows the effect of mismatch on the binding site of non-mammalian miRNA in the construct with a 1 bp central bulge, out of three different constructs having either a 1 bp central bulge, a 3 bp central bulge, or a 3' mismatch where only the miRNA seed sequence is present at the binding site. Compared to the construct with an unmodified binding site, this construct showed significantly less protein-level suppression, with all three showing the same level of suppression. This demonstrates that the strength of the feedforward control, i.e., dose sensitivity, can be fine-tuned by incorporating mismatches in the binding site of non-mammalian or synthetic miRNA. Figure 10C shows the effect of mismatch on the binding site of non-mammalian miRNA with a 3 bp central bulge, out of three different constructs having either a 1 bp central bulge, a 3 bp central bulge, or a 3' mismatch where only the miRNA seed sequence is present at the binding site. Compared to the construct with an unmodified binding site, this construct showed significantly less protein-level suppression, with all three showing the same level of suppression. The strength of the feedforward control, i.e., dose sensitivity, can be fine-tuned by incorporating mismatches in the binding site of non-mammalian or synthetic miRNAs. Figure 10D shows the effect of mismatches in the binding site of non-mammalian miRNAs in the construct with a 3' mismatch where only the miRNA seed sequence is present at the binding site, out of three different constructs with either a 1 bp central bulge, a 3 bp central bulge, or a 3' mismatch where only the miRNA seed sequence is present at the binding site. Compared to the construct with no modification of the binding site, this construct showed significantly less suppression at the protein level, with all three showing similar levels of suppression. The strength of the feedforward control, i.e., dose sensitivity, can be fine-tuned by incorporating mismatches in the binding site of non-mammalian or synthetic miRNAs. [Figure 11]This figure shows whether the non-mammalian miRNA feedforward mechanism is also effective for other related brain diseases. Here, we constructed a construct in which MECP2 was replaced with the coding sequence for the UBE3A protein (mutations in this gene cause Angelman syndrome). The 3'UTR contained three non-mammalian miRNA binding sites for the same ffluc1 miRNA used in previous experiments. Again, plasmids with non-mammalian miRNA binding sites showed reduced protein expression compared to plasmids with scrambled miRNA binding site sequences. It was suggested that UBE3A protein levels may be partially regulated by an endogenous intracellular mechanism, separate from our feedforward non-mammalian miRNA mechanism. Dose-sensitive feedforward control is also achievable for other dose-sensitive genes, in this case the UBE3A gene, which is disrupted in Angelman syndrome and Prader-Willi syndrome. [Figure 12] This diagram illustrates the workflow for incorporating feedforward gene therapy technology, where the feedforward construct is designed to incorporate an appropriate combination of functional elements (see, for example, Table 1 herein), produced by DNA synthesis, and then cloned into an AAV packaging plasmid. The plasmid containing the feedforward cassette is then transfected together with Rep / cap and helper plasmids to generate AAV particles for gene transtherapy. [Figure 13]Figure 13A shows the expression of MeCP2 in an intact nervous system after administration of a controlled cassette. It shows the predicted distribution of AAV vector-delivered protein expression. The wild-type distribution is represented as the expression of the tightly controlled native MeCP2 protein. The vector-derived (uncontrolled) distribution is a hatched region, showing a broad expression distribution brought about by the uncontrolled cassette, which included a significant proportion of cells expressing the protein above physiological levels. The vector-derived (feedforward) construct shows a hatched region that largely overlaps with the native distribution, corresponding to the restricted expression in the controlled cassette. Figure 13B (Observations) shows fluorescence intensity imaging data (a surrogate for cellular protein levels) from the somatosensory cortex of mouse brains 12 days after AAV administration of a control or feedforward controlled vector by direct injection into the brain. The mean data for 3 mice in each treatment group is shown on the left, and the data for individual animals is shown in the plot on the far right. Figure 13C is a schematic diagram of the controlled and uncontrolled feedforward AAV cassettes used in the experiment. [Figure 14] Figure 14A shows the expression of vector-derived proteins from a regulatory AAV cassette in the brain. It is a tilted confocal image showing anti-flag tag immunolabeling (to detect vector-derived proteins) of a parasagittal mouse brain section 5 weeks after AAV injection. Figure 14B shows the expression of vector-derived proteins from an unregulated AAV cassette in the brain. It is a tilted confocal image showing anti-flag tag immunolabeling (to detect vector-derived proteins) of a parasagittal mouse brain section 5 weeks after AAV injection. [Figure 15]Figure 15A is a fluorescence image showing restricted transgene expression as a result of the feedforward circuit. The image is a representative confocal image showing anti-MeCP2 transgene immunolabeling (to detect vector-derived transgene products) in the mouse somatosensory cortex 5 weeks after AAV injection. It shows the native level of MeCP2 expression. Figure 15B is a fluorescence image showing restricted transgene expression as a result of the feedforward circuit. The image is a representative confocal image showing anti-MeCP2 transgene immunolabeling (to detect vector-derived transgene products) in the mouse somatosensory cortex 5 weeks after AAV injection. This figure shows the MeCP2 immunoreactivity in wild-type (WT) mice treated with the control construct. Figure 15C is a fluorescence image showing restricted transgene expression as a result of the feedforward circuit. The image is a representative confocal image showing anti-MeCP2 transgene immunolabeling (to detect vector-derived transgene products) in the mouse somatosensory cortex 5 weeks after AAV injection. It shows the MeCP2 immunoreactivity in WT mice treated with the non-control construct. The schematic diagram at the bottom shows the feedforward-controlled and uncontrolled constructs. Figure 15D shows the quantification of vector-derived protein expression measured by quantitative anti-Mecp2 immunolabeling. Expression is shown as a relative frequency distribution (analysis of 1265–2082 cells per mouse / cohort). Mice were injected with the AAV vector into P1 at a dose of 1 x 10¹¹ vg / mouse. Figure 15E shows schematic diagrams of the controlled and uncontrolled feedforward constructs delivered to mice. [Figure 16]Figure 16A shows a toxicity study in which wild-type mice were administered an AAV9 dose of 4E × 10¹¹ vg / mouse. The controlled and uncontrolled constructs tested are shown in Figure 16A. Figure 16B shows a toxicity study in which wild-type mice were administered an AAV9 dose of 4E × 10¹¹ vg / mouse. Survival rates and phenotypes were tracked over a period of 15 weeks. The controlled construct offers a safety advantage over the uncontrolled cassette. The same figure shows an in vivo experiment in which wild-type mice were administered a high-dose vector (4 × 10¹¹ vg / mouse; direct brain injection at P1). Administration using the uncontrolled MECP2 cassette resulted in a toxicity score and death. In contrast, the controlled cassette was completely resistant, with no detectable, obvious, or harmful phenotypes. [Figure 17] Figure 17A shows a study demonstrating that administration of a controlled feedforward cassette was tolerated and therapeutically effective in Rett syndrome model mice. Figure 17B shows a study demonstrating that administration of a controlled feedforward cassette was tolerated and therapeutically effective in Rett syndrome model mice. In vivo experiments were conducted in Mepc2- / y mice with high doses of AAV9 vector (3 × 10¹¹ vg / mouse; direct brain injection at P1). Survival rate and phenotype (RTT score) were tracked for 15 weeks. [Figure 18]Figure 18A shows that a controlled feedforward cassette normalizes specific clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring of vehicle-treated Mepc2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same amount of uncontrolled cassette are not shown because they did not survive the monitoring period. Figure 18B shows that a controlled feedforward cassette normalizes specific clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring of vehicle-treated Mepc2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same unregulated cassette are not shown because they did not survive the monitoring period. Figure 18C shows that the regulated feedforward cassette normalizes certain clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring of vehicle-treated Mepc2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same unregulated cassette are not shown because they did not survive the monitoring period. Figure 18D shows that the regulated feedforward cassette normalizes certain clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring for vehicle-treated Mecp2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same procedure but without a control cassette are not shown because they did not survive the monitoring period.Figure 18E shows that a controlled feedforward cassette normalizes specific clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring of vehicle-treated Mepc2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same amount of uncontrolled cassette are not shown because they did not survive the monitoring period. Figure 18F shows that a controlled feedforward cassette normalizes specific clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring of vehicle-treated Mepc2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same unregulated cassette are not shown because they did not survive the monitoring period. Figure 18G shows that the regulated feedforward cassette normalizes certain clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring of vehicle-treated Mepc2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same unregulated cassette are not shown because they did not survive the monitoring period. Figure 18H shows that the regulated feedforward cassette normalizes certain clinical features in a Rett syndrome model mouse. This figure shows an in vivo experiment in which Mepc2- / y mice were administered a high dose of the feedforward cassette (3 × 10¹¹ vg / mouse; directly injected into the brain at P1). For comparison, scoring for vehicle-treated Mecp2- / y mice and vehicle-treated wild-type mice is shown. Mice treated with the same procedure but without a control cassette are not shown because they did not survive the monitoring period. [Figure 19]Figure 19A shows plasmids expressing ffluc1 miRNA and the mNeonGreen reporter transgene, or plasmids expressing only the mNeonGreen reporter. Figure 19B shows RNA-seq expression of 20 genes thought to contain the most likely off-target interaction sequences of the miRNA ffluc1 used in the feedforward construct. The expression levels of the top 20 predicted human target mRNA transcripts were measured using mRNA-seq. FPKM stands for FPKM, which means the number of transcript fragments per kilobase per million reads. A low FPKM value indicates a low abundance of the transcript in human HEK293 cells. [Figure 20] Figure 20 shows the effect of adding an additional element (detailed in Example 8) to the feedforward cassette on transgene expression. [Figure 21] Figure 21A shows details of a representative flattened confocal image taken from a stained lumbar dorsal root ganglion (DRG) section. The section was cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure shows the cassette administered to the mice. Figure 21B shows details of a representative flattened confocal image taken from a stained lumbar dorsal root ganglion (DRG) section. The section was cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure demonstrates staining of DRG sections from WT mice and Mecp2 knockout mice treated with control and non-control constructs. Figure 21C shows details of a representative flattened confocal image taken from a stained lumbar dorsal root ganglion (DRG) section. The section was cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure shows the quantification of MeCP2 levels measured by fluorescence microscopy. Figure 21D shows details of a representative flattened confocal image taken from stained lumbar dorsal root ganglion (DRG) sections. Sections were cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure shows the quantification of vector copy numbers in each sample. [Figure 22] Figure 22A shows the efficacy study in which Mecp2 KO mice were given an AAV9 dose of 1E × 10¹¹vg / mouse. Figure 22B shows the survival rate and phenotype (RTT score) tracked over 15 weeks. Figure 22C shows Western blot analysis of different brain regions demonstrating that MeCP2 expression was restricted in the feedforward circuit. [Figure 23] Figure 23A shows details of a representative flattened confocal image taken from stained liver sections. Sections were cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure shows the cassette administered to mice. Figure 23B shows details of a representative flattened confocal image taken from stained liver sections. Sections were cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure demonstrates staining of liver sections from WT mice treated with an uncontrolled construct and a controlled construct. Note that the developed construct restricts the expression of vector-derived transgenes compared to the uncontrolled cassette. Figure 23C shows details of a representative flattened confocal image taken from stained liver sections. Sections were cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure shows the quantification of MeCP2 levels as measured by the intensity of the fluorescence signal. Figure 23D shows details of a representative flattened confocal image taken from stained liver sections. Sections were cut to a thickness of 10 μm, stained with anti-MeCP2 antibody and DAPI, and imaged using the same confocal setting. The figure shows the quantification of the vector copy number in each sample. [Figure 24]Figure 24A shows the qRT-PCR expression of mRNA most likely to be the off-target interaction sequence for the miRNAs ffluc1, ran1g, and ran2g used in the feedforward construct. This figure shows plasmids expressing ffluc1, ran1g, or ran2g miRNA. Figure 24B shows the qRT-PCR expression of mRNA most likely to be the off-target interaction sequence for the miRNAs ffluc1, ran1g, and ran2g used in the feedforward construct. This figure shows control plasmids expressing hsa-miR-132-3p, hsa-miR-34a-5p, or hsa-miR-644a miRNA. Figure 24C shows the qRT-PCR expression of mRNA most likely to be the off-target interaction sequence for the miRNAs ffluc1, ran1g, and ran2g used in the feedforward construct. In this figure, the expression levels of the top three predicted human target mRNA transcripts were measured using qRT-PCR. Figure 24D shows the qRT-PCR expression of mRNAs most likely to be off-target interaction sequences for the miRNAs ffluc1, ran1g, and ran2g used in the feedforward construct. The figure also shows the expression levels of positive control human target mRNA transcripts, measured using qRT-PCR. [Figure 25] Figure 25A shows that dose-sensitive feedforward control can be achieved across other dose-sensitive genes. Figure 25B shows that dose-sensitive feedforward control can be achieved across other dose-sensitive genes, including the UBE3A gene which is disrupted in Angelman syndrome and Prader-Willi syndrome. Figure 25C shows that dose-sensitive feedforward control can be achieved across other dose-sensitive genes, including the CDKL5 gene which is disrupted in CDKL5 deficiency. [Figure 26]Figure 26A shows that by targeting the codon-optimized transgene sequence and using synthetic miRNA not present in the UTR, dose-sensitive feedforward control can be achieved across other dose-sensitive genes, in this case the SYNGAP1 gene which is disrupted in SYNGAP1-related intellectual disability. Figure 26B shows that by targeting the codon-optimized transgene sequence and using synthetic miRNA not present in the UTR, dose-sensitive feedforward control can be achieved across other dose-sensitive genes, in this case the SYNGAP1 gene which is disrupted in SYNGAP1-related intellectual disability. [Figure 27] Figure 27A shows that dose-sensitive feedforward control can be achieved with other dose-sensitive genes. Figure 27B shows that dose-sensitive feedforward control can be achieved with other dose-sensitive genes, in this case the SMN1 gene which is disrupted in spinal muscular atrophy. Figure 27C shows that dose-sensitive feedforward control can be achieved with other dose-sensitive genes, in this case the INS gene which is disrupted in type 1 diabetes. Figure 27D shows that dose-sensitive feedforward control can be achieved with other dose-sensitive genes, in this case the FXN gene which is disrupted in ataxia Friedreich. [Figure 28] Figure 28A shows that dose-sensitive feedforward controls can be achieved in vivo with other dose-sensitive genes. Figure 28B shows that dose-sensitive feedforward controls can also be achieved in vivo with other dose-sensitive genes, in this case the UBE3A gene which is disrupted in Angelman syndrome. [Figure 29]This figure shows CDMS data of feedforward MECP2 constructs packaged in ssAAV9. Full-length feedforward products are packaged as desired, with less abnormal or partial packaging. Hairpin-like secondary DNA structures are known to inhibit efficient packaging in AAV particles. However, in the feedforward constructs analyzed, the presence of miRNA hairpins (in EF1a or MINIX introns) did not cause significantly smaller-than-expected packaged / partially packaged particles and did not affect the quality of AAV prep vilion composition. The dominant peak corresponding to fully packaged MECP2 feedforward cassettes contrasts with the much smaller peaks representing the distribution of empty particles and partially packaged genomes.

