Process

Abstract

The present disclosure relates to a novel screening method for screening a plurality of pGREs in parallel and nucleotide constructs for use in the method. The construction body comprises a nucleotide block, the sequence of the nucleotide block is PRIMREG1-pGREE-PRIMREG2-GENEE or GENE-PRIMREG1-pGREE-PRIMREG2, and the PRIMREG1 is a primer recognition sequence 1 or an FLEx box containing the primer recognition sequence 1. PGRE is a presumed gene regulatory element, PRIMREG2 is a primer recognition sequence 2 or a FLEx cassette comprising a primer recognition sequence 2, GENE comprises nucleotide blocks REPSEQ, IRES and SSR, where REPSEQ is a reporter gene and is present or absent, IRES is an internal ribosome entry site or a 2A self-cleaving peptide, and SSR is a site-specific recombinase gene. At least one of the PRIMREG1 and the PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

Description

TECHNICAL FIELD

[0001] The present invention relates to constructs, vectors and methods for the parallel screening of genetic regulatory elements in cells, tissues and in vivo. The invention also extends to novel PGREs identified using the constructs, vectors and methods. BACKGROUND

[0002] Vectors for the delivery of recombinant DNA, such as vectors for gene therapy, take a variety of forms, including viral vectors and non-viral vectors. Viral vectors are gene delivery vehicles for delivering genetic material into cells by using viral genomes and viral packaging, such as viral capsids, whereas non-viral vectors are based on delivering genetic material into cells by using inorganic particles, lipid-based vectors, polymer-based vectors and peptide-based vectors. Furthermore, viral vectors are biology-based, whereas non-viral vectors are chemistry-based. Examples of non-viral vectors include lipid nanoemulsions, polymethacrylates, dendrimers and the like. Examples of viral vectors include lentiviruses, adenoviruses, tobacco mosaic viruses and adeno-associated viruses.

[0003] Adeno-associated viruses (AAV) have become the de facto viral vector for gene therapy applications. The combination of their excellent safety profile and relatively easy engineering has meant that they have been found useful for a wide range of disorders across tissues and cell types. AAVs contain two main components: a protein capsid and an AAV genome. Recombinant AAVs for therapeutic and research purposes typically retain only one element of the genome that is native to wild-type AAV, the inverted terminal repeat (ITR). Between the ITRs, the wild-type genome is removed and replaced with non-native nucleotides that traditionally comprise two additional elements: a gene of interest and regulatory sequences. The regulatory sequences comprise promoter and / or enhancer sequences that dictate the strength of gene expression and the cell types in which the gene is expressed.

[0004] Traditionally, AAV gene therapy vectors have used ubiquitously present regulatory elements that drive high levels of gene expression in all (or most) cell and tissue types. This approach has drawbacks, where it is desirable to have gene expression targeted to specific cell or tissue types. This is particularly important when gene expression in off-target cell types can have deleterious effects.

[0005] Many efforts have been made to identify regulatory elements that drive gene expression in a cell type-specific manner. However, the relatively small packaging capacity of AAV (4.9 kb) compared to the size of regulatory elements within the genome, coupled with the difficulty of identifying which regulatory elements drive cell-specific expression, has meant that only a limited number of these “short” regulatory elements are suitable for use in AAV vectors.

[0006] Some attempts have been made to address this issue by screening regulatory elements in AAV vectors. Initially, potential regulatory sequences were identified by the combination of next generation sequencing, for example ATAC-seq or DNAse-seq, which selectively digest accessible chromatin in a targeted cell type (which is not protected by methylation), followed by next generation sequencing to identify putative regulatory elements within that cell type. Bioinformatic analysis was then used to identify the most promising candidates, which were then further analysed using biological screens. These are typically trained on datasets in silico, and so can exhibit bias.

[0007] Despite advances in sequencing methods which have led to an explosion in the number of sequences which can drive cell type specific gene expression in AAV vectors, the biological screening of these sequences is a significant bottleneck. Typically, biological screens are performed in one of two ways, serially or in parallel:

[0008] Serial screens are performed by using a single sequence of a regulatory element upstream of a reporter construct, for example a fluorescent protein, then injecting the vector into an experimental animal and histologically identifying the cell types which express the reporter. This approach has been successfully used to identify highly specific regulatory elements, for example the hecticetic promoter in the retina (Juttner et al., 2019), or enhancer sequences in the primate neocortex (Mich et al., 2021). However, the identification of functional regulatory sequences requires testing each one in an individual experimental animal. Since tens or hundreds of sequences can need to be tested to find a suitable regulatory sequence, this approach is not scalable or practical.

