HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy

Abstract HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active ‘attB’ sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native ‘attB’ sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native ‘attB’ sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

An alternative class of tools, site-specific recombinases (SSRs), can address safety concerns and insert size limitation. SSRs catalyze recombination reactions between two specific short DNA recombination sites (RSs). The location and relative orientation of the RSs defines whether a recombination event will result in an integration, excision, inversion, or translocation reaction. The use of SSRs for targeted genome manipulation relies on their efficiency and specificity for recombining only at their respective RSs. For these reasons, an extensive repertoire of SSRs including the most used Cre, FLP and phiC31, are widely harnessed in genome manipulations, gene therapy, developmental and synthetic biology (25)(26)(27)(28). SSRs are non-viral and do not rely on host cell machinery to achieve transgenesis, hence, providing attractive genome editing tools for use in human cells. Although, for some of the SSRs, the reverse excision reaction is kinetically favored over their integration reaction (27), which leads to poor integration efficiency. This limitation is overcome by the recombinase-mediated cassette exchange (RMCE) method (29,30).
The RMCE method is based on a reciprocal exchange of DNA fragments that consists of two crossover events within two incompatible RS pairs that are localized on both sides of the cassette of interest. This strategy has the capa- bility to swap large transgene cassettes (>100 kb) (31). To achieve reciprocal DNA cassette exchange, cells with the target DNA sequence, flanked by two hetero-specific RSs, are transfected with two plasmids. The replacement cassette of interest, flanked by the compatible RSs, is introduced by a donor plasmid, while another plasmid expresses the cognate SSR ( Figure 1A) (32). RMCE can be used as a gene therapy tool by replacing a harmfully mutated genomic sequence flanked by two incompatible RSs ( Figure  1A, b) with a plasmid-borne sequence of interest flanked by matching RSs (Figure 1A, a). A molar excess of the donor plasmid over the genomic target sequence will drive the reaction towards replacement by the sequence of interest (27). A successful RMCE reaction results in a 'clean' correction of the mutation without the use of selection markers or other undesired sequences ( Figure 1A, c) (32)(33)(34). Furthermore, In recent years, RMCE has been used substantially in various research areas including the generation of induced pluripotent stem cells (35), production of therapeutic monoclonal antibodies (36), and in combination with other genome editing systems, such as ZFN, TALENs (37) and CRISPR/Cas (27,38).
In this work, we focused on the integrase (Int) site-specific recombinase of Escherichia coli HK022 bacteriophage that belongs to the tyrosine family of SSRs. It naturally catalyzes phage genome integration and excision in a mechanism very similar to that of the well-known lambda coliphage integrase ( Figure 1B). In the lysogenic cycle, a recombination reaction between the attP site of the injected and circularized phage genome and the bacterial recombination site attB results in the formation of the integrated prophage (39). In the case of phage HK022, attB (or BOB') is only 21 bp long, comprising a 7 bp central overlap (O) sequence which is the site of the recombination event. O is flanked by two palindromic 7 bp sites (B and B', see also Table 1) that are binding sites of Int. The longer attP (or POP') RS of the phage carries a core (COC') similar to attB and is flanked by two longer arms (P and P') that are essential for integration in E. coli. The 7 bp O sequences of the recombining att pairs, attB and attP, are identical (Figure 1B) (40). We have previously shown that HK022's wild type (w.t.) Int, as well as a codon-optimized for human cells Int (oInt), are active in human cells. The activity of these Int does not require any of the prokaryotic accessory proteins that are essential in E. coli (41)(42)(43). More importantly, it has also been shown that, along with the Flp site-specific recombinase, HK022 Int can catalyze an RMCE reaction in mammalian cells (44).
In previous work, we have identified active attB-like sites in the human genome that flank deleterious disease-causing mutations. These active attB-like sites with different overlaps and reduced B and B' sites were designated as 'attB's. Each att site has 7 bps in the O region that can be arbitrary if they are identical between the recombining pair. Additionally, permissible modifications in the previously assigned 7 bp in each B and B' Int binding sites (40) have led to their reduction to 5 bp requirement (Table 1) (45). These charac-teristics provide flexibility in the RS's and increase the possibility to identify such 'attB' sites in the human genome. The 'attB' sites are key to the implementation of an Intcatalyzed RMCE reaction in human cells that can cure such mutations. (45). However, neither w.t. Int nor oInt exhibit sufficient RMCE activity on the mammalian genome (44).
