Patent Application
20230323391
Gene therapy aims to deliver a therapeutic transgene to affect correction in a genetic disease. The present invention provides constructs to generate a relatively fixed level of expression of the transgene across cells receiving different levels of vector-derived transgene. Also described herein is a method of controlling gene expression wherein the control is provided using the described gene circuit.
Whilst the concept of gene therapy to deliver a therapeutic transgene to affect correction in a genetic disease is known, many genes are highly dosage sensitive whereby too little or too much expression of a gene product can have deleterious effects. Viral-mediated gene transfer is a powerful means to deliver therapeutic transgenes to target tissues and cells including cells of the nervous system. High virus titers are typically necessary to enable effective system-wide transduction for maximal therapeutic impact. However, the same high titres may cause overexpression toxicity due to supraphysiological levels of transgene expression achieved in some cells. An effective system is required to limit the expression of the vector-derived transgene within a window that alleviates the disease-causing genetic deficiency without producing overexpression toxicity.
WO2016040395 discusses the use of synthetic RNA circuits for gene transfer. The circuits include a first RNA molecule comprising at least one sequence recognized by a first microRNA specifically expressed in a cell type and a sequence encoding a protein that specifically binds to a RNA motif and inhibits protein production. A first microRNA is described as miR-21. Also provided is a second RNA molecule comprising a sequence recognized by a second microRNA that is not expressed in the cell type at a RNA motif and a sequence encoding an output molecule. A second microRNA is described as miR-141, miR-142 and miR-146. The application describes differential expression of an output protein by different cells (cancer and non-cancer cell) dependent on the endogenous miR provided by these cells.
Strovas T J, Rosenberg A B, Kuypers B E, Muscat R A, Seelig G. MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations. ACS Synth Biol. 2014; 3(5):324-331 discusses the use of a single-gene microRNA (miRNA)-based feed-forward loop. It provides an intronic miRNA that targets its own transcript. Strovas considers the difficulty of long-term stable expression of engineered genetic programs in mammalian cells. This work utilised a gene circuit in which an intron containing mouse mir-124-3 gene was inserted into a red fluorescent reporter (mCherry). The pre-mRNA is transcribed from a doxycycline inducible promoter leading to a coexpression of the mir-124 and mCherry. A repressive regulatory link between the miRNA and the mCherry transcript was provided by a truncated version of the mir-124-regulated 3′UTR of the Vamp3 gene to the mRNA.
Whilst WO2016040395 discusses the use of differently expressed endogenous miRs, in normal and cancer cells to provide for expression, this use of miRs has limited use in the treatment of non-cancer diseases. The inventors have also determined that the existing methods by Stovas would have multiple off-target effects on a variety of genes that are known to be regulated by endogenous miRNAs such as the miR124 used in this paper. Indeed, miR124 is known to be linked to a number of cancers so would be unsuitable for use in gene therapy. Thus, providing an endogeneous micro RNA may be problematic as endogenous targets in addition to the transgene may be provided.
The present inventors have sought to provide alternative constructs with advantages over the constructs provided in the art.
The inventors have determined a system to limit the expression of a vector-derived transgene within a window that alleviates the disease-causing genetic deficiency without producing overexpression toxicity, to enable what the inventors term ‘dosage-insensitivity’, whereby cells or tissues receiving more vector-derived transgene are disproportionately suppressed through an in-built single gene circuit that can regulate adaptively. That is, the vector-derived transgene is downregulated at high vector dosages so that the circuit maintains a relatively stable level of expression across a range of vector doses with the result being that the overall population of cells express a more even and controlled level of vector-derived transgene. Increasing doses of vector will result in more cells expressing the transgene within a cell population but without a concomitant increase in overexpression compared to conventional gene therapy cassettes. Sensitive cell types that often receive high vector loads such as in the heart, liver and dorsal root ganglia will also be less susceptible to superinfection-mediated overexpression by this mechanism.
The present inventors have designed synthetic or non-mammalian miRNA construct(s), which overcome disadvantages associated with mammalian-based miRNA constructs which exhibit the risk of off-target effects. The inventors have demonstrated the utility of non-mammalian or fully synthetic (not known in nature) miRNA to ensure the absence of targets within the host (human genome).
Moreover the inventors have determined how such synthetic components may be used to allow a fine-tuning of the system (number of sites and efficient intron exclusion) to achieve appropriate dosage-insensitivity.
Accordingly a first aspect of the present invention provides a construct comprising:
The miRNA binding sites discussed herein are synthetically derived to differ from mammalian sequences present in a mammalian cell or are provided from another non-mammalian species, for example insect. When the miRNA binding sites are from insect not present in a mammalian sequence, for example ffluc1, non-mammalian systems may be used. The combination of miRNA binding sites and non-mammalian or synthetic miRNA minimise the off-target regulatory effects of the construct. This allows regulation of expression of the transgene to provide a desired dosage (expression) of the transgene.
Suitably the miRNA binding sites which provide for control of the expression of the transgene may be provided within the 3′ UTR, the 5′ UTR and/or within the transgene. Suitably, when provided in the transgene, the miRNA binding site may be codon-optimised such that it provides a synthetic or non-mammalian binding site but does not impact upon the amino acid sequence of the transgene protein. The construct can be used to provide a feed forward loop which allows expression control.
Suitably, a stability element to increase transgene expression may be included. Suitably, the stability element may be located in the 3′ UTR. Suitably this stability element may be the Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) (SEQ ID NO: 74). WPRE is a tripartite regulatory element containing gamma, alpha, and beta elements. Suitably, the stability element may be a truncated version of the WPRE, retaining the stability element, but omitting the X-protein sequence, or a ribozyme stability sequence (WPRE3) (SEQ ID NO: 75). WPRE3 is a shortened WPRE sequence containing two of the three regulatory elements of WPRE (a minimal gamma and alpha elements). Suitably, the WPRE3 stability element provides a DNA sequence that creates a tertiary structure in the processed transcript, which enhances transgene expression.
Suitably different promoters can be used with a range of transgenes. In the present invention, the strength of the feed forward loop can be adjusted to allow control of the level of expression of the transgene. This provides for dosage sensitivity. Adjustment of the number of micro RNA binding sites in the single gene circuit and by using synthetic introns that are spliced out with differing efficiency also allows fine-tuning of the circuit.
The construct(s) may be adapted to express the transgene in a mammalian cell. Suitably the construct(s) may be adapted to be provided to a mammalian cell, suitably to a particular mammalian cell or cell type to which expression of the transgene is to be effected.
Advantageously, there can be provided a single gene circuit using an intron-derived microRNA in order to generate a relatively fixed level of expression across cells receiving different levels of vector-derived transgene.