[0009] Attempts have been made to screen regulatory element sequences in parallel in individual animals by placing sequences upstream of DNA barcodes. This has the advantage that multiple sequences can be tested in a single experimental animal, and has been successfully used to identify cell type specific enhancer sequences for interneuron subtypes in the visual cortex (Hrvatin et al., 2019). However, limitations in sequencing technology and difficulties in mapping DNA barcodes to the correct regulatory element also limit this technique to testing <200 sequences at a time. Additionally, the use of experimental animals is complex and often undesirable.

[0010] In view of the large amount of sequencing data available and potential useful regulatory elements, a high throughput method for screening the ability of regulatory elements to drive cell type specific gene expression would be ideal for the development of cell or tissue targeted gene therapies. It is against this background that the present invention is designed. The present invention describes a method for high throughput screening of regulatory elements across a range of cell and tissue types. It can be adapted for use in cell culture systems and animal models. SUMMARY

[0011] According to the first aspect, a nucleotide construct is provided for a method of parallel screening of multiple putative gene regulatory elements (pGREs), comprising a nucleotide block with the sequence PRIMREG1-pGRE-PRIMREG2-GENE or GEN-PRIMREG1-pGRE-PRIMREG2.

[0012] in:

[0013] PRIMREG1 is primer recognition sequence 1, or a FLEx box containing primer recognition sequence 1; pGRE is a putative gene regulatory element.

[0014] PRIMREG2 is primer recognition sequence 2, or a FLEx box containing primer recognition sequence 2;

[0015] GENE contains nucleotide blocks REPSEQ, IRES, and SSR, among which

[0016] REPSEQ is a reporter gene, and may or may not be present;

[0017] IRES is an internal ribosome entry site or a 2A self-cleaving peptide; and

[0018] SSR stands for site-specific recombinase gene;

[0019] At least one of PRIMREG1 and PRIMREG2 is an FLEx box containing a primer recognition sequence.

[0020] Advantageously, the nucleotide constructs of the present invention allow for parallel screening of large pGRE libraries. By inserting pGREs into nucleotide constructs that can produce different PCR products depending on primer design, active and inactive pGREs can be identified on a large scale.

[0021] In a second aspect, a vector is provided comprising nucleotide sequences used in a method for parallel screening of multiple putative gene regulatory elements (pGREs), comprising a nucleotide block with the sequence PRIMREG1-pGRE-PRIMREG2-REPSEQ-IRES-SSR or GENE-PRIMREG1-pGRE-PRIMREG2.

[0022] in:

[0023] PRIMREG1 is primer recognition sequence 1, or a FLEx box containing primer recognition sequence 1; pGRE is a putative gene regulatory element.

[0024] PRIMREG2 is primer recognition sequence 2, or a FLEx box containing primer recognition sequence 2; GENE contains nucleotide blocks REPSEQ, IRES, and SSR, wherein...

[0025] REPSEQ is a reporter gene, and is present or absent;

[0026] IRES is an internal ribosome entry site or a 2A self-cleaving peptide, and

[0027] SSR is a site-specific recombinase gene;

[0028] wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

[0029] The constructs and methods disclosed herein can be applied to any suitable DNA-based vector, allowing considerable flexibility.

[0030] In a third aspect, there is provided a method of parallel screening of a plurality of putative gene regulatory elements (pGREs), comprising the steps of:

[0031] a. generating or having generated a population of vectors comprising a nucleotide construct comprising a nucleotide block of sequence PRIMREG1 -pGRE-PRIMREG2- GENE or GENE-PRIMREG1 -pGRE-PRIMREG2,

[0032] wherein:

[0033] PRIMREG1 is a primer recognition sequence 1, or a FLEx cassette comprising a primer recognition sequence 1;

[0034] pGRE is a putative gene regulatory element;

[0035] PRIMREG2 is a primer recognition sequence 2, or a FLEx cassette comprising a primer recognition sequence 2;

[0036] GENE comprises a nucleotide block of REPSEQ, IRES and SSR, wherein

[0037] REPSEQ is a reporter gene,

[0038] IRES is an internal ribosome entry site or a 2A self-cleaving peptide, and

[0039] SSR is a site-specific recombinase gene;

[0040] wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

[0041] b. contacting a target cell with the population of AAV vectors under conditions suitable for transduction of the target cell;

[0042] c. isolating DNA from the transduced target cell;

[0043] d. selectively amplifying active and / or inactive pGRE using PCR; and

[0044] e. determining the sequence of the active and / or inactive pGRE. BRIEF DESCRIPTION OF DRAWINGS

[0045] For a better understanding of the present application, and to show how it can be put into effect, there will now be described, by way of example only, with reference to the accompanying drawings in which:

[0046] Figure 1 An exemplary nucleotide construct suitable for use in an AAV vector is shown. The GENE block is shown downstream (3') of the pGRE, and is indicated in the order of REPSEQ-IRES-SSR.

