Title : TALEN mediated gene targeting for CF gene therapy

Cystic Fibrosis (CF) is an inherited monogenic disorder, amenable to gene based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human CFTR minigene into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we have detected both CFTR mRNA and protein expression by qPCR and Wetern analysis, respectively. We have also confirmed CFTR function correction by flurometric Image Plate Reader (FLIPR) and iodide efflux assays. Taking together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.


INTRODUCTION
Cystic Fibrosis (CF) is an inherited autosomal recessive disease most commonly seen in the Caucasian population [1].CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.The most predominant mutation leads to the deletion of phenylalanine residue at position 508 [2].CF affects multiple organs and currently 80% of mortality is caused by lung failure [3].Conventional treatments including antibiotics, physical therapy and nutritional supplements can only alleviate the symptoms but does not provide an effective treatment for the disease.Over the past few years, new treatment strategies have been evolving; different types of CFTR channel modulators have been shown to be effective in improving channel activity in CF patients [4] [5][6] [7][8] [9].Most recently, a triple combinational therapy with VX-440, tezacaftor and ivacaftor has shown significant clinical benefits in an ongoing phase II trial [10].While substantial progress has been made in the development of CF therapies, current treatment strategies including the multiple channel modulator/corrector usage impose a heavy drug burden on patients.In addition, for a small portion of patients whose mutations lead to no CFTR protein production, there is no effective drug to slow down the disease progression.Furthermore, possibilities of drug interactions may increase the risk of adverse effects [11].
Gene therapy is an attractive strategy for CF lung disease because it treats the underlying cause of the disease rather than its symptoms.Gene replacement therapy for mutations involved in Leber's congenital amaurosis, choroideremia, achromatopsia, and retinitis pigmentosa using AAV vectors has showed correction to various degrees [12][13] [14].Many CF gene therapy trials using adenoviral (Ad) vector, adeno-associated viral (AAV) vector, liposomes as vehicles for delivery have been carried out since early 1990s, but none showed significant improvements in lung function [15][16] [17] [18].Nonviral vectors, such as liposomes, in general are not efficient in gene delivery while viral vectors have other problems [19].For example, AAV vectors have been successfully used in eye gene therapy [13], but it has a small DNA carrying capacity and thus not suitable for delivering CFTR gene expression cassette.For Ad vectors, one major obstacle is host immune responses elicited by the vectors, which eliminate the vectortransduced cells [20].To reduce the host immune responses, a helper-depended adenoviral vector (HD-Ad) vector was developed by deleting all viral coding sequences.Thus, this type of vector has a large DNA-carrying capacity up to 36 kb in addition to advantages in reduction of host immune responses [21] [22].Using HD-Ad vectors, our group has demonstrated efficient vector delivery to airway epithelia of mice and pigs with limited immune responses [22][23] [24].More recently, we showed effective delivery of HD-Ad vectors to airway basal cells of mice and pigs [25].
Engineered transcription activator-like effector nucleases (TALENs) are highly efficient in generating double-stranded breaks in cell genomes, which facilitate gene editing via the homology-directed recombination (HDR) process [26].TALENs bind and cleave their target DNA as heterodimers.Each TALEN monomer is composed of a FokI nuclease fused to a customizable DNA-binding domain [26].TALEN has been applied in gene editing both in vitro and in vivo [27][28] [28].Unlike the more popular CRISPR-cas9 system that recognizes a 20 bp target [29], TALEN recognition sequences can be designed longer to provide better specificity and they are commonly designed to be 34 bp in length [30] [31].
We have engineered a pair of TALEN that target the human AAVSI locus with assistance from Dr. Bo Zhang group at Peking University, China.Taking the advantage of the large DNA carrying capacity of the HD-Ad vector, we have used it to deliver both TALENs and donor DNA together to target cells at high efficiency.We demonstrate that a LacZ reporter donor gene expression cassette can be successfully integrated into the AAVS1 locus.Furthermore, we have also shown permanent integration of a CFTR minigene can be achieved cultured airway epithelial cells.These findings collectively demonstrated feasibility of using TALENs for future targeted CFTR gene editing in vivo.