[0085] RTT253 constructor: CMV / CBA Promote (SEQ ID NO: 76) Human EF1a intron A (SEQ ID NO: 5) ffluc1 (SEQ ID NO:9) Kozak (SEQ ID NO: 73) Human MECP2_e1 (SEQ ID NO:1) ffluc1 x3 binding site (SEQ ID NO:34) WPRE3 (SEQ ID NO: 75) SV40pA (SEQ ID NO: 70) [Modes for carrying out the invention]

[0086] The proof of concept in the gene targeting construct of the present invention relates to the neurological disease Rett syndrome. It was generated in relation to the syndrome. Rett syndrome is a loss of function in the X-linked gene MECP2. It is caused by type mutations. An attractive treatment approach for this disease is Ade Using an associated virus (AAV) vector, a functional copy of the MECP2 gene is introduced into the nervous system. The goal is delivery, but a major obstacle to this approach is that cells cannot handle multiple viral vectors. This could lead to infection with a copy of the gene, potentially resulting in overexpression of the MECP2 gene. The inventors have found that overexpression of the MECP2 gene could cause severe toxicity. As previously revealed, clinically, duplication of the MECP2 gene in humans is associated with MEC It is known to cause a distinct and severe neurological disorder called P2 overexpression syndrome.

[0087] Using the construct described in this invention, cells can take multiple viral vectors. Even if a small number of copies infect the cell, it is possible to limit the level of MECP2 expressed within the cell. This makes it possible. This significantly expands the safety window for MECP2 gene therapy interventions. This allows for the administration of larger viral doses, infecting more cells, and more It is possible to achieve a robust recovery from the disease.

[0088] In this example, the introduced gene is ME, a gene that is mutated in Rett syndrome, a neurological disorder. This is a WT copy or codon-optimized copy of the protein-coding sequence of the CP2 gene. The instruction contains two elements to control the level of the introduced gene. It is being created. The first element is located within an intron between the promoter and the introduced gene. This is a non-mammalian or synthetic microRNA sequence contained in. Introns containing microRNAs are spliced ​​during premRNA processing. Mammalian or synthetic miRNAs can then be processed to degrade their target transcripts. It produces mature miRNAs. miRNAs are either synthetic or non-mammalian. Because it originates from insect sources, there are no known off-target effects within the mammalian genome. The second element of the construct is produced from an intron located at the 3'UTR of the construct. Several non-mammalian or synthetic miRNAs that match the non-mammalian or synthetic miRNAs. These are binding sites. The presence of these binding sites allows the introduced gene to be delivered to the microRN. A becomes a target. This leads to a reduction in the level of the transgene and prevents overexpression.