[0047] Figure 2 Inactive pGRE using a construct of Figure 1 PCR products are generated using primers specific for the reverse primer recognition sequence.

[0048] Figure 3 Active pGRE using a construct of Figure 1 PCR products are generated using primers specific for the reverse primer recognition sequence.

[0049] Figure 4 A schematic of the following method is shown, wherein: 1. indicates pGRE library generation, showing multiple different pGREs in a single nucleotide construct. 2. represents constructs from 1 being packaged into a vector such as AAV. 3. represents transduction of cells / tissues of interest by the vector. 4. represents harvesting of DNA from the transduced cells / tissues. 5. represents PCR using forward and reverse primers (selected depending on whether active or inactive PGREs are being screened for). 6. represents sequencing of the PCR products.

[0050] Figure 5A An exemplary method using AAV and screening in neuro2A cells is shown. Figure 5B Output from the method of Figure 5A is shown. DETAILED DESCRIPTION

[0051] As used herein, a nucleotide construct refers to a synthetic or recombinant double stranded DNA construct comprising the nucleotide segments defined below. Typically, the nucleotide construct is described in order, i.e. in the 5' to 3' direction. However, it will be appreciated that the order of blocks is not always strictly necessary. For example, a 3' pGRE can be screened for by altering the order of the blocks, such that the gene (SSR and REPSEQ) is upstream of the pGRE and FLEx cassette.

[0052] The method of parallel screening of multiple putative gene regulatory elements (PGREs) as employed herein refers to the scalability of the method. The method advantageously employs a library of PGREs which can then be used to generate a number of different nucleotide constructs. The library of constructs is then used to generate a library of vectors. The vectors are then introduced into a population of cells or a tissue sample or even an animal where they transduce the cells and can subsequently be identified as active or inactive in that cell type.

[0053] Nucleotide segment as used herein refers to a defined segment of a nucleotide construct which performs a specific function within the construct. The nucleotide segment can be contiguous or can be distally spaced, for example, by nucleotides which can perform a function or can be spacer nucleotides.

[0054] Sequence as used herein refers to a nucleotide sequence which is conventionally read in the 5' to 3' direction.

[0055] ITR1 as used herein refers to the 5' inverted terminal repeat of a nucleotide construct.

[0056] Inverted terminal repeat is a repeated sequence in reverse that occurs at the end of a nucleotide construct. The inverted repeat sequence is a nucleotide sequence whose downstream, i.e. 3' end, is its reverse complement.

[0057] PRIMREG1 as used herein refers to the 5' primer recognition sequence block. In this context, 5' means 5' (upstream) relative to the pGRE. 5' also refers to the primer recognition sequence block closest to the 5' end of the nucleotide construct. The segment comprises a nucleotide sequence for which a PCR primer can be designed to enable amplification of active and / or inactive PGREs. The primer recognition sequence of the PRIMREG1 block can be or comprise a FLEx cassette.

[0058] pGRE as used herein means putative gene regulatory element. The term is intended to encompass promoters, enhancers and silencers of gene expression.

[0059] PRIMREG2 as used herein refers to the 3' primer recognition sequence block. In this context, 3' means 3' (downstream) relative to the pGRE. 3' also refers to the primer recognition sequence block closest to the 3' end of the nucleotide construct. The segment comprises a nucleotide sequence for which a PCR primer can be designed to enable amplification of active and / or inactive PGREs. The primer recognition sequence of the PRIMREG2 block can be or comprise a FLEx cassette.

[0060] At least one of PRIMREG1 and PRIMREG2 is a FLEx cassette. Typically, only one of PRIMREG1 and PRIMREG2 needs to be a FLEx cassette.

[0061] GENE as used herein refers to a block comprising a REPSEQ, IRES, and SSR block as described herein.