The aim of this study is to identify optimized Int recombinase with enhanced activity that can perform successful RMCE reaction within the human genome. The present study describes the construction and examination of several Int variants to perform RMCE reactions using native 'attB' sites pairs. First, HK022 Int was optimized in the context of transient in trans recombination in human cells. Then, a proof of concept successful genomic RMCE was achieved using a pair of native 'attB' sites that have been inserted as a substrate into the human genome. Finally, 'attB' sites in the human CTNS and DMD genes were targeted to demonstrate successful RMCE in their native location. Our results show that Int-based RMCE can become a safety, non-viral and sequence-specific gene therapy tool that addresses gene manipulations with large DNA fragments.

Plasmid construction
Plasmids and primers used in the work are presented in Supplementary Tables S1 and S2. The details of the cloning procedure are presented in the Supplementary Materials and Methods section. In short, the two plasmids that were used as substrates in the transient human recombination assay were constructed as described (45).
pNA1756 and pNA1757 used in the off-target assay were constructed as described (45). pNG1924 (HEXA3) and pNG1926 (ATM2) 'attP' plasmids used in the off-target assay, were constructed by RF cloning (49). The 'attP' PCR fragment was obtained using the primers 944 and 945 on pNA1285 and pNA1608 plasmids respectively as templates (Supplementary Tables S1 and S2), then cloned into the pSSK10 vector. These plasmids were propagated in S17-1 lambda pir as host.
The w.t. Int-expressing plasmid pMK22 was constructed by cloning NcoI-Int-HindIII PCR fragment obtained using primers 1074+173 on pNR69 (40) plasmid as a template into the same sites in pKK322-2. E174K Int-expressing plasmid for E. coli was constructed based on pKH70 (50) as a template using two-step PCRs procedure: In the first step, two PCR reactions were performed: the first was with primers 513+144 and the second was with 143+203. In the second step, the two PCRs from the first step were assembled by PCR with primers 513+203, producing the desired E174K Int mutated fragment. Next, this fragment cut with NdeI and HindIII was ligated into the same sites in pKH70.
All plasmid constructs were verified by DNA sequencing.

Off-target integration assays in E. coli
Cells of E. coli strain TAP114 that carried an Ap R plasmid expressing w.t. Int or the E174K mutant (pMK22 or pMK174, respectively) were transformed with the relevant attP plasmid constructed on the basis of pSSK10 (Km R ) (w.t. attP or human 'attP's: HEXA3 and 7 or ATM2 and 4). pSSK10 plasmid cannot replicate in the test strain, so Km R expression occurs only in integration products. Transformed cells were plated on LB rich medium supplemented with Km and Ap. To avoid the interference of possible false-positive colonies, the presence of the Km R gene in the Ap+Km resistant colonies was tested by PCR analysis using primers 1069+1070 (Supplementary Figure S8A, Step 1). In three independent experiments, the average growth of the cells transformed with the plasmid that carried the w.t. attP was between 35-60 colonies. When the presence of the Km R gene in 100 resistant colonies was examined by PCR analysis using primers 1069+1070, only 40 colonies yielded the positive expected PCR product but none in the absence of Int (Supplementary Figure S8C and D). The remaining 60 Ap R +Km R colonies were PCR negative, so they were considered false-positive colonies. The 40 Km R positive PCR clones were used for the Int-catalyzed integration activity analysis (Supplementary Figure S8A, Step 2). Site-specific integration of the w.t. attP Km R plasmid into the native attB was confirmed by colony PCR analysis using primers 958+1080 (for attL) and 788+1069 (for attR) followed by sequencing. Plasmids that carried different HEXA and ATM 'attP' sites yielded 5-40 Ap R +Km R colonies in the repeated independent experiments regardless of the Int plasmid presence. Km R gene PCRs of 30 such colonies (Supplementary Figure S8B, C and D) with the same primers used for w.t. attP plasmid was all negative indicating the false-positive phenotype of these colonies.

Construction of Int mutants
All Int single mutants were constructed based on the oInt expression plasmid pNA979 as a template using the twostep PCRs procedure. In the first step, two PCR reactions were needed: the first PCR was performed using 204 as a forward primer + the reverse primer that contained the desired mutation. The second PCR was performed using a for-ward primer that contained the desired mutation (complementary to the mutated reverse primer of the first reaction) + 469 as a reverse primer. In the second step, the two PCRs from the first step were assembled by PCR using primers 204+469, producing the desired Int variant fragment. Next, this fragment cut with EcoRI and HindIII was ligated into the same sites in the pCDNA3 vector. Int double mutants were constructed by using pNG1862 as a template, by the same two-step procedure described above. The triple mutant was constructed based on pAE2029 as a template, by the same two-step procedure described above.