As would be understood by those of skill in the art, features of the construct (a promoter, a synthetic miRNA expressed within an intron, a transgene, miRNA binding sites which provide for control of the expression of the transgene, a polyadenylation signal), should be provided relative to each other to allow functional expression of the transgene.
The construct may be adapted to include a modified Kozak sequence. Suitably, the modified Kozak sequence may be any Kozak sequence which includes any nucleic acid motif that functions as the protein translation initiation site. Suitably, the modified Kozak sequence may be any modified sequence which promotes an increase in translation initiation. Suitably, the Kozak sequence may be GCCACCATGG (SEQ ID NO: 73).
In embodiments a construct comprises (5′ to 3′):
In embodiments a construct comprises (5′ to 3′):
In embodiments a construct comprises (5′ to 3′):
In embodiments a construct comprises (5′ to 3′):
In embodiments a construct comprises (5′ to 3′)
In some embodiments a construct may include a promoter, at least one non-mammalian or synthetic miRNA expressed within an intron, a transgene, one or more binding sites which provide for control of the expression of the transgene within the transgene or 3′UTR, a polyadenylation signal and, optionally, any one or more features as recited in the above embodiments. In some embodiments may comprise the one or more features recited above in the order that such features are recited.
Suitably, the constructs may be modified to provide enhanced expression, regulation and stability. Suitably the constructs may contain a reporter transgene. Suitably the constructs may contain a Kozak sequence which promotes strong expression. Suitably the constructs may contain a stability element in the 3′UTR. Suitably the constructs may contain one or more binding sites which include mutations engineered to reduce the efficacy of (but not completely ameliorate) miRNA binding.
Suitably the gene of interest may be MECP2. Alternatively, the gene of interest may be any one of the following genes of interest: FMR1, UBE3A, CDKL5, FXN, SMN1, or INS. The gene of interest may be any gene which is required to be supplied using genetic therapy for treatment of a genetic condition or developmental disorder. The gene of interest may be any gene which requires controlled expression when delivered to a subject to treat a genetic condition or developmental disorder.
Suitably, the transgene is a protein-coding gene which is artificially introduced into a target cell. It is provided as part of the construct of the first aspect of the invention, for example as part of a gene therapy cassette, under the control of a selected promoter. A DNA sequence of a transgene can represent a specific isoform of a specific gene. Transgene DNA sequences may be codon optimised. Codon optimisation can provide a specific and unique DNA sequence but the DNA and subsequent mRNA changes do not affect the coding sequence of the protein; i.e. the wild-type amino acid sequence is maintained.
Suitably a transgene may be selected from
Suitably a functional variant of these transgenes may be provided wherein the functional variant retains the function provided by the transgene and has at least 60% sequence identity, 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% sequence identity. Suitably a functional variant may be a fragment of the transgene which provides the function of the transgene. Suitably where the miRNA binding sites, which provide for control of the expression of the transgene, are provided within the transgene, the miRNA binding sites are provided in the functional variant such that the miRNA can bind and control the expression of the transgene.
Sequence identity can be determined by any methods known in the art. Suitably sequence identity may be determined over the full length of the transgene.
Suitable transgenes include those based on any single gene disorders for which controlled expression of the transgene is desired. Suitable transgenes include those based on any monogenic disorder for which controlled expression of the transgene is desired. Additional exemplary transgenes include those based on single gene CNS disorders for which controlled expression of the transgene is desired. The nervous system expresses many genes that are known to be deleterious to nervous system function when overexpressed. However, the present invention is applicable to any situation in where transgene overexpression is deleterious including gene therapy for non-CNS disorders. An example would include dystrophin gene replacement in muscle cells whereby moderate overexpression does not cause deleterious adverse effects but when very high levels of overexpression leads to severe cardiac toxicity.
miRNAs Expression from within an Intron
Micro RNAs (miRNAs) are a class of small, single-stranded, non-coding RNAs of ˜22 nucleotides in length. Most miRNAs are transcribed by RNA polymerase II, either as independent transcripts or as RNAs embedded within introns of mRNAs. Primary miRNA transcripts are processed into ˜70 nt hairpin precursor miRNAs and then finally to ˜22 nt mature miRNAs by two RNase III enzymes (Drosha and Dicer). miRNAs function by regulating protein levels, targeting messenger RNAs (mRNAs) for translational repression and/or mRNA degradation.
The inventors have developed non-mammalian or synthetic miRNAs of the invention that are capable of knocking-down expression of transcripts containing the respective binding region. In some instances of the invention these are insect-derived miRNA sequences originally designed to target the firefly luciferase protein. In other instances, they are synthetic miRNA sequences, with no orthology to naturally occurring miRNAs. In some instances, synthetic miRNA sequences are designed to target codon optimised coding sequences, where the coding sequence is altered at the DNA level while retaining the same amino acid sequence. In a gene therapy context, this allows exogenously delivered transgenes to be exclusively targeted by the synthetic miRNAs, whilst endogenous genes are unaffected. In a final instance of the invention, completely novel synthetic miRNA sequences were created by in silico generation of large DNA sequences which were used with existing miRNA design tools to identify sequences suitable for miRNA targeting. Suitably, since all of these miRNAs are non-mammalian or synthetic, they have no predicted endogenous targets within the mammalian transcriptome.
Suitably a miRNA may be embedded within different introns. Examples of such introns are provided below. The human EF1a intron is the intron present in the commonly used EF1a promoter and is known to splice efficiently. The MINIX intron is also known to splice efficiently and is useful in a gene therapy context for its short sequence. The inventors have shown that that the EF1a promoter and MINIX intron can work in combination. The inventors have also shown that the JeT promoter and MINIX intron work in combination.
Suitably an intron may be selected from:
Suitably a miRNA may be provided by a non-mammalian miRNA originally targeted against firefly lucifersase (ffluc1).
Non-Mammalian miRNA: (Luciferase)
A BLAST search determined that there are no identical (21 bp) matches to this RNA in any RNA transcripts produced in human cells (thus, it is a “non-mammalian” sequence). Studies have shown that miRNAs can tolerate mismatches in target sites if there is exact complementarity to the seed sequence. The seed sequence is usually situated at positions 2-7 in the 5′ region of the miRNA and is essential from miRNA binding. However, no potential off-target RNAs contained an exact seed sequence match.
miRNAs are embedded in a hairpin loop structure to allow correct recognition and processing. Suitably an embedded non-mammalian miRNA may be selected from
Suitably a miRNA may be provided by a novel synthetic miRNA originally targeted against randomly generated sequence, with no orthology to mammalian, insect or plant miRNAs.
Suitably an embedded synthetic miRNA may be selected from:
Suitably an embedded synthetic miRNA may be targeted against the coding sequence of a target gene (i.e. a therapeutic transgene).