[0062] REPSEQ as used herein refers to a reporter gene. Suitable reporter genes are known to the skilled person, but include but are not limited to green fluorescent protein (GFP), blue fluorescent protein (BFP), mCherry, mScarlet, or bioluminescent reporter genes such as recombinant firefly luciferase or NanoLuc luciferase. Other genes that can be detected can act as reporters even if they are not fluorescent / luminescent. Other nucleotide sequences that can be detected by, for example, PCR can also be used as reporter genes. It will be appreciated that, although experimentally useful, a reporter gene is not required in all cases. Thus, the REPSEQ block can be present or absent.

[0063] IRES as used herein refers to an internal ribosome entry site. This site can be present or absent. Alternatively, the IRES segment can be a 2A self-cleaving peptide, and can be present or absent. Typically, an IRES or 2A self-cleaving peptide (or suitable equivalent) will be present.

[0064] 2A self-cleaving peptide as used herein is a class of 18-22 aa long peptides that can induce ribosomal skipping during protein translation in cells. These peptides share a core sequence motif of DxExNPGP and are found in a wide range of viral families.

[0065] SSR as used herein refers to a site-specific recombinase. Site-specific recombinases are enzymes that perform DNA fragment rearrangements by recognizing and binding to short specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA strands involved and re-ligate them. Suitable SSRs include but are not limited to cre recombinase (cre), Flp / Flp (flipping enzyme), Dre (D6 recombinase). SSRs bind DNA at target sites to induce a site-specific recombination event, for example, Cre recombinase binds loxP sites, while FLP binds FRT sites and Dre binds Rox sites.

[0066] ITR2 as used herein refers to the 3’ inverted terminal repeat of a nucleotide construct.

[0067] A FLEx cassette as used herein refers to a flip-excision switch with primer recognition sequences flanking SSR recognition sites. The FLEx switch was designed as a genetic tool for researchers to use site-specific recombination to conditionally manipulate gene expression in vivo. The FLEx switch takes advantage of the directional specificity of SSRs such as Cre and FLP. SSRs bind DNA at target sites to induce site-specific recombination events. When DNA sequences are flanked by target sites in opposite orientation (floxed), the SSRs will invert the DNA sequence between the sites. In this case, the primer recognition sequences.

[0068] For example, a flexible switch that turns off BFP expression and turns on mCherry expression can be achieved by creating a cassette with BFP coding sequence in the sense orientation and mCherry coding sequence in the antisense orientation. The DNA will be flanked (floxed) by two pairs of target sites, for example, one wild-type pair (e.g. loxP) and one mutant pair (e.g. loxl 11). This strategy necessitates the use of two different pairs of target sites to work effectively. Both loxP and loxl 11 are recognized by Cre, but loxl 11 sites can only recombine with other loxl 11 sites and not with loxP sites. Alternatively, if construct size is not a concern, one pair of loxP sites and one pair of FRT sites can be used and include both Cre and FLP recombinases. The choice of target sites is not limited as long as two different pairs are provided.

[0069] Once the chosen SSR is introduced or activated by the active pGRE in the present invention, recombination can proceed by first utilizing either the loxP sites or the loxl 11 sites. Regardless, the first recombination step will invert the inserted DNA fragment using either the loxP or loxl 11 sites, leaving two identical sites at one end of the DNA fragment (3' or 5' of the BFP / mCherry DNA). A second recombination event then excises the DNA between the same loxP or loxl 11 sites, leaving only one loxP and loxl 11 site on either side of the DNA fragment. Any additional recombination events are not possible at this stage, even in the presence of Cre recombinase. This plasmid will now specifically drive expression of mCherry and not BFP.

[0070] In the present method, the reporter sequence REPSEQ (e.g. BFP) has been moved outside the FLEx cassette, such that it is expressed in the presence of active pGRE, but not in the absence of active pGRE. Similarly, the expression of SSR is driven by active pGRE, and not expressed in the case of inactivated pGRE. In contrast to the above example, the FLEx cassette comprises primer recognition sequences fused by a pair of SSR recognition sites. When SSR is expressed, i.e. pGRE is active, the primer recognition sequences are inverted. PCR primers can then be designed to amplify either the active / inverted DNA or the inactive / non-inverted DNA.

[0071] As used herein, primer recognition sequences refer to short sequences (typically about 18 to 24 bases) that are unique to a nucleotide construct, and a PCR primer sequence complementary thereto can bind specifically. Primer design will be known to the skilled person, and thus suitable primer recognition sequences will follow equivalent parameters.

[0072] As used herein, a vector can be a viral vector or a non-viral vector.