Fluorescent-activated cell sorting (FACS) analysis
∼2 × 10 6 cells from one well of a six-well plate were collected following trypsin treatment of which 10 4 cells were selected by the FACS sorter (Becton Dickinson Instrument) for fluorescent measurements. Dot plot of side scatter (SSC) versus forward scatter (FSC) was used to gate live cells in order to separate them from aggregated and dead cells. For gated cells, the fluorescence statistics was performed using the Kaluza Analysis Software (Beckman Coulter) and represented as a quadrant analysis that clearly showed the percentage of GFP expressing/ GFP-mCherry co-expressing cells.

DNA and RNA manipulations
Plasmid DNA was prepared from E. coli using a DNA Spin Plasmid DNA purification Kit (Intron Biotechnology, Korea) or a NucleoBond™ Xtra Midi kit (Macherey-Nagel, Germany). Genomic DNA was prepared using the Genomic DNA Mini Kit (Geneaid Biotech Ltd, Taiwan). cDNA was synthesized using the Verso cDNA kit (Applied Biosystems, Foster City, CA). mRNA was prepared using Blood/Cell Total RNA Mini Kit (Geneaid Biotech Ltd, Taiwan). Gibson reaction was performed using the NEB-Builder HiFi DNA assembly master mix (NEB, MA, USA). General genetic engineering experiments were performed as described by Sambrook and Russell (48).

Identification of 'attB' sites in the human genome
A computer search for potentially active human 'attB' sites was performed as described (45). Genomic coordinates of the relevant 'attB' sites were obtained from the UCSC Genome Browser. The 'attB' distribution in the human genome was performed by NCBI Nucleotide Blast browser using human (taxid: 9606) RefSeq Genome databases.

Statistical analysis
Data were presented as the mean ± SD of at least three independent experiments, each with three repeats. P-values of <0.05 were considered statistically significant by Student's two-tailed t-test assuming equal variance.

Optimization of HK022 Integrase activity in human cells
The sequence of HK022 Int shares 70% identity with that of lambda's Int and both consist of three domains (Figure 2A). These domains facilitate assembly and function of a higher order tetrameric complex with the DNA attP and attB substrates known as the intasome (51,52). The Nterminal DNA binding domain (AB) (residues 1-63) recognizes 'arm-type' DNA sequences that flank the attP coresite. The activity of lambda integrase within the tetramer is tightly regulated so that only two diagonally positioned protomers are active at any given time. For ordered DNA strand cleavages and exchanges to form and then resolve a Holliday junction (HJ), the protomers need to switch roles during the recombination reaction, which accompanies isomerization of the HJ intermediate. It has been proposed that binding of the AB domain to the arm-type DNA, facilitates this isomerization process. The core binding domain (CB) recognizes the C and C' core binding sites of attP and those of attB (B and B'). The catalytic domain (CAT) is responsible for DNA cleavage and rejoining in the site-specific recombination reaction (51,53). To further optimize Int activity in human cells, 10 different single mutated Ints were constructed from oInt ( Figure 2A): I43F (in the AB), E174K (CB) and E264G, R319G, D336V (CAT). These variants were selected based on previously described mutations used for lambda and HK022 Ints (46,(54)(55)(56)(57). We noted that two of the known lambda mutations that improved Int activity (E174K and E218K) carry Glu to Lys changes (56,58). Therefore, we tested additional replacements of acidic residues (Glu/Asp) potentially close enough to make DNA backbone contacts that could be changed to Lys. Thus, the mutants E134K, D149K (CB), and D215K, D278K, E309K (CAT) were constructed.