Target genes may be codon optimized and synthetic miRNAs, with no orthology to mammalian, insect or plant miRNAs, screened for ability to target the codon optimized transgene without targeting endogenous transcripts of the same gene. Suitably an embedded synthetic miRNA targeting a coding optimised sequence may be selected from:
miRNAs work by binding to specific sequences complementary to the mature miRNA sequence. These binding sites may be located in the 3′ untranslated region (3′UTR) of endogenous mRNAs. The binding sites may alternatively be located in the 5′UTR, exons, and introns. In further alternative embodiments a binding site may be located within a codon optimised transgene sequence. Suitably the miRNA binding sites which provide for control of the expression of the transgene may be provided within the 3′ UTR, the 5′ UTR or within the transgene.
Suitably, a ‘seed’ sequence in the binding site forms Watson-Crick pairs with bases at the 5′ end of the miRNA, at positions 2 through 7/8. However, the skilled person would understand the way in which binding specificity and strength, for example based on sequence conservation, strong base-pairing at the 3′ end of the miRNA, local AU content and location of miRNA binding sites within the 3′ UTR may be altered.
Suitably, different numbers of binding sites can be used to alter the strength of transgene control. In addition, mismatches introduced into the binding site can be used to lower the level of transgene control. Such changes enable setting the level of dosage insensitivity.
Suitably, the binding sites may be mutated to reduce, but not completely inhibit, miRNA-target binding. Suitably, these mutations may be used to enhance expression of the transgene, whilst still maintaining regulatory control of transgene expression, by having some target miRNA still bind to binding sites.
Successful miRNA-target binding usually results in a knock-down of protein levels, either via translational repression or mRNA degradation mechanisms.
Suitably a non-mammalian or synthetic miRNA binding site may be selected from
Any suitable promoter, constitutive or conditional, can be used to drive expression of the transgene. Suitably a promoter may comprise an Ef1a promoter, CAG promoter, Jet promoter, CMV promoter, CBA promoter, CBH promoter, Synapsin1 promoter, Mecp2 promoter, U1a promoter, U6 promoter, ubiquitin C promoter, neuron-specific enolase promoter, oligodendrocyte transcription factor 1 or GFAP promoter. In embodiments the feedforward miRNA can be incorporated into an intronic sequence coupled to suitable, for example any of the above promoters.
The exact promoter used will be dependent on the strength of expression needed and, in cases of larger genes, the amount of packaging capacity available, for example in an AAV delivery vector. Suitable promotors may be provided by:
The approach can be used with synthetic polyA sequences or truncated fragments of native polyA sequences. In embodiments the feed forward miRNA binding sites can be incorporated within the 3′UTR. Suitably the miRNA binding sites can be incorporated within the 3′UTR unless embedded within the transgene sequence.
Any suitable polyadenylation signal as known in the art may be utilised. Suitably, the polyA signal may be
Suitably, a stability element to increase transgene expression may be included. Suitably, the stability element may be located in the 3′ UTR. Suitably the stability element may be
The miRNA feed-forward construct of the invention is designed to work in vivo. To deliver these constructs to the requisite tissues/organs, any suitable viral vector can be utilised. In an embodiment, a viral vector may be an adeno-associated virus (AAV) delivery system or other therapeutic viral vector systems including lentivirus, adenovirus, herpes simplex virus, retrovirus, alphavirus, flaviviruses, rhadboviruses, measles virus, picornaviruses and poxviruses. For AAV, the entire construct (promoter, miRNA, transgene, binding site, polyA) can be cloned into an AAV-compatible plasmid where it is flanked by inverted terminal repeat (ITR) sequences. AAV production has strict size limits, so the entire construct must be no more than 4.4 kb (excluding ITRs). This size limit can restrict the use of certain transgenes, which would take up the bulk of the available space. Alternative, smaller promoters and polyA's can be used to accommodate larger transgenes. Suitably, in constructs, the 3′UTR region could be removed, and a synthetic miRNA targeted to the codon-optimised sequence of the transgene. As the codon-optimised transgene has a different DNA/mRNA sequence, endogenous mRNA from the gene of interest (G01) would not be targeted.
According to a second aspect of the present invention there is provided a vector comprising a construct of the first aspect of the invention.
Suitably the construct may be provided in a viral vector to allow delivery of the construct to target cells. A target cell may be cells of the central nervous system and peripheral nervous system including neurons, neuronal subtypes, oligodendrocytes, astrocytes, Schwann cells. Advantageously a viral vector may be selected from; adeno-associated virus (AAV), in particular AAV9, AAV1, 2, 4, 5, 6, 6.2, 8, 9, rh10, PHP.B, PH P.S, PH P.eB vectors can be used.
According to a third aspect of the present invention, there is provided a method of using a construct of the first aspect to express a transgene. Suitably the second aspect encompasses a method of expressing a transgene in a cell which may be provided to a subject. Suitably constructs can effectively be screened in vitro to assess the required level of dosage regulation. In vitro, the transgene can be contained within a plasmid and introduced into cell lines via lipid-mediated transfection. Robust transgene expression can be seen after 24 hours. Thereafter, the feed-forward transgene cassette suitably can be vectorized by insertion onto a rAAV expression vector which can then used to generate AAV particles.
According to a fourth aspect of the present invention, there is a method of treating a disorder caused by insufficient expression of a gene in a subject, the method comprising the steps of providing a construct of the first aspect of the invention or a vector of the second aspect with a wild type or codon optimised or modified copy of a transgene to be expressed in the subject to treat the condition caused by insufficient expression of the gene in the subject. Suitably, AAV viral vector packaged with the transgene will be introduced into the subject by various methods including systemic intravenous injection or by intra CSF routes of administration including intrathecal lumbar, intracerebroventricular, intra cisterna magna injection or by injection into neuropil.
Suitably the transgene may be a gene that is under-expressed in a subject who has the neurological disorder Rett Syndrome. Typically, Rett Syndrome is caused by loss-of-function mutations in the gene X-linked gene MECP2. Suitably, the transgene may be a functional copy or copies of the MECP2 gene. Suitably the construct provides for delivery of the transgene to the nervous system using adeno-associated virus (AAV) vectors.
The construct provides for expression of a transgene within a narrow/desired range in a target cell. For example where the transgene is a wild type or codon optimised copy of the protein coding sequence of the MECP2 gene, it is considered that the construct can provide the transgene at an expression level which provides a suitable therapeutic effect but which is less than a level at which adverse effects are observed. In the case of MECP2, FMR1 and UBE3A, overexpression of the gene is known to be deleterious.
For example, in Rett syndrome, the inventors have previously shown that low levels of expression can ameliorate disease phenotypes in mice. Conversely, overexpression (duplication of the gene locus) in patients, as well as in experimental animals, (2× or more) result in adverse neurological outcomes. This defines a narrow therapeutic window for genetic therapy for which the feed forward technology is well suited. The FMR1, UBE3A and SYNGAP1 genes are also considered to be dosage sensitive. In such circumstances, the level of expression of the transgene to ameliorate disease, but minimise adverse effects could be determined and then the expression level suitably provided to a patient using the present invention.