[0073] Suitable non-viral vectors include, but are not limited to, the use of inorganic particles, lipid-based vectors, polymer-based vectors, and peptide-based vectors.

[0074] Suitable viral vectors include any suitable DNA-based virus, including but not limited to AAV, lentivirus, adenovirus, and tobacco mosaic virus.

[0075] As used herein, an AAV vector is a viral particle of adeno-associated virus, comprising a nucleotide construct packaged within an AAV viral capsid, which is capable of transducing cells. The AAV capsid can be capable of preferentially infecting some cell types over others.

[0076] AAV is a small virus belonging to the dependovirus genus, containing a single strand of DNA of up to about 4.9 Kb. The AAV genome comprises three capsid proteins, VP1, VP2 and VP3, all of which are translated from one mRNA by alternative splicing. In wild-type, multiple serotypes of AAV have been identified, each with a unique sequence of the capsid gene, and thus with different tropisms, although wild-type serotypes tend to be able to infect a variety of tissues and cell types. These serotypes are denoted by numbers: AAV1, AAV2, etc. It has been shown that modification of the capsid sequence by DNA recombination methods can produce non-natural sequences with tailored properties and tropisms for (or against) specific cells or tissues, and to evade the immune system (Vandenberghe et al., 2009).

[0077] As used herein, producing or having produced a population of vectors, for example AAV vectors, refers to obtaining a pre-prepared population of vectors from a suitable source or producing them while carrying out the methods disclosed herein. Typically, producing a population of vectors comprises the following steps: producing a pGRE library using any suitable method, producing nucleotide constructs according to the application using molecular biology techniques, wherein the pGRE blocks are those from the pGRE library, cloning into a suitable vector genome (for example an AAV genome flanked by ITRs), and packaging the vector genome into a suitable vector, for example an AAV capsid, to produce a population of AAV vectors.

[0078] The pGRE library can be produced by any suitable method, including but not limited to oligo- printing of rationally / bioinformatically designed constructs, DNA extracted from ATAC-seq or DNAse-seq protocols, which selectively digest accessible chromatin in a targeted cell type, random mutagenesis or DNA shuffling of previously published pGREs or previous pGRE libraries.

[0079] In the case of ATAC-seq libraries, the requirement to produce candidates in silica is replaced by the bio-methods disclosed herein. Advantageously, this means that any potential bias introduced by in silico training data is removed and the potential to identify new PGREs is increased.

[0080] As used herein, contacting a target cell means introducing a population of vectors to a target cell using a method appropriate to the vector and the cell type to be transduced. This will vary depending on whether the target cell is a cell in culture, a tissue or an organoid or a model animal.

[0081] As used herein, conditions appropriate for transduction of a target cell refer to experimental conditions and will vary depending on the target cell. For example, conditions in cell culture can involve controlling temperature, pH and available nutrients. Conversely, in an animal model, conditions can simply be met by injecting the vector into the host animal.

[0082] It is important that only one vector transduces each cell, because for example, if two vectors (one active and one inactive) transduce the same cell, the SSR will act on both constructs (one active and one inactive), leading to the inactive pGRE being wrongly identified as active.

[0083] When the vector is a non-viral vector, conditions appropriate for transfecting a target cell will vary depending on the target cell and the transfection protocol. For example, conditions in cell culture for electroporation transfection can involve applying an electric field to form pores in the cell membrane and allow molecules to pass through. Conversely, for chemical vector transfection protocols, conditions can require inorganic nanoparticles, for example gold or superparamagnetic iron oxide coated to facilitate DNA binding.

[0084] As used herein, isolating DNA from transduced cells refers to the process of extracting DNA from transduced cells. Typically, the three basic steps involved will be lysis of the cells, precipitation of the DNA, and purification of the precipitate. These techniques are well known in the field of molecular biology, but are not intended to limit the scope of methodology, as all suitable methods are applicable.

[0085] As used herein, selective amplification refers to the process by which a nucleic acid molecule is replicated to produce multiple copies of the same sequence. The most widely used method of amplifying nucleic acid sequences is PCR (polymerase chain reaction). The methods described herein employ PCR techniques to amplify active and / or inactive PGREs, but it will be understood that other methods can be employed without departing from the spirit of the invention, including not amplifying the PGRE at all. For example, the entire nucleotide construct or a portion thereof can be sequenced without amplification.