Co-delivery of TALEN and donor DNA using HD-Ad vectors
The human AAVS1 locus has been extensively explored for targeted gene integration studies as it leads to abundant transgene expression with no adverse consequences [32] [33].To test if large gene constructs, such as the CFTR expression cassette [34], can be integrated into this locus, we first tested integration of a LacZ reporter gene expression cassette (Figure 1).For homologydirected gene targeting, homology arms of 4 kb in length were added to either end of the LacZ gene expression cassette (Figure 1A, Supplementary Figure 1).The LacZ HD-Ad vector that harbors both the donor and TALEN genes was produced as described [35].As shown in Fig. 1B, the transduced human IB3-1 cells showed abundant transgene expression.The TALEN cleavage efficiency was evaluated using the T7E1 assay (Figure 1C).It was around 36% when cells transduced at 50 MOI or 66% when cells transduced at 100 MOI.

Analysis of the LacZ reporter gene integration into the AAVS1 locus
Junction PCR assays indicated precise on-target integration of the LacZ reporter gene into the AAVS1 locus (Figure 2A).Junction PCR products were further subjected to analyses by restriction enzyme (RE) digestion and Sanger sequencing (Figure 2A), both of which further confirmed the identity of junctional PCR products.To evaluate potential off-target cleavage activity of LacZ integration vector, T7E1 assays were performed for the top three potential offtarget locations (Figure 2B).Results indicated that there was no detectable off-target activity in transduced cells.
To determine the integration efficiency of LacZ vector, transduced cells were first continuously passaged for 18 generations (Supplementary Figure 2).Passaging was implemented to dilute out residual vector genomes in transduced cells.The LacZ gene integration efficiency was determined by staining for percentage of LacZ-positive cells at passage 18 (Figure 3A,B).The addition of a DNA ligase VI inhibitor, SCR7, has been shown to enhance gene integration by promoting cellular repair via HDR pathway [36].In line with this finding, we have also observed an increase of 1.5 fold in integration efficiency with SCR7 treatment (Figure 3C).To verify the gene integration efficiency with an independent method, Imagene Green substrate was used to fluorescently label LacZ positive cells at passage 18, and percentage of LacZ positive cells was quantified by flow cytometry (Figure 3D).The integration efficiency determined from LacZ staining was 3% for 50 MOI transduction, 5% for 100 MOI transduction; while flow cytometry showed higher integration efficacy of 6.37% for 50MOI transduction and 8.23% for 100MOI transduction.This discrepancy of integration efficiency seen from two LacZ quantifying methods may have been caused by low level of background substrate reaction since the negative control (NC) from substrate reaction also showed 1.48% of fluorescent cells (Figure 3C).

Integration of CFTR gene into the AAVS1 locus allowed persistent CFTR expression and
presence of CFTR channel activity.For CFTR gene integration, an HD-Ad vector that contains CFTR expression cassette [34] flanked by homology arms in addition to TALEN genes was constructed and produced (Figure 4A, Supplementary Figure 1).Following transduction, CFTR protein was expressed in transduced IB3-1 cells (Figure 4A, B, Supplementary Figure 3).Junction PCR on either end of the integration site showed positive products, indicating integration was at the precise location (Figure 4C).PCR product was validated by restriction enzyme digestion and Sanger sequencing (Figure 4C).We further examined CFTR mRNA expression at multiple time points after transduction by qPCR, and found that the CFTR mRNA level was significantly higher in cells transduced with the integration vector than that in cells transduced with the control (nonintegrating) vector (Figure 5A).More importantly, CFTR protein was detectable at passage 6 and passage 12 for cells transduced with CFTR integration vector; whereas cells transduced with non-integrating vector failed to show detectable CFTR protein expression at passage 12 (Figure 5B).
To assess the level of CFTR channel activity, we performed flurometric Image Plate Reader (FLIPR) assays in 96 well-plates with cells treated with CFTR integration vector.Results indicated significant CFTR channel activity both at initial transduction and at passage 12 (Figure 5C).This result was consistent with CFTR channel activity measured using iodide efflux assay (Supplementary figure 4).Looking at these findings, it is evident that our vector can achieve effective integration of the CFTR expression cassette into the AAVS1 locus and allow persistent CFTR expression at mRNA, protein, and functional level.Because TALEN is a foreign protein and is not naturally expressed in humans, its prolonged expression in hosts may cause unwanted antigenic responses.We therefore examined TALEN expression over time in cells transduced with CFTR integration vector.We first monitored the GFP fluorescence level following vector transduced for 12 days and noticed significant decline in GFP fluoresce after day 3 (Figure 6A).We then performed qPCR and Western blotting to measure TALEN expression in transduced cells at multiple time points following integration vector transduction.Results showed that TALEN protein and mRNA expression was undetectable after 5-6 passages (Figure 6B, C, Supplementary Figure 5).In addition, the relative quantity of HD-Ad vector genome measured by qPCR also gradually declined and was undetected after 6 passages (Figure 6B, Supplementary Figure 5).These results collectively indicate that although high level of TALEN expression can be achieved upon initial administration of gene integration vector in cells, its expression was lost gradually in transduced cells.Hence, we conclude that the HD-Ad integration vector does not show prolonged presence of TALEN or vector in transduced cells.