Many other genes associated with monogenic disorders are dosage sensitive and would benefit from use of a construct and system of the present invention to regulate expression of such exogenous transgenes. Human copy number variants (CNVs) can be an indication of dosage sensitive genes, and studies have implicated the dosage sensitivity of individual genes as a common cause of CNV pathogenicity. Gu W & Lupski JR. CNV and nervous system diseases—what's new? Cytogenet Genome Res. 2008; 123:54-64 cite several examples of dosage sensitive genes and their associations with neurodevelopmental disorders. Examples include MECP2 duplication syndrome (involving the gene MECP2), adult-onset autosomal dominant leukodystrophy (ADLD, involving the LMNB1 gene), isolated lissencephaly sequence (ILS, involving the PAFAH1B1/LIS1 gene), Miller-Dieker syndrome (MDS, involving the YWHAE gene).
Rice AM & McLysaght. Dosage sensitivity is a major determinant of human copy number variant pathogenicity. Nature Communications. 2017; 8:14366|DOI: 10.1038 show that solitary pathogenic genes involved in CNVs associated with disease are enriched for roles in neurodevelopment and identify many dosage sensitive genes, for example: PRKCZ, TTC34, PRDM16, ARHGEF16, PARK7, PRDM2, IGSF21, PTCH2, NFIA, ST6GALNAC3, DPYD, COL11A1, PDZK1, GPR89A, NBPF11, GPR89B, KCNT2, CFHR2, ASPM, PTPRC, GPATCH2, DUSP10, GPR137B, RYR2, CHRM3, RGS7, AKT3, KIF26B, SMYD3, LPIN1, EPCAM, MSH2, NRXN1, XPO1, LRP1B, ZEB2, ACVR2A, MBD5, KIF5C, SCN1A, COL3A1, PMS1, PLCL1, SATB2, PARD3B, EPHA4, SPHKAP, CHL1, GRM7, TRANK1, DOCK3, FAM19A1, FOXP1, ROBO1, CADM2, FOXL2, SOX2, LPP, RASGEF1B, GRID2, FAT4, NR3C2, LRBA, FGA, GALNTL6, WWC2, TLR3, IRX2, IRX1, CDH12, CDH9, NIPBL, HEXB, MEF2C, GRAMD3, FBN2, PRELID2, TCOF1, GABRG2, MSX2, NSD1, FOXC1, CDYL, TBC1D7, RUNX2, MUT, RIMS1, NKAIN2, LAMA2, ARID1B, PARK2, 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, KANK1, GLIS3, PTPRD, MLLT3, ROR2, PTCH1, AL162389.1, ARRDC1, EHMT1, PCDH15, CTNNA3, ADK, BMPR1A, PAX2, BTRC, INPP5A, MRPL23, ELP4, PAX6, CPT1A, DYNC2H1, KIRREL3, WNK1, CACNA1C, PPFIBP1, TBX5, MED13L, NALCN, CHD8, MYH7, TTC6, DAAM1, NRXN3, MTA1, SNRPN, UBE3A, OCA2, HERC2, CHRFAM7A, ARHGAP11B, OTUD7A, FBN1, HEXA, SNUPN, NRG4, AC112693.2, IGF1R, LRRC28, HBA2, HBQ1, CREBBP, RBFOX1, CDR2, CDH13, CYBA, NXN, YWHAE, SMG6, METTL16, PAFAH1B1, ADORA2B, NT5M, RAI1, NF1, C17orf67, PITPNC1, ACOX1, TCF4, DOCK6, CACNA1A, LPHN1, ZSCAN5A, BMP2, MYT1, PEX26, USP18, DGCR6L, USP41, UBE2L3, NF2, LARGE, BRD1, SHANK3.
The inventors consider that any suitable gene, in particular any dosage sensitive gene, for example as discussed above, may suitably be utilised in the present invention as required. For example, as would be understood in the art, the constructs and systems of the present invention may be used in the expression of any suitably protein for treatment of a disease or a condition, particularly wherein control of the expression level of the protein being provided is of importance.
The inventors consider the concept, constructs with suitable transgenes therein and methods of expressing the transgene to be applicable to any other clinically relevant and dosage sensitive genes.
Suitably the construct may be used in other gene therapy programmes including Fragile X syndrome (using FMR1 transgene), Angelman syndrome (using for example UBE3A transgene), or Syngap-related intellectual disability (using SYNGAP1).
It can be envisaged that particular vectors may be used to provide a vector to a specific cell type dependent on disease.
For example, SYNGAP1 is a neuronal gene and expressed only in neurons, but UBE3A, MECP2 and FMR1 are ubiquitously expressed across multiple tissues. However, the dominant disease features occur in loss of expression in the nervous system and therefore the nervous system is the dominant target for the therapeutic feed-forward transgenes.
The inventors have developed constructs in which the synthetic components have been considered to fine-tune the system (number of sites and efficient intron exclusion) to achieve appropriate dosage-insensitivity. Dosage-insensitivity in the context of the present invention is intended to infer a range of protein expression that does not result in undesired effects that are observed when there is too much expression of a therapeutic transgene, for example, two copies of the MECP2 gene in an individual are known to result in a severe MECP2 duplication syndrome, with symptoms as severe as Rett syndrome, in which MeCP2 levels are drastically reduced, or absent.
In embodiments, the construct can contain two elements that allow the transgene levels to be controlled. Suitably, the first element may be a micro RNA sequence contained within an intron located between the promoter and transgene. This micro RNA containing intron will be spliced out during pre-mRNA processing. The miRNA will then be processed to produce a mature miRNA capable of degrading its target transcripts. An important element of the design is that the miRNA is designed not to target the mammalian genome in order to prevent off-target effects. In some examples the miRNA can be insect-derived (e.g. one from the Lampyridae group, but any suitable insect or other suitable non-mammalian miRNA could be optimized for this use). In alternative examples the sequence can be completely synthetic (designed such that it does not bind to the mammalian genome and is not a naturally occurring sequence) and is therefore devoid of known off-target effects within the mammalian genome. The second element can be a number of non-mammalian or synthetic miRNA binding sites in the 3′UTR of the construct that match the miRNA produced from the intron. The presence of these binding sites causes the transgene to be a target for the delivered micro RNA. This leads to reduced levels of the transgene and prevents overexpression, providing for the desired dosage insensitivity effect of the system.