[0089] In an alternative embodiment of the feedforward principle, non-mammalian or synthetic microRNAs are used. It can be delivered within the gene therapy synthesis cassette intron. Micro within the 3'UTR Instead of targeting RNA binding, non-mammalian or synthetic microRNAs are used as transgenes. It is created within the codon-optimized protein coding sequence, and the corresponding binding site is located within the mammalian genome. It binds to a unique microRNA binding region (within the mammalian genome); that is, m The iRNA binding region will be a unique synthetic binding region. This version of feedforward The vector system can be made more compact. This is the packaging for the viral vector. This is particularly advantageous for large genes that are close to the ing capacity.

[0090] A single-gene loop allows for a certain level of expression, thereby enabling the circuit to handle a wide range of gene expression. It can maintain a relatively constant expression level across doses (i.e., desirable doses) (Shows dose insensitivity). This experimental system shows the relative effects of changes in gene dosage on gene expression. To create regimens that result in smaller changes. This is being introduced in gene therapy. This is an important characteristic when aiming for broad and uniform expression throughout the entire cell population, and is not associated with It is possible to increase the dose to achieve a high transduction rate without the effects of overexpression. ru. [Examples]

[0091] Example 1 Synthetic non-mammalian miRNA (ffluc1) or synthetic mi that cannot bind to the mammalian genome The binding site of non-mammalian miRNA combined with RNA or the binding site of synthetic miRNA By utilizing this method, it becomes possible to control the expression of transgenes while also ensuring no off-target effects. It can indeed be omitted. Preferably, constructs such as those described in Table 1 It may be provided.

[0092] [Table 1]

[0093] Table 1. Overview of gene therapy constructs for major indications, demonstration trials, and design considerations. Selection of feedforward components based on limitations.

[0094] These embodiments relate to major dose-sensitive genes, but those skilled in the art will understand As can be understood, the same feedforward design is applied in relation to known or specific conditions. This can also be applied to other dose-sensitive genes that will be determined.

[0095] As discussed herein, the feedforward system includes CAG, UBC, SV40, P GK, synapsin 1, neuron-specific enolase, U6, GFAP, MAG, MPZ It can be constructed using any alternative ubiquitous cell type-specific promoter. Ron may be any synthetic or endogenous sequence that can harbor non-mammalian or synthetic miRNA sequences may comprise an intron, which may be an intron upstream of or within a protein coding sequence or a combination where more than one non-mammalian or synthetic miRNA is produced from a single transgene cassette. Any non-mammalian or synthetic miR NA may target a recognition site within the transgene cassette, including translated and untranslated regions and may be any non-mammalian or synthetic miRNA. The gene may be any dose-sensitive gene where gene dosage confounds transfection efficacy. The number of binding sites can be fine-tuned to the desired level of dosage insensitivity, and may be 1, 2, 3, 4, 5, 6 or any number within the capacity of the transgene cassette. The polyA signal is preferably , for example, SV40, BGH, or a commonly used natural or synthetic polyA sig nal.

[0096] Neuro2a cells were transfected with various constructs with or without incorporation of a feedforward mechanism , and the expression level of the MECP2 transgene was analyzed by flow cytometry. A separate fluorescent marker on the construct was used to monitor the level of construct delivered to each cell (a surrogate for dose). Constructs comprising the feedforw ard control element showed a much narrower range of MECP2 transgene expression than constructs that did not comprise the element. Promisingly, as the amount of delivered construct increases , the attenuating effect of these elements increases, which means that the control element mitigates toxicity without interfering with therapeutic levels of gene expression This suggests that fine-tuning of dosage sensitivity levels can be provided .

[0097] Example 2 A feedforward cassette is administered to mice to limit transgene expression in cells . Wild-type mice are administered with a flag-tagged Mecp2 transgene, and somatosensory transgene expression in neurons of the cortex was monitored. The transgene was administered in an AAV vector with or without a feedforward regulatory system. The feedforward regulatory system utilized miRNA ffluc1 (SEQ ID NO: 9) and the EF1a promoter . Three ffluc1 binding sites (SEQ ID NO: 34) were provided after the Mecp2 sequence . Figure 13C shows a schematic diagram of the viral vector administered to mice. It shows the observed expression of MeCP2 in mice treated with a regulatory (feedforward) cassette and a non-regulatory (no feedforward mechanism) cassette . The regulatory cassette consistently results in limited expression levels (protein levels) , preventing the outgrowth of cells expressing very high levels of vector-derived protein . The inset shows representative micrographs from unregulated mouse brain (bright but variable expression) and regulated mouse brain (more uniform expression between cells) . Advantageously, this feedforward mechanism can be used to reliably limit protein expression of a transgene administered using a viral vector .

[0098] Example 3 Using a feedforward regulatory mechanism to achieve appropriate transgene expression throughout the tissue Distribution can be ensured. Figure 14 shows a more consistent MeCP2-FLA in controlled samples. This demonstrates the G expression level. The distribution of vector-derived proteins is shown in both samples. Although broad, the control cassette has expression hotspots and compared to the non-control version. This indicates that there is almost no gradient. Therefore, a feedforward mechanism is preferable. This involves the appropriate concentration of transgene protein expression across a group of cells, tissues, or organs. It can be used to control.

[0099] Example 4 Feedforward regulatory mechanisms ensure that the expression of transgenes is restricted throughout the neocortex. It can be used for this purpose. Figure 15 shows exogenous M administered to mice using an AAV cassette. Compared to eCP2, the difference between using and not using the feedforward control mechanism. It naturally exhibits MeCP2 expression.

[0100] Single-stranded AAV (ssAAV) particles are AAVs packaged in an AAV9 capsid. Includes a construct sandwiched between 2 ITRs, and a UPV virus vector generation unit (Un Produced by gene transfer of HEK293 cells at iversitat Autonoma de Barcelona .

[0101] The miRNA used was ffluc1 (SEQ ID NO:9), and 3x ffl The uc1 binding site (SEQ ID NO:34) is provided after the Mecp2 gene sequence. Expression was uniform across cells in the controlled image (15B) (however, in the case of native + vector). —Slightly higher due to the combination of signals from different sources—demonstrates restriction of expression. In contrast, , the uncontrolled cassette sample (15C) is a cell population that expresses very high levels of MeCP2 and shows variable levels of immunoreactivity across the entire cell population, including the population. Quantitation of these sam ples (15D) demonstrates restriction of expression to a narrow range by the feedforward cassette.

[0102] Example 5 Preferably, the feedforward control cassette can be administered in vivo without ad verse effects on health. Phenotypic evaluation was performed in wild-type mice administered with the feedforward control cassette. A regulatory construct expressing ffluc1 (SEQ ID NO: 9) miRNA and a codon-optimized human MECP2 transgene was administered. The uncontrolled con struct only expressed the codon-optimized human MECP2 transgene . The MeP426 uncontrolled construct was previously described in Gadalla KKE, Vudhironarit T, Hector RD, Si nnett S, Bahey NG, Bailey MES, Gray SJ, Cobb SR. Development of a Novel AAV Gene Therapy Cassette with Improved Safety Features and Efficacy in a Mouse Model of Rett Syndrome. Mol Ther Methods Clin Dev. 2017 Jun 16;5:180-190, and expressed wild-type human MECP 2 under the control of an endogenous mouse Mecp2 promoter described therein.

[0103] Figure 16 depicts a high-dose study, showing that transgene expression is restricted using the feedforward circuit. This restriction of transgene expression is superior to that of uncontrolled cassettes It is also advantageous in terms of safety. This figure shows a high-dose vector (4x10) administered to wild-type mice. 11 This shows in vivo experiments in which vg / mouse (direct injection into the brain in P1) was administered. In administration using an uncontrolled MECP2 cassette (16A), a toxicity score was observed, and it was fatal. (16B). In contrast, the control cassette had no detectable, obvious, harmful phenotypes. It was completely acceptable.

[0104] Example 6 Preferably, the feedforward mechanism does not interact with other sequences in the mammalian genome.

[0105] The miRNAs expressed in the feedforward construct are derived from insect miRNAs. Column (ffluc1; SEQ ID NO:9) or novel synthetic miRNA sequence (ran1 g;SEQ ID NO:17 and ran2g;SEQ ID NO:18) However, there are no predicted endogenous targets within mammalian transcripts.

[0106] To verify this, we used mirDB's off-target prediction tool to analyze miRNA The most likely human mRNA targets for the sequences ffluc1, ran1g, and ran2g are predicted. We measured the candidate human target genes / transcripts, and the genes that matched the miRNA seed sequences. The transcripts were ranked based on the number of target sites they contained.