DISCUSSION
In this study we showed insertion of large 8 kb gene constructs into the human AAVS1 locus can be achieved at an efficiency of 5%.Because of the large carrying capacity and high transduction efficiency, both TALEN and donor genes can be simultaneously delivered by a single vector.
This method of co-delivery of donor and gene editor cannot be achieved by other viral vectors due to their limited DNA carrying capacity.In addition, our laboratory has also demonstrated that HD-Ad vectors can target the residential progenitor cells in the airway system [25].This will allow us to explore the possibility of CFTR gene editing in vivo in progenitor cells for sustained CFTR gene expression in the airway system in our future work.
In gene editing studies, one of the crucial safety criteria is the targeting precision.Off-target cleavage at essential genes may cause deleterious consequences.Studies revealed small point mutations such as substitutions and deletions caused by DSB may allow cell transformation into an oncogenic nature [37][38].The challenge is faced by all the endonuclease-mediated gene editing systems, including, zinc fingers, TALENs and CRISPRs [39].The CRISPR-cas9 system has been used widely as a versatile tool for gene editing [40].Compared to the CRISPR-cas9 system which shows off-target effects due to the tolerance for mismatched binding of its short 20 nt recognition sequence [40], TALEN recognition requires a longer binding sequence which minimizes the chance of off-target cleavage [41].Genome wide off-target studies by looking for cleavage at sequences similar to on-target site revealed lower off-target cleavage for TALEN than for CRISPR-cas9 system [42][43][44] [45].Technical advancements in the field will likely improve the safety profile of all these gene editing systems.
We chose to integrate the CFTR expression cassette into the AAVS1 locus, instead of editing a CFTR mutation as our strategy for CFTR gene correction because this approach is not mutationspecific and it is applicable to correcting all CFTR mutations.We selected the AAVS1 locus for the test because it is known that transgenes integrated in this locus can be efficiently expressed.
However, future work should consider targeting the CFTR locus with this approach since integration of a CFTR expression cassette in the locus can stop the mutant CFTR gene expression.This may be considered an advantage over gene integration into a non-CFTR locus.The efficiency of site-specific gene integration is important for achieving therapeutic effects in gene therapy.SCR7 treatment has been used previously by other groups to increase CRIPSR-cas9 mediated integration efficiency.We tested different concentrations of SCR7 for its effect on efficiency of gene integration.We did see an increase in integration efficiency on TALEN mediated gene editing although the effect was not as significant as mentioned previously by other groups [36][46].One reason could be the difference in gene editors utilized as other groups mainly applied SCR7 in combination with the CRISPR-cas9 system.Another reason could be the difference in the cell lines used.We are currently working on enhancing the efficiency of CFTR gene integration.We plan to explore the possibility of combining protein factors [47] that facilitate the HDR pathway with TALEN integration vectors.
In this study, we examined the time course of TALEN expression in transduced cells.Since TALEN was derived from a pathogenic bacterial species from plant Xanthomonas (genus) [30], we worry that it may cause antigenic responses in host, which are a significant problem seen in studies of in vivo gene therapy [21].To our favor, this study has shown a rapid clearance of TALEN in the transduced cells, examined at levels of mRNA and protein.This greatly limits the chance of host immune responses that may cause elimination of gene corrected cells.Taking all of these together, our study demonstrated successful TALEN-mediated gene correction in a human CFTR mutant cell line as well as rescue of CFTR channel activity.This provided new insights in TALEN-mediated gene editing, and opened opportunity for further enhancing the efficiency and performance in future studies.