In a separate embodiment of the feed-forward principle, the synthetic micro RNA is delivered within the gene therapy synthetic cassette intron, but instead of targeting a miRNA binding site contained within the 3′UTR, it is targeted against the coding sequence of the transgene itself. Crucially, in such an embodiment the transgene sequence is codon optimised such that the sequence is altered at the DNA level while remaining the same at the amino acid level. This creates a novel DNA sequence that allows synthetic miRNAs to be uniquely targeted to the transgene without targeting endogenous mammalian sequences. This version of the feed-forward system, being more compact, is advantageous for larger genes (for example Syngap1) which approach the packaging capacity of the viral vector. Overall, the single gene loop enables constant levels of expression whereby the circuit can maintain a relatively fixed level of expression across a broad range of gene dosages (i.e. this relatively fixed or constant expression level is what results in the desired dosage insensitivity). The experimental systems produced a regimen in which changes in gene dosage lead to much smaller relative changes in gene expression. This is an important feature when applied to gene therapy where one is aiming to achieve broad even expression across the transduced cell population and enables increased viral vector dosing to achieve higher transduction rates without concomitant overexpression effects.
In embodiments, the construct is suitable for expression in cells and/or tissues which are sensitive to AAV genetic therapy. In embodiments, the construct allows for control of transgene expression in cells which typically over-express transgenes delivered using AAV vectors. In embodiments, the construct prevents cellular toxicity in these cells and/or tissues. In embodiments, the construct may prevent cellular toxicity in dorsal root ganglions. In embodiments, the construct may prevent cellular toxicity in liver cells. In embodiments, the construct may prevent cellular toxicity in cardiac cells. In embodiments packaging of the construct in a viron does not affect or only minimally affects the quality of the construct.
In embodiments, the construct can be used to reduce the severity of clinical symptoms caused by certain genetic conditions or developmental disorders. In embodiments, the construct can be used to completely reverse clinical symptoms caused by certain genetic conditions or developmental disorders. In embodiments, the construct can be used to treat certain genetic conditions or developmental disorders. In embodiments, the construct can be used to treat Rett syndrome. In embodiments, the construct can be administered in vivo to reduce the clinical presentation of Rett syndrome.
In embodiments, the construct can be used to reduce toxicity of genetic therapy. In embodiments, the feed-forward mechanism regulates transgene expression, reducing the toxicity to cells. In embodiments, the construct can be administered in vivo without adverse health effects.
Embodiments of the invention will now be described by way of example only with reference to the accompanying figures in which:
RTT253 construct:
CMV/CBA promote (no SEQ ID 76)
Human EF1a intron A (SEQ ID NO: 5)
ffluc1 (SEQ ID NO: 9)
ffluc1×3 binding sites (SEQ ID NO: 34)
A proof-of-concept in the transgene targeting construct of the present invention has been generated in relation to the neurological disorder Rett Syndrome. Rett Syndrome is caused by loss-of-function mutations in the X-linked gene MECP2. Although an attractive therapeutic approach for this disorder is to deliver functional copies of the MECP2 gene to the nervous system using adeno-associated virus (AAV) vectors, a major obstacle to this approach is that cells can be infected with multiple copies of the virus vector leading to over-expression of the MECP2 gene. The inventors have previously determined that over expression of the MECP2 gene can lead to severe toxicity. Clinically it is known that duplication of the MECP2 gene in humans leads to MECP2 over-expression syndrome, a distinct and severe neurological disorder.
Using a construct as described by the present invention, the levels of MECP2 expressed in a cell can be limited, even when the cell has been infected with multiple copies of the viral vector. This greatly increases the safety window of MECP2 gene therapy interventions and allows higher viral doses to be administered, enabling a greater number of cells to be infected and a more robust disease reversal to be achieved.
In this example, the transgene is a WT or codon optimised copy of the protein coding sequence of the MECP2 gene, a gene mutated in the neurological disorder Rett Syndrome. The construct contains two elements that allow the transgene levels to be controlled. The first element is a non-mammalian or synthetic micro RNA sequence contained within an intron located between the promoter and transgene. This non-mammalian or synthetic micro RNA containing intron will be spliced out during pre-mRNA processing. The mammalian or synthetic miRNA will then be processed to produce a mature miRNA capable of degrading its target transcripts. As the miRNA is either synthetic or derived from a non-mammalian, insect source, it is therefore devoid of known off-target effects within the mammalian genome. A second element of the construct is a number of non-mammalian or miRNA binding sites in the 3′UTR of the construct that match the non-mammalian or synthetic miRNA produced from the intron. The presence of these binding sites causes the transgene to be a target for the delivered micro RNA. This leads to reduced levels of the transgene and prevents overexpression.
In an alternative embodiment of the feed-forward principle, the non-mammalian or synthetic micro RNA can be delivered within the gene therapy synthetic cassette intron. Instead of targeting micro RNA bindings within the 3′UTR, the non-mammalian or synthetic micro RNA instead binds to a unique (within the mammalian genome) micro RNA binding region that is created within the codon optimized protein coding sequence of the transgene, and has no corresponding binding site within the mammalian genome; i.e. the miRNA binding region is a unique synthetic binding region). This version of the feed-forward system, can be made more compact. This can be particularly advantageous for larger genes which approach the packaging capacity of a viral vector.
The single gene loop enables constant levels of expression whereby the circuit can maintain a relatively fixed level of expression across a broad range of gene dosages (i.e. exhibiting a desired dosage insensitivity). The experimental systems produce a regimen in which changes in gene dosage lead to much smaller relative changes in gene expression. This is an important feature when applied to gene therapy where one is aiming to achieve broad, even expression across the transduced cell population and enables increased dosing to achieve higher transduction rates without concomitant overexpression effects.
Non mammalian miRNA binding sites or synthetic miRNA binding sites in combination with synthetic non mammalian miRNA (ffluc1) or synthetic miRNA which are not capable of binding to the mammalian genome can be utilised to ensure a lack of off-target effects, whilst enabling regulation of transgene expression. Suitably constructs as described by Table 1 may be provided.
As discussed, herein, the feed-forward system can be constructed using alternative ubiquitous and cell-type specific promoters including CAG, UBC, SV40, PGK, Synapsin1, neuron-specific enolase, U6, GFAP, MAG, MPZ. The intron may include any synthetic or endogenous intron capable of hosting the non-mammalian or synthetic miRNA sequence and may be upstream of the protein coding sequence or an intron within the protein coding sequence or a combination where more than a single non-mammalian or synthetic miRNA is generated from a single transgene cassette. The non-mammalian or synthetic miRNA may be any non-mammalian or synthetic miRNA that targets recognition sites within the transgene cassette including the translated and untranslated regions. The gene may be any dosage sensitive gene where gene dosage is confounding to the effectiveness of gene transfer. The number of binding sites may be fine-tuned to the level of desired dosage insensitivity and may range of 1, 2, 3, 4, 5, 6 or any number within the capacity of the transgene cassette. The polyA signal may suitably, for example be SV40, BGH or any commonly used native or synthetic polyA signal.