[0086] As used herein, active PGRE refers to a PGRE that turns on expression of the genes of the nucleotide construct, such as the reporter gene and / or SSR gene (encoded by REPSEQ and SSR block, respectively). An active PGRE can be identified by the fact that the primer recognition sequence of the FLEx cassette has been inverted. When a PCR method is employed, the amplified nucleotide sequence produced depends on the choice of primers. If primers directed to active and active pGRE are used, the active pGRE produces an amplified nucleotide sequence that will be shorter than the nucleotide sequence produced when the primer recognition sequence is not inverted (i.e., when the pGRE is inactive). This is because one of the 5' SSR recognition sites is excised by a site-specific recombinase such as ABC1 or ABC2 (see Figure 3 ). In addition, primer selection enables selective amplification of active or inactive (or both) PGREs. Figure 1 Exemplary primers for the exemplary primer recognition sequences shown in

[0087] Forward primer: GTACCCAGTCAG (binds to 5' primer recognition sequence)

[0088] Reverse primer 1 : TAGTTGACGACT (binds to 3' primer recognition sequence when pGRE is inactive)

[0089] Reverse primer 2: AGTCGTCAACTA (binds to 3' primer recognition sequence when pGRE is active and flex cassette is inverted).

[0090] It will be understood that the primer sequences are not limited by the foregoing examples.

[0091] Inactive PGRE as used herein refers to a PGRE that does not turn on the expression of a nucleotide construct, e.g. a reporter gene and / or a SSR gene (encoded by REPSEQ. and SSR block, respectively). Inactive PGREs can be identified by the fact that the primer recognition sequence of the FLEx cassette is not inverted. When a PCR approach is taken, the amplified nucleotide sequence that is produced depends on the choice of primers. If primers are used that are directed against an inactive and an active pGRE, the amplified nucleotide sequence that is produced will be longer than when the primer recognition sequence is inverted, i.e. when the pGRE is active. This is because no 5' SSR recognition site is excised by the site-specific recombinase, e.g. both ABC1 and ABC2 are still present (see Figure 2 ). Furthermore, primer selection enables selective amplification of active or non-active (or both) PGREs.

[0092] Determination sequence as used herein refers to any suitable method to sequence the amplified nucleotide sequence. Once the sequence of the active PGREs is known, they can be further studied. Similarly, patterns in the inactive PGREs can be identified using bioinformatics techniques.

[0093] ABC1 as used herein refers to the first SSR recognition site. This is the site that is recognized by the SSR encoded by the SSR block. The SSR cuts the DNA at the ABC1 site and reattaches it to the 1CBA site during the method. The ABC1 block will be different from the ABC2 block.

[0094] ABC2 as used herein refers to the second SSR recognition site. This is the site that is recognized by the SSR encoded by the SSR block. The SSR cuts the DNA at the ABC2 site and reattaches it to the 2CBA site during the method. Although the same SSR is employed for both inversions, different SSRs can be employed.

[0095] 1CBA is the reverse copy of ABC1, located downstream (3') of the primer recognition sequence.

[0096] 2CBA is the reverse copy of ABC2, located downstream (3') of the primer recognition sequence.

[0097] SSR inversion is a two-step process. There is an inversion at the ABC1 and 1CBA sites and another inversion at the ABC2 and 2CBA sites. The order in which the ABC1-1BCA and ABC2-2CBA inversions occur is not important. The first inversion will remove the inverted primer recognition sequence, leaving one of the SSR recognition sites on the 5' or 3' side of the primer recognition sequence and three SSR recognition sites on the other side. The second inversion will remove two of the SSR recognition sites, leaving one of the pair in ABC1-1CBA and one of the pair in ABC2-2CBA. As a result, further inversion of the SSR cannot occur.

[0098] PRSEQ is the primer recognition sequence within the FLEx cassette.

[0099] Loxl as used herein is a Lox sequence comprising a site recognized by a cre SSR. 1xoL is the reverse copy of Loxl, downstream of PRSEQ.

[0100] Lox2 as used herein is a different Lox sequence with a different site recognized by a cre SSR. 2xoL is the reverse copy of Lox2, downstream of PRSEQ.

[0101] FRT1 as used herein is a FRT sequence comprising a site recognized by a FLP SSR. 1TRF is the reverse copy of FRT1, downstream of PRSEQ.

[0102] FRT2 as used herein is a different FRT sequence with a different site recognized by a FLP SSR. 2TRF is the reverse copy of FRT2, downstream of PRSEQ.

[0103] Roxl as used herein is a Rox sequence comprising a site recognized by a Dre SSR. 1xoR is the reverse copy of Roxl, downstream of PRSEQ.