[0107] We created a plasmid that expresses ffluc1 miRNA and a reporter transgene. Figure 19A). The plasmid drives the expression of the mNeonGreen reporter transgene. The hEF1a promoter was included. The ffluc1 miRNA was used with the hEF1a promoter. It was expressed within the EF1a intron located between the data and the transgene. HEK 293 100 μg of each plasmid was introduced into cells using lipofectamine. 48 hours later... , lyse the cells and use the MagMAX-96 total RNA isolation kit (Thermo Fisher Total RNA was isolated using ). The samples were pooled, and three samples were taken for each test plasmid. We created biological copies. RNA sequencing was performed on each biological copy, and read counts were calculated. Using FPKM (Fragments per kilobase of transcript per million reads), each The expression levels of human target transcripts were compared.

[0108] Figure 19 shows the analysis of the top 20 predicted human mRNA targets for ffluc1. This indicates that there is no significant difference in expression levels between the sample set and the control. From this result, f Overexpression of fluc1 has off-target effects on predicted human target genes. It was confirmed that this was not the case.

[0109] Therefore, preferably, the present invention affects endogenous gene expression in mammalian host cells. This invention provides a method for controlling the expression of an introduced gene without providing any external stimulus.

[0110] Example 7 Preferably, a feedforward mechanism is used to improve the phenotypic characteristics of the clinical state. We can provide complete and effective treatment.

[0111] AAV vector expressing a feedforward MECP2 construct, CBA / Wild-type (WT) and Mecp2 knockout strains maintained in a C57 mixed background. Tested with KO mice. Regulated (ffluc1; SEQ ID NO:9) or non-regulated mice. Intraventricular administration of ssAAV expressing the genus MECP2 into the brains of males at birth (P)0 / 1 (I Injections were administered bilaterally via CV. For the control injection, the same diluent without the vector was used. Vehicle control). Injected pups were returned to their home cages and evaluated weekly from 4 weeks of age. Uss was monitored until 15 weeks of age or until the human endpoint was reached. Figure 17B This shows the clinical scores and survival of WT and KO mice under all treatment conditions. Administration using the MECP2 cassette resulted in toxicity and reduced survival. In contrast, Administration using cassettes is perfectly acceptable, and mice are given uncontrolled feedforward cassettes. Compared to mice treated with a tonic or vehicle, mice with lower clinical severity scores (Rett-like table) (Regarding the current form) it exhibited. Therefore, the control mechanism is safe administration and the phenotype of KO mice. This shows both the correction of clinical severity and the improvement of clinical severity.

[0112] Figure 18 further develops this data, showing the specific characteristics observed in the Rett syndrome model mouse. This measures typical clinical characteristics. Mecp2 - / y Mouse (KO) with ffluc1 Administration of the control cassette resulted in partial improvement within the range of the Rett-like phenotype. This was not observed in KO mice treated with an uncontrolled construct. This is because the mice This is because they did not survive long enough for their phenotypes to be studied.

[0113] Example 8 The construct has been modified to provide enhanced expression, control, and stability. It may be provided. The construct may be provided to include a reporter transgene. The construct can contain a Kozak sequence that promotes potent expression. The 3'UTR can further include a stability element. The construct is mi It was engineered to reduce (but not completely eliminate) the effectiveness of RNA binding. It may further include one or more binding sites containing mutations. The constructs are described in detail below in Table 2.

[0114] [Table 2]

[0115] Table 2: Designed to enhance the expression of the transgene while maintaining strict control of expression levels. Summary of the gene therapy construct. Feed based on validation trials and design limitations. Elements of the forward mechanism. These embodiments relate to important dose-sensitive MeCP2. However, as those skilled in the art will understand, the same feedforward design is known This applies to other dose-sensitive genes that will be determined in relation to or specific conditions. This is possible. In generating a feedforward construct, the special mentioned above It should be understood that any combination of symbols can be used. Furthermore, these The instruct is illustrative and does not include any of the features mentioned above in Tables 3 and 4. Combine with any of the elements mentioned to generate a feedforward cassette. We should also understand that this is a good thing.

[0116] Figure 20 shows the effect of the above additional elements on MeCP2 expression in the control cassette. The incorporated features are related to the dosage of the cassette administered to HEK293T cells. This shows the effect on the level of introduced gene expression.

[0117] Any suitable promoter, constitutive or conditional, drives the expression of the transgene. It can be used. Preferably, the promoter is an Ef1a promoter, a CAG promoter, or a Je t promoter, CMV promoter, CBA promoter, CBH promoter, Synapsin 1 Promoter, Mecp2 Promoter, U1a Promoter, U6 Promoter, Ubiquitin C Promoter, neuron-specific enolase promoter, oligodendrocyte transcription factor 1 Or it may include a GFAP promoter. For the constructs in Table 2, any appropriate It should be understood that promoters can be used.

[0118] The miRNA used is any suitable synthetic miRNA that does not bind to the mammalian genome. It may be present. Preferably, the miRNA used is a synthetic sequence or a mammalian miRNA. It may be derived from a non-mammalian genome with no homology. Preferably, the mi used The RNA may be derived from an insect genome. An exemplary miRNA is shown in the table below. (As described in section 3).

[0119] [Table 3]

[0120] Table 3: m that can be used in feedforward constructs that control transgene expression The sequence of iRNA elements. It can bind to binding sites, but it does not bind to the mammalian genome. It is understood by those skilled in the art that any synthetic or non-mammalian miRNA may be used. Figure 20 shows the effects of various miRNAs on transgene expression.

[0121] The construct may be adapted to include a modified Kozak sequence. Preferably, modified A Kozak sequence is any Kozak sequence containing a nucleic acid motif that functions as a protein translation initiation site. It may also be a Kosack sequence. Preferably, a modified Kosack sequence is optional to promote increased translation. It may also be a modified sequence. Preferably, the Kozak sequence is GCCACCATGG(SEQ ID NO:73) may also be used. Figure 20 shows SEQ ID N as the Kozak sequence. This demonstrates the effect of using O:73 on the expression of the transgene.

[0122] In this embodiment, the target gene may be any one of the following target genes. i: MECP2, FMR1, UBE3A, CDKL5, FXN, SMN1, or INS or supplied using gene therapy for the treatment of a genetic condition or developmental disorder. A gene for which treatment is required. In particular, the target gene is for the treatment of a genetic condition or developmental disorder. It may be any gene whose expression is required to be regulated when it is delivered to the target.

[0123] Examples of binding mutations can be seen in Table 4 below.

[0124] [Table 4]

[0125] Table 4: Exemplary examples of miRNAs that can be introduced into the binding site to partially reduce miRNA binding. Sequence of binding mutants. Figure 20 shows how the mutant miRNA binding site can be altered in the introduced gene. This shows the effect on expression.

[0126] Preferably, it may include a stabilizing element to increase the expression of the transgene. Preferably, the stabilizing element may be located at 3'UTR. Preferably, this stabilizing element The ment is a post-transcriptional regulatory element (WPRE) of woodchuck hepatitis virus (WHV) ( SEQ ID NO:74) may also be used. Preferably, the stabilizing element is a stabilizing element. A cleaved version of WPRE that retains the rement but omits the X protein sequence, or A bozyme-stabilized sequence (WPRE3) may also be used. Figure 20 shows the stability element WPR. This shows the effect of E3 (SEQ ID NO:75) on the expression of the transgene.

[0127] Example 9 Evaluation of gene expression restriction in AAV-sensitive tissues

[0128] Dorsal root ganglion The dorsal root ganglia (DRG) are highly sensitive to AAV. After AAV delivery, the DRGs are highly sensitive to AAV. Transduction occurs repeatedly, potentially leading to toxicity. Feedforward circuits can combine these. To investigate whether it weakens expression in the weave, DRG was measured in 4 × 10⁻⁶ units. 11 vg / mouse At this dose, CBE-controlled and CBE-uncontrolled MECP2 feedforward ssAAVs are cured. It was dissociated from treated wild-type mice. The lumbar DRG was used to express MeCP2 from a vector ( n=3 per mouse, 3 mice per group) and in vivo distribution of the vector (1 per mouse) DRG (3 mice per group) was used for treatment. Mice treated with CBE-uncontrolled MECP2. The study was terminated at 3-4 weeks of age for toxicity / humanitarian endpoints. CBE-controlled MECP The mice treated in step 2 were terminated at 20 weeks of age. DRG was used as a control and age-matched. It was also isolated from WT and KO mice.