To examine the activity of these Int variants, we performed an assay in human HEK293T cells of a transient trans integrative recombination reaction. The assay utilized two plasmids as recombination substrates (42) (Figure 2B), the first substrate (pGH790) carried the attB site downstream to the CMV promoter. The second plasmid (p1331) carried attP located upstream to the open reading frame (ORF) of the green fluorescent protein (GFP) and downstream to transcription terminator (Stop). A successful attB × attP reaction forms a dimer plasmid encoding CMV-promoted GFP expression ( Figure 2B). HEK293T cells were co-transfected with these two substrate plasmids, with or without an Int-expressing plasmid (oInt or one of its Int mutant derivatives, Supplementary Table S1d). 48 hours post-transfection GFP expression was analyzed by fluorescence-activated cell sorting (FACS). The quantified FACS data ( Figure 2C and Supplementary Figure S1) showed that only two single Int mutants (E174K and D278K) demonstrated an increased activity, of 7% and 6.5% GFP expression, respectively, compared to 5.5% by oInt. All other 8 single mutants demonstrated similar or lower activity (between 5.5% and 0.2%) compared to 5.5% by oInt (Supplementary Figure S2). Next, we combined each of the two more efficient mutations (E174K and D278K) with some of the moderately active ones and assayed their activity. The double mutants E174K+I43F, E174K+R319G, and E174K+D278K showed elevated activity of 7.5%, 8% and 9.5% of GFP expression, respectively, compared to oInt ( Figure 2C and Supplementary Figure  S1). However, E174K+E264G and E174K+D336V showed a significantly lower activity (Supplementary Figure S2). Lastly, a constructed E174K+I43F+R319G triple mutant showed increased activity of 10% ( Figure 2C and Supplementary Figure S1).
The various Int variants were examined on 8 different active 'attB' sites with their compatible 'attP's ( Table 1, Figure S3), using the same recombination assay described above ( Figure 2B). Two of the 'attB' sites (HEXA3 and ATM4, Figure 3A and Supplementary Figure S3A) were previously reported (45), while another three 'attB' pairs flank known mutations in the genes CTNS (chromosome 17), DMD (chromosome X) and CFTR (chromosome 7) that respectively cause the following diseases: Cystinosis, Duchene muscular dystrophy and Cystic fibrosis (59)(60)(61). Notably, different Int-mutants showed various efficiencies with the different attB sites. For instance, the triple mutant Int and the double mutant (E174K+D278K) were the most efficient Ints with the w.t. att sites ( Figure 2C) but a lower efficiency with the CTNS4 and DMD3 sites ( Figure 3B). Thus, examining the optimal Int variant toward the targeted 'attB' pair may give the prospect to achieve more efficient RMCE reaction.

Int-catalyzed RMCE reaction using human native 'attB' sites
We designed an assay to examine if genomic 'attB' sites that flank human deleterious mutations can enable successful Int-catalyzed genomic RMCE reactions. First, we constructed a target DNA docking platform that carried the mCherry reporter gene in the Flp-In™-293 cells. This 'docking' RMCE substrate plasmid ( Figure 4A) carries two different human 'attB's (HEXA3 and ATM4, Table 1) that are 2.7 kb apart. attB1 represents the HEXA3 'attB' located downstream to the EF1α promoter, and attB2 represents the ATM4 'attB' located upstream to promoter-less ORF of the mCherry ( Figure 4A). This docking plasmid also carried an frt-Hyg R ORF sequence to be inserted into the chromosome of the Flp-In™-293 cells by an Flp-catalyzed SSR reaction followed by hygromycin selection. In addition, an 'incoming' plasmid was constructed ( Figure 4B), that carries the relevant compatible 'attP' sites (attP1 and attP2 for HEXA3 and ATM4, respectively) 4.3 kb apart. attP1 is located upstream to the promoter-less ORF of EGFP, and attP2 is located downstream to the CMV promoter ( Figure 4B). A successful Int-catalyzed RMCE reaction between these two plasmids is expected to form a recombinant product that co-expresses both green GFP and red mCherry fluorescent products ( Figure 4C). The validity of these two plasmids was initially tested transiently by co-transfecting HEK293T cells with both docking and incoming plasmids with and without Int (oInt and its variants), followed by 48 hours post-transfection FACS analysis. The quantified FACS data showed a mean increase of 5.5% of GFP and mCherry co-expression in the presence of oInt and 6.4% in the presence of E174K compared to the cells without Int ( Figure 4D and Supplementary Figure  S4A). E174K+I43F, E174K+R319G, E174K+D278K, and E174K+I43F+R319G Ints showed lower or non-significant RMCE activity compared to oInt (data not shown). To further verify that the GFP and mCherry co-expression properly indicated the occurrence of the expected RMCE reaction, extrachromosomal DNA was extracted from the  transfected cells, and tested by PCR ( Figure 4C). The treated cells demonstrated the formation of the expected recombination junctions: EF1α-attL1-EGFP (500 bp) (Figure 4C and E), CMV-attL2-mCherry (486 bp) ( Figure 4C and F), and of the complete RMCE product (4.6 kb) (Figure 4G), which were confirmed by sequencing.