[0104] Rox2 as used herein is a Rox sequence comprising a site recognized by a Dre SSR. 2xoR is the reverse copy of Rox2, downstream of PRSEQ.

[0105] Method

[0106] First, a population of nucleotide constructs is generated that contain a library of putative gene regulatory elements (PGREs), such as promoters and enhancers. Each individual nucleotide construct contains one pGRE. Multiple constructs are combined and packaged into recombinant AAV virions to generate a population of AAV vectors containing the library of pGREs. Each virion contains one construct. The library of PGREs can contain sequences generated by rational design or by direct amplification from mammalian genomes, such as by tagging with Tn5 transposase, as used in methods such as ATAC-Seq.

[0107] In the nucleotide construct, the pGRE sequence is flanked by DNA regions that bind PCR primers (primer recognition sequences). Primer recognition sequences are found at both the 5' and 3' of the pGRE. One (or more) of the primer recognition sequences is flanked by two sets of heterotypic antiparallel recombinase recognition sites, such as LoxP sites, which in combination are sometimes referred to as a flip excision (FLEx) switch. When a site-specific recombinase, such as cre, acts on the region, the DNA between the recognition sites flips orientation, and then the now parallel LoxP sites are excised, preventing any further flipping of orientation (see Figure 3 ).

[0108] The pGRE region is upstream of the SSR, which acts on the recognition sites, such as cre and LoxP. In the case of cre-LoxP, if the pGRE region is sufficient to drive expression of the cre gene, then cre protein is produced, leading to recombination of the LoxP sites and inversion of the primer recognition sequences.

[0109] PCR primer pairs can be designed such that they recognize the primer recognition sequences in the un-recombined (inactive) or recombined (active) case, allowing for selective PCR of active and inactive PGREs in separate PCR reactions.

[0110] Transduction of rAAV pGRE libraries into cells in culture or whole tissues in vivo allows for the parallel screening of large numbers of pGREs. In this case, harvesting the cell population and then performing PCR will yield PCR products that can be subjected to next-generation or Sanger sequencing to ultimately determine the sequences of active (and inactive) GREs across cell and tissue types.

[0111] In an extension of the method, multiple (two) primer recognition sequences can flank (be floxed) recombinase recognition sites. For example, the 5' primer recognition sequence can flank an FRT site (recognized by FLP recombinase), while the 3' primer recognition sequence flanks a LoxP site (recognized by cre); or vice versa. Other recombinases and recognition sites can also be used, such as Dre-Rox. In this case, certain combinations of PCR primers can be used to detect pGREs in the presence of cre and flp recombinases.

[0112] One example for this is the determination of pGRE in animals expressing recombinant enzymes, such as cre transgenic mice. Here, the nucleotide construct will encode the FLP-recombinase required for inversion of one initiation region, and the target cell type will provide cre for inversion of the other initiation region. For example, injection of the vector into ChAT-Cre test animals expressing cre in cholinergic cells, such as motor neurons, will allow selective harvesting and identification by PCR of pGRE active in motor neurons.

[0113] pGREs giving positive results from these types of screens can be subjected to further analysis, for example by individual screening or by the DNA-barcode approach.

[0114] As used herein, about means ± 10%.

[0115] In the context of the present specification, “comprising” is to be interpreted as “including”.

[0116] Aspects of the application comprising certain elements are also intended to extend to alternative embodiments consisting of or consisting essentially of the said elements.

[0117] Embodiments of the application can be combined where technically appropriate.

[0118] Embodiments are described herein as comprising certain features / elements. The disclosure also extends to separate embodiments consisting of or consisting essentially of the said features / elements.

[0119] Patents and applications, and other technical references, are incorporated herein by reference.

[0120] Any embodiment specifically and explicitly recited herein can form the basis of a disclaimer, either alone or in combination with one or more other embodiments.