Neuro2a cells were transfected with various constructs, with or without the feed-forward mechanisms built-in, and the level of MECP2 transgene expression was assessed by flow cytometry. A separate fluorescent marker on the construct was used to monitor the level of construct delivered to each cell (surrogate for dose). Constructs in which the feed-forward control elements were included showed a much narrower range of MECP2 transgene expression than those which did not include these elements. Promisingly, the dampening effect of these elements increased as the amount of construct delivered increased suggesting that the control elements can mitigate toxicity without impeding expression of the gene at the therapeutic level. Fine tuning of the level of dosage sensitivity can therefore be provided.
The feedforward cassettes may be administered to mice to provide constrained transgene expression in cells. Wild-type mice had transgene flag tagged Mecp2 administered and transgene expression monitored in somatosensory cortex neurons. The transgene was delivered in an AAV vector which either did or did not contain a feedforward regulation system. The feedforward regulation system utilised miRNA ffluc1 (SEQ ID NO: 9) and EF1a promoter. Three ffluc1 binding sites (SEQ ID NO: 34) were provided after the Mecp2 sequence.
The feedforward regulation mechanism may be used to ensure appropriate distribution of transgene expression throughout a tissue.
The feedforward regulation mechanism may be used to ensure constrained expression of a transgene throughout the neocortex.
Single-stranded AAV (ssAAV) particles, comprising constructs flanked by AAV2 ITRs packaged into AAV9 capsids, were produced by transfection of HEK293 cells at the UPV Viral Vector Production Unit (Universitat Autònoma de Barcelona).
The miRNA utilised was ffluc1 (SEQ ID NO: 9), and 3×ffluc1 binding sites (SEQ ID NO: 34) were provided after the Mecp2 gene sequence. Expression is even across cells in the regulated image (15B) (but slightly higher due to combined native plus vector-derived signal), demonstrating constrained expression. In contrast, the unregulated cassette sample (15C) shows variable levels of immunoreactivity across cell population including populations of cells expressing very high levels of MeCP2. The quantification of these samples (15D) shows narrowly constrained expression with the feed forward cassette.
Suitably, the feed-forward cassettes may be administered in vivo without adverse health effects. Phenotypic assessment was carried out on wild-type mice administered with a feed-forward regulated cassette. Regulated constructs expressing the ffluc1 (SEQ ID NO: 9) miRNA and a codon-optimized human MECP2 transgene were administered. Unregulated constructs expressed only the codon-optimized human MECP2 transgene. The MeP426 unregulated construct expressed wild-type human MECP2 under the control of an endogenous mouse Mecp2 promoter, previously described by Gadalla K K E, Vudhironarit T, Hector R D, Sinnett S, Bahey N G, Bailey M E S, Gray S J, Cobb S R. 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.
Suitably, the feed forward mechanism does not interact with other sequences in the mammalian genome.
The miRNAs expressed in the feed-forward constructs, either insect derived miRNA sequence (ffluc1; SEQ ID NO: 9) or novel synthetic miRNA sequence (ran1g; SEQ ID NO: 17 and ran2g; SEQ ID NO: 18), have no predicted endogenous targets within the mammalian transcriptome.
To verify this, the mirDB off-target prediction tool was used to predict the most likely human mRNA targets of the miRNA sequences ffluc1, ran1g and ran2g. Potential human target genes/transcripts were ranked based on the number of target sites in the gene/transcript sequence matching the seed sequence of the miRNA.
Plasmids were generated that expressed the ffluc1 miRNA and a reporter transgene (
Suitably, therefore, the invention provides a method of regulating transgene expression without impacting upon endogenous gene expression in a mammalian host cell.
Suitably, the feed forward mechanism can be used to provide safe and effective treatment to ameliorate the phenotype of clinical conditions.
AAV vectors expressing feed-forward MECP2 constructs were tested in wild-type (WT) and Mecp2 knock-out (KO) mice maintained on a mixed CBA/C57 background. ssAAV expressing regulated (ffluc1; SEQ ID NO: 9) or unregulated MECP2 was injected bilaterally into the brains of postnatal day (P)0/1 males by intracerebroventricular (ICV) administration. Control injections used the same diluent without vector (vehicle control). Injected pups were returned to the home cage and assessed weekly from 4 weeks of age. Mice were monitored until 15 weeks of age, or until reaching their human endpoint.
Constructs can be provided wherein the constructs are modified to provide enhanced expression, regulation and stability. The constructs can be provided such that they contain a reporter transgene. The constructs can contain a Kozak sequence which promotes strong expression. The constructs can further contain a stability element in the 3′UTR. The constructs can further contain one or more binding sites which include mutations engineered to reduce the efficacy of (but not completely ameliorate) miRNA binding. Some exemplary constructs are detailed below in Table 2.
Any suitable promoter, constitutive or conditional, can be used to drive expression of the transgene. Suitably a promoter may comprise an Ef1a promoter, CAG promoter, Jet promoter, CMV promoter, CBA promoter, CBH promoter, Synapsin1 promoter, Mecp2 promoter, U1a promoter, U6 promoter, ubiquitin C promoter, neuron-specific enolase promoter, oligodendrocyte transcription factor 1 or GFAP promoter. It should be understood for the constructs Table 2, any suitable promoter may be used.
The miRNA used may be any suitable synthetic miRNA which does not bind to the mammalian genome. Suitably, the miRNA used may be derived from a synthetic sequence or a non-mammalian genome with no orthology to mammalian miRNAs. Suitably, the miRNA used may be derived from an insect genome. Exemplary miRNAs are provided in Table 3, below.)
The construct may be adapted to include a modified Kozak sequence: Suitably, the modified Kozak sequence may be any Kozak sequence which includes a nucleic acid motif that functions as the protein translation initiation site. Suitably, the modified Kozak sequence may be any modified sequence which promotes an increase in translation. Suitably, the Kozak sequence may be GCCACCATGG (SEQ ID NO: 73).
In embodiments, the gene of interest can be any one of the following genes of interest: MECP2, FMR1, UBE3A, CDKL5, FXN, SMN1, or INS or a gene required to be supplied using genetic therapy for treatment of a genetic condition or developmental disorder. In particular, the gene of interest may be any gene which requires controlled expression when delivered to a subject to treat a genetic condition or developmental disorder.
Examples of binding mutations may be seen in Table 4 below.
Suitably, a stability element to increase transgene expression may be included. Suitably, the stability element may be located in the 3′ UTR. Suitably this stability element may be the Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) (SEQ ID NO: 74). Suitably, the stability element may be a truncated version of WPRE retaining the stability element, but omitting the X-protein sequence, or a ribozyme stability sequence (WPRE3).