These results demonstrated the validity of the two plasmids as substrates in proceeding towards a chromosomal RMCE reaction ( Figure 5). Hence, the Flp-In™-293 cell line ( Figure 5B) that carries a chromosomal frt recombination site downstream to the SV40 promoter was cotransformed with the docking plasmid ( Figure 5A) along with a Flp-expression plasmid (pOG-Flp) followed by hygromycin selection. The correct insertion of the docking plasmid in selected Hyg R cells ( Figure 5C) was confirmed by sequencing of the PCR product (415 bp, Figure 5C and G). Next, these dual 'attB'-docked cells were co-transfected with the dual 'attP' incoming plasmid ( Figure 5D), with and without different Int-expressing plasmids. An Int-catalyzed chromosomal RMCE product ( Figure 5E) is expected to demonstrate both promoter-trapped GFP and mCherry fluorescence products resulting from both recombination reactions. The FACS analysis of the Int-treated cells, 48 hours post-transfection, showed a mean of 0.45% increase in dual fluorescence by oInt activity and 0.7% by E174K compared to the untreated Int cells ( Figure 5F and Supplementary Figure S4B). E174K+I43F, E174K+R319G, E174K+D278K and E174K+I43F+R319G Ints showed lower or non-significant RMCE activity compared to oInt (data not shown). PCR and sequencing analyses by the appropriate primers and the chromosomal DNA of the transfected cells as a template confirmed the presence of the expected recombination junction products EF1α-attL1-EGFP (500 bp) ( Figure 5H), and CMV-attL2-mCherry (273bp) ( Figure 5I) only in the Int-treated cells. The PCR analysis for the expected full 4.6 kb RMCE product (Figure 5E, primers 635+1191) revealed a weak band for the expected product ( Figure 5J), dominated by the shorter 3.2 kb PCR product of the non-recombined docking chromosomal cassette ( Figure 5C). Therefore, this weak 4.6 kb product was gel-purified and used as a template for a nested PCR reaction that confirmed the presence of both expected recombination junctions only in the Int-treated cells (Figure 5K). The expected sequence of these PCR products was confirmed by sequencing. These results are a proof of concept that Int variants can successfully catalyze chromosomal RMCE with two different active 'attB' sites in the absence of any selective pressure or fluorescence-based sorting.

Int-catalyzed RMCE reactions in native 'attB' sites at the human genes CTNS and DMD
Our next aim was to demonstrate a successful Int-based RMCE reaction in native locations of the human genes. We targeted genes whose disease-causing mutations are flanked by two active native 'attB' sites. Therefore, we examined Intcatalyzed RMCE reactions in the CTNS and DMD genes using their appropriate flanking 'attB' sites in HEK293 cells by GFP trap assays. In the CTNS case, we chose the CTNS1 and CTNS4 'attB' sites (Table 1) which are 7.6 kb apart and flank a region containing the CTNS promoter and exons 1 to 3 ( Figure 6B). Deleterious mutations in this region are associated with Cystinosis disease (59). The compatible constructed incoming plasmid ( Figure 6A) carried a CMVpromoted EGFP ORF (lacking polyA) followed by a P2A ribosomal skipping sequence and the sequence of the CTNS exon 3 splice donor (for RNA splicing), all flanked by the relevant 'attP's (CTNS1 and CTNS4), 1.7 kb apart. Successful Int-catalyzed RMCE is expected to replace the genomic sequence between the two CTNS 'attB's with the incoming cassette between its two 'attP's ( Figure 6C). Thus, the RMCE genomic recombinant is expected to transcribe an mRNA of the EGFP-P2A-Exons 4-12 sequence (Figure 6D). The P2A ribosomal skipping site will lead to the translation of two peptides, GFP and a distal peptide of CTNS. HEK293 cells were co-transfected with this incoming plasmid along with a plasmid expressing one of the Int variants (oInt, E174K, E174K+I43F, E174K+R319G, E174K+D278K and E174K+I43F+R319G). FACS analysis of transformed cells have shown that the E174K+I43F Int variant gave the highest RMCE efficiency with a mean 0.6% GFP fluorescence compared to 0.45% by oInt and the untransformed cells ( Figure 6E and Supplementary Fig-Figure 6. ure S5A). E174K, E174K+R319G, E174K+D278K and E174K+I43F+R319G Ints showed lower or non-significant RMCE activities compared to oInt (data not shown). Chromosomal DNA and mRNA extracted from the transfected cells were used as templates for PCR reactions with the proper primers ( Figure 6C and D). PCR results demonstrated the formation of the expected recombinant junctions CTNS-'attL'CTNS4-CMV of 500 bp ( Figure 6C and F), EGFP-2A-SD-'attL'CTNS1-Intron3 of 400 bp ( Figure  6C and G), as well as the mRNA expected EGFP-P2A-Exon4 junction of 177 bp ( Figure 6D and H). The correct sequence of all PCR products was confirmed by nextgeneration sequencing (NGS).