[0129] After completion, the mice were perfused with 4% paraformaldehyde (PFA), and then the tissue was separated. The tissue was fixed overnight in 4% PFA at 4°C and then stored in 30% sucrose until processing. It is embedded in a mixture of 30% sucrose and an optimal cutting temperature (OCT) compound on dry ice. The frozen tissue blocks were stored at -20°C until sectioning. The frozen sections were 12μm. Cut into m pieces, place on a coated slide, air dry at room temperature for 30 minutes, then proceed to staining. They were stored at -20°C. The frozen slides were rinsed with 0.1 MPBS and the tissue freezing matrix was used. After removing the kus, in an 85°C water bath, 10 mM sodium citrate buffer, 0 Antigen retrieval was performed for 30 minutes in 0.05% Tween-20 (pH 6.0). After cooling the slides at room temperature for 30 minutes, fill them with 0.3M PBS / TritonX-100 solution. Rinse and incubate in a humidifier at room temperature for 1 hour with 5% goat serum in 0.3M PBS / T solution. The sample was incubated to inhibit nonspecific binding. The slide was then treated with the primary antibody (monoclorose). (Naru, mouse anti-MECP2, M7443, Sigma, 1:500) in buffer at 4°C, humidified. The slides were incubated overnight in a container. After rinsing (with 0.3M PBS / T solution), the slides were... Secondary antibody (Alexa Fluor® 488 goat anti-mouse (H+L), cel (L signaling, 1:500) incubated in a humidifier at room temperature for 2 hours. After rinsing further with 0.3M PBS / T solution, the slide was treated with Hoechst333. 42. Dip in DNA dye staining solution (1:2000 in 0.1M PBS) at room temperature for 30 minutes. I incubated it. I used fade-resistant mounting fluid and nail polish to cover the slide. The samples were enclosed in a container and imaged using a confocal microscope.

[0130] Figure 21 shows the development of repressed transgenes in a DRG with a feedforward circuit. This is a diagram of a high-dose trial showing that the expression is restricted. This restriction of transgene expression is not controlled. It is safer than a cassette. The figure shows a high-dose vector (4×) administered to wild-type mice. This shows in vivo experiments in which 10¹¹vg / mouse (direct brain injection in P1) was administered. When a controlled MECP2 cassette was administered (21A), MeCP2 was significantly over-produced in the DRG. This manifested as toxicity and lethality. In contrast, the control cassette was perfectly acceptable, and DR MeCP2 expression levels were significantly lower in G. In vivo distribution analysis of the vector was performed using DR. G showed the same level of AAV in CBE-controlled and CBE-uncontrolled mice. This was shown (Figure 21D), and the difference observed at the MeCP2 level in DRG was found to be We confirmed that this was due to the attenuation effect of the forward circuit (Figures 21B-C).

[0131] liver The liver is also highly sensitive to AAV. Liver cells undergo high transduction after AAV administration. This can be toxic. Feedforward pathways reduce expression in these tissues. To investigate whether it causes deterioration, 1 × 10 12 CBE control or Wild-type cells treated with CBE-uncontrolled MECP2 feedforward ssAAV systemically (intravenously). The liver was dissected from a mouse. The liver was then subjected to vector-derived MeCP2 expression (n per mouse). (3 intercepts, 3 mice per group) and in vivo distribution of the vector (3 mice per group) They were treated for the following reasons. Mice treated with CBE-controlled and CBE-uncontrolled MECP2 showed the following results after injection. The procedure was completed in 4 weeks. In addition, as a control, liver cells were obtained from age-matched, non-injected WT mice. The organs were isolated.

[0132] After completion, the mice were perfused with 4% paraformaldehyde (PFA), and then the tissue was separated. The samples were fixed overnight in 4% PFA at 4°C, and then stored in 30% sucrose until processing. The fabric is wrapped in a mixture of 30% sucrose and an optimal cutting temperature (OCT) compound on dry ice. The tissue blocks were buried. The frozen tissue blocks were stored at -20°C until sectioning. The frozen sections were divided into 12 sections. Cut into μm pieces, place on a coated slide, and air dry at room temperature (RT) for 30 minutes. The slides were stored at -20°C until staining. The frozen slides were rinsed with 0.1 MPBS and the tissue was frozen. After removing the matrix, the 10 mM sodium citrate bath was placed in an 85°C water bath. Antigen retrieval was performed for 30 minutes in Farr, 0.05% Tween-20, pH 6.0. After cooling the mixture in the same buffer for 30 minutes, use 0.3M PBS / Triton X-10. Rinse with solution 0, then leave in a humidified room for 1 hour with 5% goat serum in 0.3M PBS / T solution. Incubation was performed to inhibit nonspecific binding. Subsequently, the primary antibody (mouse anti-MeCP2) was used. Incubated overnight in a humidifier at 4°C with (1:500). After rinsing (0.3M PBS / T solution), slides show secondary antibody (Alexa Fluor(registered trademark) 488Y The mice were incubated with anti-Gi mice (H+L, 1:500) at room temperature for 2 hours in a humidified chamber. After rinsing further with 0.3M PBS / T solution, the slide was treated with Hoechst 33. 342. Dip in DNA dye staining solution (1:2000 in 0.1M PBS) at room temperature for 30 minutes. I incubated it. I covered the slide with fade-resistant mounting liquid and nail polish. The samples were mounted in a container and imaged using a confocal microscope.

[0133] Figure 23 shows that gene expression in the liver is restricted by the feedforward circuit. This is a diagram of a high-dose trial. This restriction of transgene expression is safer than the uncontrolled cassette. This is advantageous in that respect. The figure shows wild-type mice given a high dose of vector (2 × 10⁻¹⁰). 12 vg / mouse This is an in vivo experiment in which the drug was administered intravenously at 5.5 to 6.5 weeks of age. However, when using the uncontrolled MECP2 cassette (23A), MeCP2 levels in the liver are significantly increased. Overexpression occurred. In contrast, the control cassette showed a significantly higher expression level of MeCP2 in the liver. Low levels were shown. In vivo distribution analysis of the vector showed that the liver controls and CBE It was shown that the same level of AAV was transduced in uncontrolled mice, and observed in the liver. The difference in MeCP2 levels was confirmed to be due to the attenuation effect of the feedforward circuit. ru.

[0134] This indicates that feedforward constructs are highly susceptible to AAVs. In tissues, suppressing the overexpression of introduced genes reduces the possibility of tissue damage / toxicity. This demonstrates that it is superior to conventional gene therapy structures because it allows for this.

[0135] Preferably, the feedforward construct is a combination that is highly sensitive to AAV. It can also be used in tissues to suppress the overexpression of introduced genes, causing tissue damage / toxicity. This approach is advantageous over conventional gene therapy constructs because it reduces the likelihood of sexual dysfunction.

[0136] Example 10 Constructs sandwiched between AAV2 ITRs packaged in AAV9 capsids Single-stranded AAV (ssAAV) particles containing this material were introduced at Virovek (California, USA). Manufactured using Iward's baculovirus introduction system.

[0137] About AAV vectors expressing a modified feedforward MECP2 construct Mecp2 knockout (KO) mau, maintained in a CBA / C57 mixed background. The study tested ssAAVs expressing controlled or uncontrolled MECP2 postnatally (P)0 / The male brain of 1 was injected bilaterally via intracerebroventricular venous injection (ICV). In the control injection, the vector was not included. The same dilution was used (vehicle control). The injected pup mice were returned to their home cages. Mice were evaluated weekly starting at 4 weeks of age. They were evaluated until 15 weeks of age, or until they reached the human endpoint. I monitored it until then.

[0138] Figure 22 shows that administration of a modified controlled feedforward cassette is acceptable for Rett syndrome. A study demonstrating therapeutic effects in a mouse model (Mecp2 KO mouse) was presented. This is a diagram showing a modified AAV-packaged construct used in in vivo studies. The (cassette) design is illustrated (Figure 22A). The control construct is ffluc1 mi RNA (SEQ ID NO:9) and a wild-type human MECP2 transgene were expressed. The unregulated construct expressed only the wild-type human MECP2 transgene. Protein expression is regulated by post-transcriptional effects of woodchuck hepatitis virus (WHV) at the 3'UTR. The presence of Rement (WPRE3) (SEQ ID NO:74) facilitated this process. The vector was administered to Mecp2 KO mice (1 × 10⁻¹⁰ 11 vg / mouse; P1 in the brain (Direct injection), followed by weekly evaluations from 4 weeks of age. Based on survival rate and RTT score data, In Rett syndrome model mice (Mecp2 KO mice), the control feedforward Docassette administration was tolerated and demonstrated therapeutic efficacy. CBE uncontrolled + WPR Mice administered with the E3 construct showed severe overexpression toxicity 2-3 weeks after injection. They had to be weeded out in between.