Dorsal root ganglions (DRGs) are highly susceptible to AAV. DRGs are highly transduced after AAV delivery and can result in toxicity. To test if the feed-forward circuit dampened expression in these tissues, DRG were dissected from wild-type mice treated with CBE-regulated and CBE-unregulated MECP2 feed-forward ssAAV at a dose of 4×1011 vg/mouse. Lumbar DRGs were processed for vector derived MeCP2 expression (n=3 per mice, 3 mice per group) and vector biodistribution (1 DRG per mouse, 3 mice per group). Mice treated with CBE-unregulated MECP2 were terminated at 3-4 weeks old due to toxicity/humane endpoint. Mice treated with CBE-regulated MECP2 were terminated at 20 weeks. DRGs were also isolated from age-matched WT and KO mice as controls.
Upon termination, mice were perfused with 4% paraformaldehyde (PFA) then tissues were dissected and post-fixed in 4% PFA overnight at 4° C. then stored in 30% sucrose until time of processing. Tissues were embedded in a mixture 30% sucrose and Optimal cutting temperature (OCT) compound on dry ice. Frozen tissue blocks were stored at −20° C. until time of sectioning. Cryostat sections were cut at 12 μm and mounted on coated histological slides, air dried for 30 minutes at room temperature, then stored at −20° C. until time of staining. Frozen slides were rinsed in 0.1 MPBS to remove the tissue-freezing matrix then antigen retrieval was performed in 10 mM sodium citrate buffer, 0.05% Tween-20, pH 6.0) for 30 minutes in a water bath at 85° C. After cooling the slides for 30 minutes at room temperature in the same buffer slides were rinsed in 0.3M PBS/Triton X-100 solution then incubated with 5% goat serum in 0.3M PBS/T solution for 1 hour at room temperature in a humidified chamber to block non-specific binding. Slides were then incubated with the primary antibody (monoclonal, mouse anti-MECP2, M7443, Sigma, 1:500) in a buffered solution, overnight at 4° C. in a humidified chamber. After rinsing (0.3M PBS/T solution), slides were incubated with the secondary antibody (Alexa Fluor® 488 goat anti-mouse (H+L), cell signalling, 1:500) for 2 hours at room temperature in a humidified chamber. After further rinsing in 0.3M PBS/T solution, slides were incubated in Hoechst 33342, DNA dye staining solution (1:2000 in 0.1M PBS) for 30 minutes at room temperature. Slides were sealed to coverslips using anti-fade mounting solution and nail polish, then imaged by confocal microscope.
The liver is also highly susceptible to AAV. Liver cells are highly transduced after AAV delivery and can result in toxicity. To test if the feed-forward circuit dampened expression in these tissues, liver was dissected from wild-type mice treated systemically (intravenous) with CBE-regulated or CBE-unregulated MECP2 feed-forward ssAAV at a dose of 1×1012 vg/mouse. Liver was processed for vector derived MeCP2 expression (n=3 sections per mouse, 3 mice per group) and vector biodistribution (3 mice per group). Mice treated with CBE-regulated and CBE-unregulated MECP2 were terminated at 4 weeks post-injection. Liver was also isolated from non-injected age-matched WT mice as controls.
Upon termination, mice were perfused with 4% paraformaldehyde (PFA) then tissues were dissected and post-fixed in 4% PFA overnight at 4° C. then stored in 30% sucrose until time of processing. Tissues were embedded in a mixture 30% sucrose and Optimal cutting temperature (OCT) compound on dry ice. Frozen tissue blocks were stored at −20° C. until time of sectioning. Cryostat sections were cut at 12 μm and mounted on coated histological slides, air dried for 30 minutes at room temperature (RT), then stored at −20° C. until time of staining. Frozen slides were rinsed in 0.1 MPBS to remove the tissue-freezing matrix then antigen retrieval was performed in 10 mM sodium citrate buffer, 0.05% Tween-20, pH 6.0 for 30 minutes in a water bath at 85° C. After cooling the slides for 30 mins at RT in the same buffer slides were rinsed in 0.3M PBS/Triton X-100 solution then incubated with 5% goat serum in 0.3M PBS/T solution for 1 hour at room temperature in a humidified chamber to block non-specific binding. Slides were then incubated with the primary antibody (mouse anti-MeCP2, 1:500) in a buffered solution, overnight at 4° C. in a humidified chamber. After rinsing (0.3 M PBS/T solution), slides were incubated with the secondary antibody (Alexa Fluor® 488 goat anti-mouse (H+L), 1:500) for 2 hours at room temperature in a humidified chamber. After further rinsing in 0.3M PBS/T solution, slides were incubated in Hoechst 33342, DNA dye staining solution (1:2000 in 0.1 M PBS) for 30 minutes at room temperature. Slides were sealed to coverslips using anti-fade mounting solution and nail polish, then imaged by confocal microscope.
This further demonstrates that feed-forward constructs can constrain transgene over-expression even in tissues highly susceptible to AAV, reducing the probability of tissue damage/toxicity, and therefore providing an advantage over conventional gene therapy constructs.
Suitably, the feed-forward constructs can be used to constrain transgene over-expression even in tissues highly susceptible to AAV, reducing the probability of tissue damage/toxicity, and therefore providing an advantage over conventional gene therapy constructs.
Single-stranded AAV (ssAAV) particles, comprising constructs flanked by AAV2 ITRs packaged into AAV9 capsids, were produced by a baculovirus transfection system at Virovek (Hayward, CA, USA).
AAV vectors expressing modified feed-forward MECP2 constructs were tested in Mecp2 knock-out (KO) mice maintained on a mixed CBA/C57 background. ssAAV expressing regulated or unregulated MECP2 was injected bilaterally into the brains of postnatal day (P)0/1 males by intracerebroventricular (ICV) administration. Control injections used the same diluent without vector (vehicle control). Injected pups were returned to the home cage and assessed weekly from 4 weeks of age. Mice were monitored until 15 weeks of age, or until reaching their human endpoint.
Western blot analysis was performed (
The results demonstrated constrained MeCP2 expression with the feedforward circuit. Combined with enhanced survival and a lower RTT phenotype score, this constrained transgene expression further demonstrates safety advantages over the unregulated cassette.
To further verify that insect derived miRNA sequence (ffluc1; SEQ ID NO: 9) or novel synthetic miRNA sequence (ran1g; SEQ ID NO: 17 and ran2g; SEQ ID NO: 18), have no predicted endogenous targets within the mammalian transcriptome, quantitative RT-PCR of predicted mRNA targets was performed.