A second RMCE reaction targeted the DMD gene, using DMD2 and DMD3 'attB' sites ( Table 1) that are 23 kb apart located in introns 43 and 44 respectively, and flank exon 44 ( Figure 7B). Deleterious mutations in this region are associated with Duchene muscular dystrophy (60). Here we used a GFP promoter trap whose incoming plasmid (Figure 7A) carried a splicing acceptor (SA), a ribosomal skipping site (P2A), and the ORF of EGFP with a polyA sequence, all flanked by the two relevant 'attP's (DMD2 and DMD3), 1.4 kb apart. Like CTNS, a processed mRNA is expected to be translated to an active GFP and a proximal DMD peptide. FACS analysis of transformed cells have shown that the E174K+I43F+R319G Int variant gave the highest RMCE efficiency with a mean 0.55% GFP fluorescence compared to 0.3% by oInt and the untransformed cells ( Figure 7E and Supplementary Figure S5B). E174K, E174K+I43F, E174K+R319G and E174K+D278K Ints showed lower or non-significant RMCE activities compared to oInt (data not shown). Chromosomal DNA and mRNA extracted from the transfected cells served as templates for PCR reactions with the proper primers. PCR results confirmed and demonstrated the expected recombinant junctions: Intron43-'attL' DMD2-SA-P2A-EGFP (700 bp) ( Figure 7C and F), EGFP-'attR' DMD3-Intron44 (800 bp) ( Figure 7C and G) and the mRNA Exon43-P2A-EGFP junction (229 bp) ( Figure 7D and H). The correct sequence of all PCR products was confirmed by NGS. RMCE reactions were also demonstrated in exons 45 and 52 in the DMD gene ( Supplementary Figures S6 and S7). The used DMD4 and DMD5 'attB' sites (Table 1) Figures S6E-F and S7E-F). Other Int variants showed lower or non-significant RMCE activities compared to oInt (data not shown).

DISCUSSION
Despite the progress achieved in the last two decades in the field of gene therapy, human genome-editing tools still need to improve in efficiency, safety, sequence-specificity, and large DNA fragments targeting. In this study, we aimed to demonstrate a novel potential gene therapy tool based on the Int-catalyzed RMCE. Previously we identified pairs of human native active 'attB' sites that can be used to correct 'attB'-flanked mutations by the Int-catalyzed RMCE reactions. The 'attB' sites have a short 17 bp recombining site and a promiscuous 7 bp overlap sequence. In contrast to the 'attB' sites, the RSs of commonly used tyrosine recombinases like Cre and Flp were not identified in the human genome. Consequently, additional strategies were utilized to carry out a genome editing, either by constructing a variety of recombinases with altered target specificities (62,63), or by the insertion of their specific recombination sites into the targeted DNA sequence (27). HK022 Int is active in human cells without the need to supply the prokaryotic IHF accessory protein unlike lambda's w.t. Int (42,43). These features of HK022 Int offer a simpler means for correcting human mutations by RMCE.
We hypothesize that the mechanism of the Int-catalyzed RMCE reaction is sequential. The reaction begins with a single trans integration reaction that takes place between one 'attP' of the incoming plasmid and its compatible chromosomal 'attB'. This step creates an integrated intermediate of the incoming plasmid. Thereafter, a cis reaction occurs between the second 'attP' × 'attB' pair resulting in the fully-integrated intermediate to the final RMCE product (44,64). According to our hypothesis, we predict that the efficiency of the first trans integration reaction is a major limiting step towards the formation of the RMCE product. Therefore, an optimal Int variant for RMCE must either improve the first step of the integration in trans reaction activity, and/or reduce the cis reverse excision activity of the first recombination intermediate. Hence, to identify a more efficient Int variant we examined the constructed Int variants in a single 'attP' × 'attB' trans reaction ( Figure 2). The integration analysis revealed that only two Int variants (E174K and D278K) increased the integration activities compared to oInt ( Figure 2C).