[0139] Western blot analysis was performed in different brain regions (cortex, hippocampus, thalamus, brainstem) (Figure) (22°C). The frozen tissue sample was mixed with 300 μL of NE1 buffer in a bead mill. The samples were modified and stored on ice. Each sample was given 250 U of benzonase nuclea. After adding the gel, the sample was shaken, incubated at room temperature for 15 minutes, and stored on ice. The sample was diluted 1:20 in NE1 buffer to obtain the protein. 4 100 μL Add x Laemmli Sample Buffer to each bead mill tube, Sun The pull was boiled for 10 minutes and then stored at -80°C. The samples were thawed, and each sample was 25 μg of the dye was electrophoresed on a 10% acrylamide gel at 150V until the dye reached the bottom of the gel. The gel was then transferred to a nitrocellulose membrane and maintained at 85V for 2 hours. Total protein was measured. Determined. Removed total protein staining and the membrane with LI-COR blocking buffer. The membrane was incubated at room temperature for 1 hour on a shaking incubator. Next, the membrane was subjected to a 1:1000 ratio. Along with the primary antibody anti-MECP2 at the specified dilution, 20 mL of LI-COR blocking buff Incubated overnight at 4°C in a fertilizer. Washed with TBS-T buffer for 10 minutes (x3). ) After that, in 20 ml of LI-COR® blocking buffer, 1:100 The membrane was incubated with a secondary dilution of 00 at room temperature for 2 hours. The membrane was then treated with TBS-T buffer. The samples were washed for 10 minutes (x3), rinsed with TBS buffer, and then imaged.

[0140] The results demonstrated that MeCP2 expression is restricted by the feedforward circuit. This was achieved. Along with improved survival rates and a decrease in RTT phenotype scores, the expression of this transgene was The limitations were further demonstrated to be safer than uncontrolled cassettes.

[0141] Example 11 Insect-derived miRNA sequence (ffluc1; SEQ ID NO:9) or novel synthesis miRNA sequences (ran1g; SEQ ID NO: 17 and ran2g; SEQ I D NO:18) does not have a predicted endogenous target within the mammalian transcriptome. To further verify this, we performed quantitative RT-PCR on the predicted mRNA target.

[0142] ffluc1(SEQ ID NO:9), ran1g(SEQ ID NO:18) Alternatively, run2g (SEQ ID NO:18) miRNA under the hEF1a promoter A plasmid was constructed to be expressed from the intron of the gene (Figure 24A). hsa-miR-1 32-3p, hsa-miR-34a-5p, or hsa-miR-644a miRN We also created a control plasmid that expresses A from the intron downstream of the hEF1a promoter. (Figure 24B). miRNAs expressed by the control plasmid are recognized human mRNs. Endogenous human miRNs possessing target A (MECP2, HSPA1B, and ACTB, respectively) It is A.

[0143] In human embryonic kidney 293 cells (HEK 293), Lipofectamine (registered trademark) Using the specified method, 100 μg of each plasmid was introduced into the cells, and after 48 hours, the cells were lysed and the total R NA was isolated. The quality and quantity of the isolated RNA were analyzed. 500 ng of whole RNA template Single-chain synthesis was performed using a 20 μL reaction solution containing 500 nM random hexamer. The SYBR Green PCR reaction involved 1 / 10 the amount of the first-chain synthesis reaction and a 300 nM residue. PCR was performed using gene-specific primers in 20 μL of reaction solution. The procedure was performed under Kling conditions: initial denaturation at 95°C for 3 minutes, followed by 95°C for 10 seconds and 55°C for 30 seconds. The experiment was conducted for 40 cycles of 30 seconds at 60°C, and the dissociation curve was continuously analyzed. The results were as follows: -ΔΔCt Using the law The analysis was performed to determine the relative difference between the sample and a control sample containing only Lipofectamine. The gene expression rate was calculated.

[0144] Using quantitative RT-PCR (qRT-PCR), ffluc1(IRF2BP2, H NRNPH1 and RPP30), ran1g (FASN, ETAA1 and MAIP1) ) and human mRNA labels of ran2g (MCFD2, SLC38A2 and FZD6) The transcription levels of the top three candidate plasmids were quantified. qRT-PCR was performed using the control plasmid: hsa- miR-132-3p (MECP2), hsa-miR-34a-5p (HSPA1B) , or recognition of miRNA expressed by hsa-miR-644a(ACTB) It was also used to quantify the transcription levels of identified endogenous mRNA targets.

[0145] Evaluation by qRT-PCR showed that ffluc1, ran1g, or ran2g were very Even when expressed at high levels, the detectable off-target effects are minimal. This was shown (Figure 24C). The only exception is FZD6, which is strongly controlled by ran2g. It was firmly downregulated. In contrast, the on-target positive control deeply suppressed the target and the assay. This demonstrates its robustness (Figure 24D).

[0146] Example 12 In vitro evaluation of feedforward effects in other CNS signs.

[0147] The inventors believe that the present invention is also effective in treating other diseases affecting the central nervous system (CNS). It was identified that MECP2 is linked to the UBE3A gene (a mutation in this gene is associated with Angel). (leading to Mann syndrome and Prader-Willi syndrome), and the CDKL5 gene (this We created a construct in which the gene mutation (which leads to CDKL5 deficiency) was replaced. .

[0148] ffluc1 miRNA (SEQ ID NO:9) and mNeonGreen Plasmids expressing target genes (GOIs) fused to porter genes were constructed. Regarding OI, there are constructions with a feedforward mechanism and constructions without one. A signal was generated (Figure 25A). In the control construct, at 3'UTR, in previous experiments, Three results for the same ffluc1 miRNA (SEQID NO:34) used The binding site for non-mammalian miRNA was included. In the unregulated construct, at the 3'UTR, The ffluc1 miRNA contained a scrambled (scr) sequence that was incompatible with its binding. In embryonic kidney 293 cells (HEK293), Lipofectamine 3000 was used. Then, 100 μg of each plasmid was introduced. After 48 hours, the cells were harvested and the introduced genes were analyzed. The expression level of the offspring was evaluated by flow cytometry. Another fluorescent marker on the construct - (mRuby) is used to determine the level of construct delivered to each cell (as a substitute for dose). We monitored it.

[0149] Figures 25B-C show that a dose-sensitive feedforward control is compared to other dose-sensitive controls. This diagram shows that this can also be achieved with sex genes. The feedforward control is U BE3A gene (25B) (Angelman syndrome and Prader-Willi syndrome) (and destroyed) and the CDKL5 gene (25C) (destroyed in CDKL5 deficiency disorder) This was observed in both (and)

[0150] The expression of these proteins (UBE3A and CDKL5) is measured by flow cytometry. Determined by NeonGreen protein levels, which are evaluated by [the relevant mechanism]. The feedforward construct is a non-controlled control construct lacking miRNA regulation. A comparison was made with the ct (Figures 25A-C). The graph is mRuby (x-axis - positive to cell). (A measure of midi levels, unaffected by miRNA regulation) vs. UBE3A-Neon Green or CDKL5-mNeonGreen (y-axis miRNA controlled) This shows the level of the protein (that is being produced). The results are shown by the difference in the slope of the linear regression line. Therefore, ffluc1 miRNA was found in the feedforward sample compared to the control. This has shown that it is effective in controlling the expression of UBE3A and CDKL5.

[0151] As observed in MECP2, the attenuation effect of the feedforward element is delivered As the amount of construct increases, this increases, and this is because the control element is treating This suggests that toxicity can be mitigated without interfering with gene expression levels.

[0152] Example 13 In vitro feedforward evaluation of other CNS signs - codon optimization Targeting introduced genes

[0153] The inventors have found that codon-optimized protein coding sequences serve as miRNA binding sites. They discovered that it could be used. By delivering synthetic miRNA within the gene therapy cassette, 3 Instead of targeting miRNA binding sites within the UTR, the goal is codon optimization of the transgene. This synthesis targeted a unique miRNA binding region created within the protein-coding sequence. miRNAs do not have a corresponding binding site within the mammalian genome. This method is used by viruses. This is particularly advantageous for larger genes, which are close to the packaging capacity of the gene.

[0154] Figure 26A-B shows the case where the miRNA binding site is located in the transgene protein-coding sequence. This figure shows that dose-sensitive feedforward control can be achieved. The figure shows the SYNGAP1 gene (destroyed in SYNGAP1-related intellectual disability) using this method. It shows the control of (removed). Codon optimization S fused to the mNeonGreen reporter gene YNGAP1 transgene and synthetic syn3i miRNA (SEQ ID NO: 29) Plasmi expresses either a controlled construct or no miRNA (uncontrolled construct). A sample was prepared (Figure 26A). Lipofe was added to human embryonic kidney 293 cells (HEK293). Each plasmid was transfused using ctamine3000 at a dose of 100 μg. (48 hours) After a period of time, the cells were harvested and the level of transgene expression was evaluated by flow cytometry. Using another fluorescent marker (mCherry) on the struct, the delivered cone to each cell was identified. The struct level (dosage substitute) was monitored.