Plasmids were generated that expressed the ffluc1 (SEQ ID NO: 9), ran1g (SEQ ID NO: 18) or ran2g (SEQ ID NO: 18) miRNAs from an intron downstream of the hEF1a promoter (
Human embryonic kidney 293 cells (HEK 293) were transfected with 100 μg of each plasmid using Lipofectamine®. After 48 hrs, cells were lysed and total RNA isolated. The quality and quantity of isolated RNA was analysed. First-strand synthesis was performed, in 20 μl reactions containing 500 ng of total RNA template and 500 nM random hexamers. SYBR Green PCR reactions were carried out, in 20 μl reactions using 1/10th of the first-strand synthesis reaction and 300 nM gene-specific primers. PCR was performed under the following cycling conditions: an initial denaturation at 95° C. for 3 min, then 40 cycles of 95° C. for 10 s, 55° C. for 30 s and 60° C. for 30 s, followed by a dissociation curve. Results were analysed using the 2−ΔΔCt method to calculate the relative fold gene expression of samples relative to the lipofectamine-only control sample.
Quantitative RT-PCR (qRT-PCR) was used to quantify transcript levels of three of the top predicted human mRNA targets of ffluc1 (IRF2BP2, HNRNPH1 and RPP30), ran1g (FASN, ETAA1 and MAIP1) and ran2g (MCFD2, SLC38A2 and FZD6). qRT-PCR was also used to quantify transcript levels of recognised endogenous mRNA targets of miRNAs expressed by control plasmids: hsa-miR-132-3p (MECP2), hsa-miR-34a-5p (HSPA1B) or hsa-miR-644a (ACTB).
qRT-PCR assessment shows that, even when ffluc1, ran1g or ran2g are expressed at very high levels, there is minimal detectable off-target effects (
The inventors identified that the present invention was also effective in treatment of other disorders affecting the central nervous system (CNS). Constructs were made, replacing MECP2 with the the UBE3A gene (mutations in this gene lead to Angelman syndrome and Prader-Willi syndrome), and the CDKL5 gene (mutations in this gene lead to CDKL5 deficiency disorder).
Plasmids were generated that expressed the ffluc1 miRNA (SEQ ID NO: 9) and a gene-of-interest (GOI), fused to a mNeonGreen reporter gene. For each GOI, a construct with and without the feedforward mechanism was generated (
The expression of these proteins (UBE3A and CDKL5) is determined by NeonGreen protein levels as assessed by flow cytometry. Regulated feed-forward constructs were compared against unregulated control constructs absent of miRNA regulation (
As seen with MECP2, the dampening effect of the feed-forward elements increased as the amount of construct delivered increased suggesting that the control elements can mitigate toxicity without impeding expression of the gene at the therapeutic level.
The inventors determined that a codon-optimised protein coding sequence can be utilised as the miRNA binding site. A synthetic miRNA was delivered within a gene therapy cassette to target a unique miRNA binding region created within a codon optimized protein coding sequence of a transgene, instead of targeting miRNA binding sites within the 3′UTR. The synthetic miRNA has no corresponding binding site within the mammalian genome. This approach can be particularly advantageous for larger genes, which approach the packaging capacity of a viral vector.
The expression of SynGAP protein was determined by NeonGreen protein levels as assessed by flow cytometry. Regulated feed-forward constructs were compared against unregulated control constructs absent of miRNA regulation (
The dampening effect of the feed-forward elements increased as the amount of construct delivered increased suggesting that this alternative embodiment of the feed-forward principle can also mitigate toxicity without impeding expression of the gene at the therapeutic level.
The non-mammalian miRNA feedforward mechanism was also effective in other disorders where the primary phenotype is peripheral rather than the central nervous system (CNS). Constructs were made with MECP2 replaced with the coding sequence for other proteins: the SMN1 gene (mutations in this gene lead to spinal muscular atrophy), the INS gene (mutations in this gene lead to type 1 diabetes) and the FXN gene (mutations in this gene lead to Friedreich's ataxia). The 3′UTR contained 3 non-mammalian miRNA binding sites for the same ffluc1 miRNA (SEQ ID NO: 9) used in previous experiments (SEQ ID NO: 34).
Plasmids were generated that expressed the ffluc1 miRNA and one of the genes-of-interest (GOI) above. The GOI was fused to a mNeonGreen reporter gene. For each GOI, a construct with and without the feedforward mechanism was generated (
The expression of these proteins (SMN1, insulin and Frataxin) are determined by NeonGreen protein levels as assessed by flow cytometry. Regulated feed-forward constructs were compared against unregulated control constructs absent of miRNA regulation (
As seen with MECP2, the dampening effect of the feed-forward elements increased as the amount of construct delivered increased suggesting that the control elements can mitigate toxicity without impeding expression of the gene at the therapeutic level.
The inventors further demonstrated the use of a non-mammalian miRNA feedforward mechanism in treating other dosage sensitive disorders which affect the central nervous system (CNS). The UBE3A gene, disrupted in Angelman syndrome and Prader-Willi syndrome, was shown to be regulated in vivo by the feedforward mechanism.
Constructs were generated that expressed the ffluc1 miRNA (SEQ ID NO: 9) and human UBE3A, fused to a 3×FLAG tag. A construct with and without the feedforward mechanism was generated (
This demonstrates that UBE3A (i.e., transgenes other than MECP2) can be regulated in vivo under control of the non-mammalian miRNA feedforward mechanism. This reduces the probability of tissue damage/toxicity from overexpression of transgenes where the gene/disorder is known to be dosage sensitive.
Feed-Forward Constructs Package Efficiently in ssAAV
Feed-forward constructs expressing the MECP2 transgene were prepared as single-stranded AAV (ssAAV) particles, comprising constructs flanked by AAV2 ITRs packaged into AAV9 capsids and were produced either by a HEK293 process (Viral Vector Production Unit, Universitat Autonoma Barcelona, Spain) or by a baculovirus based infection system at Virovek (Hayward, CA, USA). Using both processes, the inventors demonstrate that the feed-forward gene therapy constructs can be produced efficiently, to scale and to very high titer (up to 1.94×1014 viral genomes/ml). Therefore, the inventors have identified that the feed-forward regulated gene therapy technology has been configured for efficient manufacture. Importantly, the inventors demonstrate that the feed-forward synthetic circuit constructs package efficiently in AAV.
Following AAV production, CDMS (charge detection mass spectrometry) analysis was performed to determine the size of the AAV particles based on charge and mass. This tool helps in determining the quality of packaging and if there are any partially packaged species that might potentially affect the potency of the AAV product.
It is known that genetic sequence containing secondary structure such as stem loops, hairpins and miRNA generating sequence very commonly result in aberrant packaging and the encapsulation of heterogeneous species that adversely compromise product purity (Xie et al., 2017).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010024.4 | Jun 2020 | GB | national |
| 2107990.0 | Jun 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2021/051653 | 6/29/2021 | WO |