The construction of HK022 Int variants was based on lambda's Int structural information. The E174K mutant of lambda Int (also known as Int-h) proved to be IHFindependent in E. coli, and more active in trans site-specific recombination reactions (56). Furthermore, it showed an enhanced DNA binding activity (65) that supports its enhanced activity in trans. This mutation has also been used to allow lambda Int to be active in human cells (66)(67)(68)(69). The E174K mutation in HK022 Int is in the inter-domain linker (I160-R176), which corresponds with the location of the mutation in lambda Int. It is likely that this CB-CAT linker is highly flexible when Int is not bound to DNA, which would affect the entropic cost of DNA binding and thereby DNA binding affinity. Thus, we predict that lysine residue substitution might enhance the DNA binding affinity by stabilizing interaction with the DNA and/or by constraining the movement of the inter-domain linker (70). We suppose that E174K and D278K, which replaced the negatively charged Glu/Asp by the positively charged lysine near the DNA, enhances Int activity, most likely, by introducing new ionic interactions with the DNA backbone ( Figure 8). We could have expected the same for E309K as it is also near the DNA backbone. Yet, E309 is close to the active site and is hydrogen-bonded to R179, an important residue for positioning Tyr342 in the active site. This might explain why the E309K mutation significantly compromises Int activity.
The constructed Integrase variants based on E174K Int showed higher recombination activities with the different 'attB' sites compared to oInt (E174K+I43F, E174K+E264G, E174K+R319G, E174K+D278K, E174K+I43F+D336V) ( Figure 3A, B and Supplementary Figure S3). Based on the crystal structure (Figure 8), I43 is far away from the arm-site DNA and faces the adjacent N-terminal domain within the Int tetramer ( Figure 8B). It was suggested earlier that the I43F mutation in lambda Int affects the allosteric rearrangements that drive resolution of the intermediate Holliday junction towards recombined products (55). The R319G mutation is located on the CAT surface ( Figure 8E) which plays a key role in the catalytic activity and the regulation of site-specific recombination. Notably, it has been previously described that mutations in lambda Int E319 reduce substrate specificity and increase its activity toward HK022 sites (46). In the present work, the single I43F and R319G mutations did not increase transient trans integrative recombination activity (Supplementary Figure S2) but they did enhance E174K activity in a double mutant context ( Figure 2C and Supplementary Figure S1). Moreover, the I43F and R319G mutations contributed mostly toward improving in trans integrative recombination in the triple mutant context ( Figure 2C and Supplementary Figure S1). However, in the context of transient RMCE ( Figure 4D), and in stable genomic RMCE in the human cell-line Flp-In™-293 ( Figure 5F), the most active Int variant was E174K showing 6.4% and 0.7% RMCE activity, respectively (Supplementary Figure S4). Although, in the CTNS and DMD genes the most efficient mutants were E174K+I43F and E174K+I43F+R319G reaching 0.6% and 0.55% RMCE activity, respectively ( Figures 6E, 7E, and Supplementary Figure S5A-B). The differences in stable RMCE efficiency between the three cases could be due to the distance between their targeted 'attB' pairs (2.7 Kb, 7.6 Kb and 23 Kb, respectively), the 'attB' pairs activity, the different Int variants efficiency, and/or the genomic location accessibility.
The RMCE reactions within the CTNS and DMD genes were initially performed in their native sequence contexts (in HEK293 strain). Hence, probable reduction in cell viability compared to the unrecombined cells may have originated from replacing the native sequence with the EGFP reporter in either gene. Although, the FACS data of the Int-treated cells showed elevations of GFP expression by 0.2-0.7% compared to the untreated control, and the occurrences of the expected RMCE reactions were confirmed by PCRs and sequencing. These achieved preliminary efficacies are encouraging in the light of the already successfully used gene therapy approach based on the Adeno-associated virus system that have similar efficacies (between 0.2% and 0.5%) (71,72). To further study and increase the efficiency of the HK022 Int recombination reaction, we plan to perform sequence consensus analysis (73)(74)(75) with structurebased examination of potential scaffold-optimizing mutations (76)(77)(78). This will help identify and manipulate critical residues that are important for the att sites' recognition and binding. Additionally, the correction of targeted mutated genes by Int-based RMCE approach in tissue cultures (ex vivo) and mice models (in vivo) will be performed using systemic treatment (72,79). Such an approach of mutated genes correction with the appropriate sequence may favor cured cells and even show increased efficiencies.