[0155] SynGAP protein expression was evaluated by flow cytometry in NeonGree Determined by n protein levels. A controlled feedforward construct This was compared to an unregulated control construct lacking miRNA regulation (Figure 26A-B). The graph shows mRuby (x axis - measure of the amount of plasmid into cells and miRNA regulation). (Not affected by) vs. SynGAP-NeonGreen (y-axis - miRNA) This indicates the level of the protein that is controlled by it. The results are shown by the difference in the slope of the linear regression line. As such, syn3i miRNA (SEQ ID NO:29) was compared to the control. It is effective in controlling SynGAP expression in feedforward samples. show.

[0156] The attenuation effect of the feedforward element increases as the amount of constructs being delivered increases. This increases, and this is an alternative embodiment of this feedforward principle, which is genetic at the therapeutic level. This suggests that toxicity can be mitigated without hindering the expression of offspring.

[0157] Example 14 In vitro feedforward evaluation of other non-CNS signs

[0158] The feedforward mechanism for non-mammalian miRNAs is peripheral, not central nervous system (CNS). It was also effective in other disorders with a predominantly nervous system phenotype. MECP2 encodes other proteins. Sequence: SMN1 gene (mutations in this gene lead to spinal muscular atrophy), INS gene (Mutations in this gene lead to type 1 diabetes), FXN gene (Mutations in this gene lead to fr I created a construct that replaced it with (leading to Dreich's dysphasia). 3'UTR is , the same ffluc1 miRNA (SEQ ID NO:9) used in the previous experiment It contained binding sites for three non-mammalian miRNAs (SEQ ID NO:34).

[0159] Plasmid expressing ffluc1 miRNA and one of the target genes (GOI) mentioned above. We created the following. The GOI was fused with the mNeonGreen reporter gene. Each GOI Regarding this, constructs with a feedforward mechanism and constructs without one Generated (Figure 27A). In the control construct, at 3'UTR, SEQ ID NO: 34 miRNA binding sites were included. In the unregulated construct, f was added to the 3'UTR. Human embryos were treated with a scrambled (scr) sequence that is incompatible with fluc1 miRNA binding. In the kidney 293 cells (HEK293), Lipofectamine 3000 was used to treat each A plasmid of 100 μg was introduced into the cells. After 48 hours, the cells were harvested and the expression of the introduced gene was measured. The level was evaluated by flow cytometry. Another fluorescent marker (mR) on the construct. We use UBY to monitor the level of construct delivered to each cell (dose surrogacy). —did.

[0160] Figures 27B-D show other dose-sensitive genes (in this case, (27B) disruption in spinal muscular atrophy). The SMN1 gene is destroyed (27C), and the INS gene is destroyed in type 1 diabetes (27D). Dose-sensitive feed across the FXN gene (which is destroyed in Friedreich's ataxia) This diagram shows what can be achieved with forward control.

[0161] The expression of these proteins (SMN1, insulin, and frtaxin) is flusa Determined by NeonGreen protein levels assessed by iteometry. It controls the feedforward construct, which lacks miRNA regulation and is unregulated. A comparison was made with the illuminated construct (Figures 27A-D). The graph was generated using mRuby (x-axis - thin). (A measure of the amount of plasmid in the cell, unaffected by miRNA regulation) vs. SMN 1-NeonGreen, insulin-mNeonGreen, or Frataxin- This indicates the level of mNeonGreen (y-axis - miRNA-regulated protein). The results are shown by the difference in the slope of the linear regression line, ffluc1 m iRNAs, compared to the control, were found in the feedforward sample, specifically in SMN1 and insulin. It has been shown to be effective in controlling the expression of and Frataxin.

[0162] As seen in MECP2, the attenuation effect of the feedforward element is the delivered con As the amount of struct increases, this increases, and this is because the control element is at the therapeutic level. This suggests that toxicity can be mitigated without interfering with gene expression in the body.

[0163] Example 15 Control of UBE3A in glucose

[0164] The inventors have further developed a treatment for other dose-sensitivity disorders affecting the central nervous system (CNS). In this treatment, we demonstrated the use of a non-mammalian miRNA feedforward mechanism. The UBE3A gene, which is disrupted in Germain syndrome and Prader-Willi syndrome, It was shown that this mechanism is controlled in vivo by a forward drive mechanism.

[0165] ffluc1 miRNA (SEQ ID NO:9) fused to 3xFLAG tag A construct expressing human UBE3A was generated. It has a feedforward mechanism. We generated constructs that have and constructs that do not (Figure 28A). Control construct In this study, the 3'UTR contained the miRNA binding site SEQ ID NO:34. The AAV2 ITR is sandwiched between the AAV9 capsid. Single-stranded AAV (ssAAV) granules containing controlled and uncontrolled UBE3A constructs. The child was introduced into Virovek (Hayward, California, USA) baculovirus system It was generated by [the specified method / system].

[0166] Figure 28B shows the UBE3A controlled feedforward cassette and the uncontrolled UBE3A cassette. Compared to [another method], it has been demonstrated to provide in vivo control. Anti-FLAG antibody Immunoblot analysis using this method provides readout of UBE3A expression levels in cells. A feedforward UBE3A construct-expressing AAV vector is used in CBA Tested in wild-type mice maintained in a C57 mixed background. Controlled or Unregulated UBE3A-expressing ssAAV was introduced into the brains of male infants 1 day postnatally (P) (ICV). The drug was administered by injection to both sides. PBS (vehicle control) was used as the control injection. The injected pup mice were culled on the 7th day after injection, and tissue samples were taken for analysis. The pull was homogenized with 300 μL of buffer NE1 using a bead mill and stored on ice. After adding 250 U of benzonase nuclease to each sample, the samples were then... The samples were shaken, incubated at room temperature for 15 minutes, and stored on ice. The samples were then subjected to protein analysis. For volume control, dilute with NE1 buffer at a 1:20 ratio. 100 μL of 4 bead mill tubes were added. Add x Laemmli Sample Buffer and boil the sample for 10 minutes. Afterward, the samples were stored at -80°C. The samples were thawed, and 25 μg of each sample was mixed with 10% acrylamide. The dye was electrophoresed at 150V on a mid-gel until it reached the bottom of the gel. The gel was then covered with nitrocellulose. The sample was transferred to a membrane and held at 85V for 2 hours, after which the total protein was measured. The total protein was stained. Remove and shake the membrane with LI-COR® blocking buffer. The membrane was incubated in an incubator at room temperature for 1 hour. Next, the membrane was placed in 20 mL of LI-COR (Registered Trademark) Blocking Buffer and 1:2000 dilution of primary antibody anti-FLAG for 4 The membrane was incubated overnight at °C. The membrane was washed with TBS-T buffer for 10 minutes (x3). Incubate in 20 mL of LI-COR® blocking buffer at room temperature for 2 hours. Then, add the secondary antibody IRDye 800CW anti-mouse at a dilution of 1:10000. The membrane was washed with TBS-T buffer for 10 minutes (x3), and then rinsed with TBS buffer. After that, it was converted into an image.

[0167] This is because UBE3A (i.e., a transgene other than MECP2) is a non-mammalian miRN. This demonstrates that it can be controlled in vivo under the control of the feedforward mechanism of A. Yes. This allows for the introduction of genes / disorders that are known to be dose-sensitive. This can reduce the potential for tissue damage / toxicity caused by gene overexpression.

[0168] Example 16 Efficient package of feedforward constructs in ssAAV

[0169] A feedforward construct expressing the MECP2 transgene is used with AAV9 caps. A single-stranded A2 containing a construct sandwiched between AAV2 ITRs packaged in a sid. Prepared as AV(ssAAV) particles and processed using the HEK293 process (Viral Vector Production). n Unit, Universitat Autonoma Barcelona, ​​Spain) or Virovek (Hayward, CA) It was generated by a baculovirus-based infection system in the USA. Both processes Using this, the inventors have demonstrated that a feedforward gene therapy construct can be efficiently constructed. In terms of scale, it has a very high titer (maximum 1.94 × 10⁻⁶). 14 Can be produced using viral genome / ml. This demonstrates that the present inventors have developed a feedforward controlled gene therapy technique. We confirmed that the technique is configured for efficient manufacturing. Importantly, the inventors The feedforward synthesis circuit construct can be efficiently packaged into AAV. This demonstrates that.

[0170] After AAV production, CDMS (charge detection mass spectrometry) analysis is performed, and based on the charge and mass, AA The size of the V particle was determined. This tool is used to determine the quality of the packaging and the potency of the AAV product. To determine if there are any partially packaged species that could have an impact It is useful for the eyes.

[0171] Figure 29 shows the feedforward MECP2 constraint packaged with ssAAV9. This is a typical CDMS analysis of the tract. The full-length feedforward product was as expected. Caged with minimal abnormal or partial packaging. Secondary DNA structures like this inhibit efficient packaging in AAV particles. It is known that... However, in the feedforward construct that was analyzed, miRN The presence of hairpin A (in EF1a or MINIX intron) is less significant than expected. This does not result in packaging / partially packaged particles, and AAV preparations It does not affect the quality.