[0121] References

[0122] Juttner et al., 2019 Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans. Nature Neuroscience volume 22, pages 1345-1356 (2019)

[0123] Mich et al., 2021 Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. Cell Reports 34, 108754

[0124] Hrvatin et al., 2019 A scalable platform for the development of cell-type-specific viral drivers. eLife 8:e48089

Claims

1. A nucleotide construct for use in a method of parallel screening of a plurality of putative gene regulatory elements (pGRE), comprising a nucleotide block of sequence PRIMREG1-pGRE-PRIMREG2-GENE or GENE-PRIMREG1-pGRE-PRIMREG2, wherein: PRIMREG1 is a primer recognition sequence 1, or a FLEx cassette comprising a primer recognition sequence 1; pGRE is a putative gene regulatory element; PRIMREG2 is a primer recognition sequence 2, or a FLEx cassette comprising a primer recognition sequence 2; GENE comprises a nucleotide block of REPSEQ, IRES, and SSR, wherein REPSEQ is a reporter gene, and is present or absent; IRES is an internal ribosome entry site or a 2A self-cleaving peptide; and SSR is a site-specific recombinase gene; and wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence. wherein:

2. A vector comprising a nucleotide sequence for use in a method of parallel screening of a plurality of putative gene regulatory elements (pGRE), comprising a nucleotide block of sequence PRIMREG1-pGRE-PRIMREG2-REPSEQ-IRES-SSR or GENE-PRIMREG1-pGRE-PRIMREG2, wherein: PRIMREG1 is a primer recognition sequence 1, or a FLEx cassette comprising a primer recognition sequence 1; pGRE is a putative gene regulatory element; PRIMREG2 is a primer recognition sequence 2, or a FLEx cassette comprising a primer recognition sequence 2; GENE comprises a nucleotide block of REPSEQ, IRES, and SSR, wherein REPSEQ is a reporter gene, and is present or absent; IRES is an internal ribosome entry site or a 2A self-cleaving peptide; and SSR is a site-specific recombinase gene; and wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

3. A method of parallel screening of a plurality of putative gene regulatory elements (pGRE), comprising the steps of: a. generating or having generated a population of vectors comprising a nucleotide construct comprising a nucleotide block of sequence PRIMREG1-pGRE-PRIMREG2-GENE or GENE-PRIMREG1-pGRE-PRIMREG2, wherein: PRIMREG1 is a primer recognition sequence 1, or a FLEx cassette comprising a primer recognition sequence 1; pGRE is a putative gene regulatory element; PRIMREG2 is a primer recognition sequence 2, or a FLEx cassette comprising a primer recognition sequence 2; GENE comprises a nucleotide block of REPSEQ, IRES, and SSR, wherein REPSEQ is a reporter gene, IRES is an internal ribosome entry site or a 2A self-cleaving peptide, and SSR is a site-specific recombinase gene; and wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence; b. introducing the population of vectors into a plurality of cells; c. allowing the cells to express the nucleotide construct; d. allowing the cells to express the reporter gene; e. detecting the expression of the reporter gene; and f. identifying the cells that express the reporter gene. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ b. contacting the target cells with a population of AAV vectors under conditions suitable for transduction of the target cells; c. isolating DNA from the transduced target cells; d. selectively amplifying active and / or inactive pGRE using PCR; and e. determining the sequence of the active and / or inactive pGRE.

4. The vector of claim 2 or the method of claim 3, wherein the vector is a viral vector.

5. The vector or method of claim 4, wherein the viral vector is an AAV vector.

6. The construct, vector or method of any one of the preceding claims, wherein the FLEx cassette comprises the sequence ABC1-ABC2-PRSEQ-1CBA-2CBA, wherein: - ABC1 is a recognition site of a recombinase protein encoded by a SSR segment; - ABC2 is a different recognition site of a recombinase protein encoded by a SSR segment; - PRSEQ is primer recognition sequence 1 or primer recognition sequence 2; - 1CBA is reverse ABC1; and - 2CBA is reverse ABC2.

7. The construct, vector or method of claim 6, wherein: - ABC1 is Lox1 ; - ABC2 is Lox2; - SSR is cre; - 1CBA is 1xoL; and - 1CBA is 2xoL.

8. The construct, vector or method of claim 6, wherein: - ABC1 is FRT1, - ABC2 is FRT2, - SSR is Flp, - 1CBA is 1TRF; and - 1CBA is 2TRF.

9. The construct, vector or method of claim 6, wherein: - ABC1 is Rox1 ; - ABC2 is Rox2; - SSR is dre; - 1CBA is 1xoR; and - 1CBA is 2xoR.

10. The construct, vector or method of any one of the preceding claims, wherein the nucleotide construct has a 5’ ITR1 segment and a 3’ ITR2 segment.

11. The method of any one of claims 3 to 10, wherein PRIMREG1 and PRIMREG2 are both FLEx cassettes, and the target cells are capable of expressing a site-specific recombinase that is different from the site-specific recombinase encoded by the SSR segment of the nucleotide construct.

12. A novel pGRE identified using the method of any one of claims 3 to 11.

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