Off-target events interfere with successful gene targeting attempts and present a safety concern. Therefore, we performed a blast search for possible identical copies of the identified minimal 17 bp active 'attB' sequences in the human genome. The analysis of human 'attB' sites showed a varying number of repetitions within the human genome, ranging from a single copy to several copies. DMD2, DMD4, CTNS1 and CTNS4 have a single copy, on the other hand, CF12, DMD7, HEXA3, DMD5, DMD3, DMD6, CF10 and ATM4 all have additional copies (Supplementary Table S3). However, none of these additional copies of the paired sites are on the same chromosome, except for the single site of CF12 and one of the CF10 additional sites that are located on chromosome No.18. The distance between the two sites is 18,000 kb, deeming it irrelevant for a productive RMCE reaction. Despite the presence of additional sites, we successfully detected productive RMCE reactions even for the case that uses the ATM4 'attB' site out of 41 additional sites. Even though single-site integration is less likely to occur due to its kinetically favored reverse excision reaction (27), for future gene therapy applications, the selection of a pair of 'attB' sites is essential to avoid potential off-target effects. Therefore, we suggest choosing a pair of 'attB' sites, each having a single copy in the human genome, as in the case of CTNS, or ones that do not have additional sites on the same non-targeted chromosome.
In our previous publication (45), we tested Int-catalyzed off-target integration activity of various 'attP' sites in the E. coli genome by using a selection force. While all tested w.t. attP integrations occurred exclusively with their matching w.t. attB site, in 8.5% selected colonies of four different 'attP's (HEXA3 and 7, ATM4 and 8) integration were reported as off-targeted. However, we later realized that this assay did not take into consideration false-positive colonies. Thus, to re-examine this alleged off-target activity, we applied a more restrictive two-step assay (described in Materials and Methods and in Supplementary Figure S8). The assay confirmed a complete absence of off-target integration in the E. coli genome catalyzed either by w.t. or E174K Int using the same four 'attP' sites. This further demonstrates the low risk of off-target recombination based on 'attB' sites. In addition, the use of Integrase-based binary system as an anti-cancer treatment did not show any broad toxicity effects as known in alternative technologies in the lungs and spleen of the healthy mice. TUNEL assay that detects double strand breaks, did not show any DNA damage in the healthy mice tissue biopsies taken 45 days after systemic injections (twice a week) (79). These findings strengthen our claim that HK022 Int-based gene therapy is Interaction between the four N-terminal domains, sandwiched between two arm-type DNAs. I43 is located at the protein-protein interface. (C) D278 positioned close to the distal region of core-type DNA, that is, away from the center of the Holliday junction. (D) The position of lambda Int E174 in the linker between core-binding and catalytic domains, close to the DNA backbone. Note that E174K mutant was used in the crystallographic studies, and due to the flexibility of the Lys side chain that residues were modeled as an alanine. (E) Y342 covalently bonded to the cleaved DNA 3' end, and surrounding residues E309, E319 (R in HK022 Int), D336 (modeled as A due to side chain flexibility) from the Int's catalytic domain. a safe strategy. In the future, we aim to examine off-target effects by using unbiased, high-throughput assay as the linear amplification-mediated high-throughput genome-wide translocation sequencing (80), to detect and quantify DNA double-stranded breaks (DSBs) in the context of much larger human genome in mutated tissue culture and mice models. These experiments will help provide a better understanding regarding off-target activity.
The HK022 Int-RMCE approach based on native 'attB' sites that flank deleterious mutations along with improved variants of Int, is a promising human genome-editing and gene-therapy tool. Future studies of this approach will aim to further optimize HK022 Int-based RMCE efficiency in the human genome and demonstrate gene correction in appropriate mutated tissue cultures and in mice models using cDNA of the targeted gene. Since many human disorders arise from loss-of-function mutations, this strategy could be applied to a wide variety of diseases. Hence, presenting a universal therapeutic approach that could restore normal expression levels of any targeted gene, regardless of the specific deleterious mutation. This method holds the potential to treat diseases such as Cystinosis, Duchene muscular dystrophy and Cystic fibrosis that are caused by several mutations in the same gene (81)(82)(83). Furthermore, the Int-based RMCE addresses the insert size limitation; in the present work, we demonstrated its capability to exchange a human chromosomal fragment over 20 kb long. Moreover, this strategy allows a promoter-less stable transgene expression by relying on the endogenous promoter expression of the targeted gene as in the case of the DMD gene. This is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion (84) and oncogenesis (15). Here, we described a novel genome editing method that relies on the ability of HK022 Int to recombine with native RSs within the human genome. Thus, providing the basis for a promising gene therapy tool that is universal, easy to deliver, safe, stable and reliable.