[0172] Gene sequences including secondary structures such as stem-loops, hairpins, and miRNA-generating sequences. This is very commonly the case of unusual packaging and heterogeneous species that conversely impair the purity of the product. It is known to cause capsule formation (Xie et al., 2017). Figure 29 shows the relevant technology. It displays profiles that are considered to be very clean within the context. Therefore, The inventors have developed a feedforward AAV construction that enables large-scale production of high-purity products. By developing the ct, we provided a solution to the purity problem.

Claims

1. Promoter and; At least one non-mammalian or synthetic miRNA expressed within an intron, prior Synthetic miRNAs are sequences that do not exist in nature, and are composed of at least one non-mammalian or synthetic miRNA. With adult miRNA; Transgenes and; At least one non-mammalian or synthetic gene that provides control over the expression of the aforementioned transgene The binding site of the miRNA, the binding site of the synthetic miRNA, is a compound that does not exist in nature. A column with at least one binding site for non-mammalian or synthetic miRNA; A construct including a polyadenylation signal.

2. The miRNA binding site that provides control over the expression of the aforementioned transgene is located at 3'UTR Or the construct according to claim 1, provided within 5'UTR.

3. The binding site for the non-mammalian or synthetic miRNA is provided within the transgene. The construct described in Item 1.

4. The relative performance of the transgenes between cells receiving vector-derived transgenes at different levels. A single gene circuit to provide a fixed level of expression (i.e., dose-insensitive) A construct according to any one of claims 1 to 3, which provides the following.

5. The aforementioned at least one synthetic or non-mammalian miRNA exhibits an off-target binding effect. The construct according to any one of claims 1 to 4.

6. The aforementioned non-mammalian or synthetic miRNA is: [Array 5-6] The construction according to any one of claims 1 to 5, expressed in an intron provided by to.

7. The aforementioned miRNA is a non-mammalian miRNA derived from insect miRNA, and optionally, miRNA specifically binds to the miRNA binding site of firefly luciferase (ffluc1). A construct according to any one of claims 1 to 6 that can be combined.

8. Multiple miRNA binding sites within the construct, optionally three miRNA binding sites. Site, binding sites for at least four miRNAs, binding sites for at least five miRNAs , providing at least six miRNA binding sites, as described in any one of claims 1 to 7. A constructed structure.

9. In the aforementioned construct, there are multiple non-mammalian or synthetic miRNAs that are expressed. The construct according to any one of claims 1 to 8.

10. The aforementioned non-mammalian firefly luciferase miRNA is as follows: [Arrays 9-12] A construct according to any one of claims 1 to 9, wherein the array is selected from the following.

11. The aforementioned synthetic miRNA is as follows: [Array 13-20] A construct according to any one of claims 1 to 6 or 8 to 9, which is an array selected from 。

12. The aforementioned synthetic miRNA targets the coding sequence of the target gene, and the following [Arrays 21-32] A construct according to any one of claims 1 to 11, selected from the above.

13. The binding sites of the aforementioned non-mammalian or synthetic miRNA are as follows: [Arrays 33-55] A construct according to any one of claims 1 to 12, selected from the above.

14. The synthetic miRNA targets the coding sequence of the target gene, and the binding portion of the synthetic miRNA The rank is as follows [Arrays 56-67] A construct according to claim 12, selected from the above.

15. The promoter is selected from constitutive or conditional promoters, and optionally the promoter The construct according to any one of claims 1 to 14, wherein the substance is tissue-specific.

16. The aforementioned promoter, [Arrays 68-69] A construct according to any one of claims 1 to 15, selected from the above.

17. The aforementioned polyA sequence is as follows: [Arrays 70-72] A construct according to any one of claims 1 to 16, selected from the above.

18. The claim further includes a stability element, the stability element being located at 3'UTR. The constructs described in items 1 to 16.

19. The aforementioned stability element is as follows: [Arrays 74-75] A construct according to claim 17, selected from the above.

20. [Array 73] A construct according to any one of claims 1 to 19, further comprising:

21. The aforementioned miRNA binding site is designed to partially reduce miRNA binding. a construct according to any one of claims 1 to 20.

22. A vector comprising the construct according to any one of claims 1 to 20.

23. The vector is an AAV or lentiviral vector, and optionally the vector is It is an AAV vector, and optionally the construct activates the expression control element. They are linked together, and the expression control element and the construct are together approximately 5' The vector according to claim 21, flanked by a 3'AAV terminal inversion sequence (ITR). —.

24. A vector according to any one of claims 21 to 22, packaged in a billion. And, optionally, the vector when packaged in the aforementioned billion is the construct A vector that does not affect the quality of the output.

25. A vector according to any one of claims 21 to 22, formulated into nanoparticles.

26. The construct according to any one of claims 1 to 20 or the construct according to claims 21 to 24 A method for expressing an introduced gene using a vector, wherein the introduced gene can optionally be expressed in a specific vector. A method for expression in mammalian cell type.

27. A method for treating a disorder in a subject, the method described in any one of claims 1 to 20. The step of providing the struct or the vector according to claims 21 to 24 to the target Includes, method.

28. To be used to treat disorders caused by insufficient gene expression in the target organism. The construct according to any one of claims 1 to 20 or the construct according to claims 21 to 24 A composition containing a vector.

29. The aforementioned disorder is any monogenic disorder for which control of the expression of the corrective gene is desired. And, optionally, the aforementioned monogenic disorder is Rett syndrome, fragile X syndrome, or Angelman syndrome. Group, Syngap-related intellectual disability, CDK15 deficiency, Fredrich ataxia, spinal muscular dystrophy The method according to claim 26, selected from the group consisting of trophy, hemophilia and diabetes. The composition according to claim 27.

30. The aforementioned faults are: PRKCZ, TTC34, PRDM16, ARHGEF16, P ARK7, PRDM2, IGSF21, PTCH2, NFIA, ST6GALNAC3, DPYD, COL11A1, PDZK1, GPR89A, NBPF11, GPR89B, KCNT2, CFHR2, ASPM, PTPRC, GPATCH2, DUSP10, GP R137B, RYR2, CHRM3, RGS7, AKT3, KIF26B, SMYD3, LPIN1, EPCAM, MSH2, NRXN1, XPO1, LRP1B, ZEB2, A CVR2A, MBD5, KIF5C, SCN1A, COL3A1, PMS1, PLCL1 , SATB2, PARD3B, EPHA4, SPHKAP, CHL1, GRM7, TRA NK1, DOCK3, FAM19A1, FOXP1, ROBO1, CADM2, FOXL 2, SOX2, LPP, RASGEF1B, GRID2, FAT4, NR3C2, LRB A, FGA, GALNTL6, WWC2, TLR3, IRX2, IRX1, CDH12, CDH9, NIPBL, HEXB, MEF2C, GRAMD3, FBN2, PRELID 2, TCOF1, GABRG2, MSX2, NSD1, FOXC1, CDYL, TBC1 D7, RUNX2, MUT, RIMS1, NKAIN2, LAMA2, ARID1B, P ARK2, PACRG, QKI, TNRC18, FBXL18, SUGCT, GLI3, AUTS2, MLXIPL, COL1A2, PPP1R9A, CFTR, TSPAN12 , GRM8, CNTNAP2, MNX1, CSMD1, MCPH1, LPL, ANK1, IMPAD1, CHD7, VCPIP1, TRPS1, PARP10, DOCK8, KA NK1, GLIS3, PTPRD, MLLT3, ROR2, PTCH1, AL16238 9.1, ARRDC1, EHMT1, PCDH15, CTNNA3, ADK, BMPR1 A, PAX2, BTRC, INPP5A, MRPL23, ELP4, PAX6, CPT1 A, DYNC2H1, KIRREL3, WNK1, CACNA1C, PPFIBP1, T BX5, MED13L, NALCN, CHD8, MYH7, TTC6, DAAM1, NR XN3, MTA1, SNRPN, UBE3A, OCA2, HERC2, CHRFAM7A , ARHGAP11B, OTUD7A, FBN1, HEXA, SNUPN, NRG4, A C112693.2, IGF1R, LRRC28, HBA2, HBQ1, CREBBP, RBFOX1, CDR2, CDH13, CYBA, NXN, YWHAE, SMG6, ME TTL16, PAFAH1B1, ADORA2B, NT5M, RAI1, NF1, C17 orf67, PITPNC1, ACOX1, TCF4, DOCK6, CACNA1A, L PHN1, ZSCAN5A, BMP2, MYT1, PEX26, USP18, DGCR6 L, USP41, UBE2L3, NF2, LARGE, BRD1, SHANK3 CDK Expression of genes selected from a list including L5, FXN, SMN1, F8, and INS. The method according to claim 26 or the composition according to claim 27, which is treated by...

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