Plasmid constructs
TALEN plasmids were generated by Dr. Bo Zhang's lab using the Unit Assembly method [27].
The  The same methods as described above were used to add the 4 kb homology arms to the K18CFTR expression cassette carried by pBSII-SK(+) backbone plasmid.Similarly, the expression cassette and the homology arms were cloned into pC4HSU backbone (NotI/SalI).The DNA fragment for TALEN expression was inserted to the pC4HSU-K18CFTR-L4-R4 by AscI digestion and ligation.The resulting plasmid was denominated as pHD-Ad-K18CFTR-TALEN.

Vector production
HD-Ad-UBCLacZ-TALEN and HD-Ad-K18CFTR-TALEN vectors were produced as previously described [35].Briefly, HD-Ad vectors were packaged by transfecting 116 producer cells with the linearized pHD-Ad vector and tansduced with NG163 helper virus.In 116 cells, HD-Ad vectors were amplified by serial passage and then purified by CsCl density gradient ultracentrifugation.Viral particle numbers of HD-Ad preparations were determined by spectrophotometry.

Transduction
IB3-1 cells were seeded in 6-well dishes and cultured until 70% confluency.Before transduction, cells were washed with pre-warmed PBS, pH 7.4.HD-Ad vectors were added to the cells at 50 MOI and 100 MOI in 0.5 mL of serum-free media.Cells were incubated for 1 hour before adding pre-warmed media to make a final volume of 2 ml per well.Transduced IB3-1 cells were cultured for 5 days before the first passage.After the first passage, the cells were split twice per week at a ratio of 1:7.

Off-target analysis
Top 3 off-target sites in the human genome were predicted by the TALEN offer programme (http://galaxy.informatik.uni-halle.de/root?tool_id=TALENoffer). PCR primers were designed to amplify regions around the predicted sites.Primers sequences and the corresponding length of the amplicons are: 5' GTA CAG CAA TCT AAG GAA GTA GAC TCT TAG G 3' (forward primer) and 5' GTT TCA CTA TGT TGG TCA GGC TGC TC 3' (reverse primer) for off-target site #1 (738 bp), 5' GTC TGA TTG TGC AGG TTG TGT AGG ATC 3' (forward primer) and 5' CAC CCA AGT GCT GAC CTT ACT GC 3' (reverse primer) for off-target site #2 (687 bp), and 5' CAG CAG TCA GGG TTG TTC AGT TTG TTC 3' (forward primer) and 5' CCT AAA CCT TGG TGG CTA AAC ACA GTA AAA G 3' (reverse primer) for off-target site #3 (763 bp).PCR products at the three selected sites were then analyzed using T7E1 assays as described in the previous section.
Treated cells were cultured until day 5 after the transduction and passaged as normal.

β-galactosidase staining for integration efficiency
Transduced IB3-1 cells were washed with PBS, pH 8.0 and fixed in 0.5% glutaraldehyde in PBS for 15 min.After PBS washing, β-galactosidase staining solution (0.1% X-gal, 2 mM MgCl2, 5 mM K-ferricyanide, 5 mM K-ferrocyanide in PBS) was added to cover the cell monolayer.Cells were incubated at 37°C overnight in dark.To terminate the reaction, the staining solution was removed from the dish and the cells were washed with PBS.
To monitor the transduction efficiency, IB3-1 cells were stained 3 days post-transduction.To assess the integration efficiency, IB3-1 cells were passed for 18 passages before staining.Twenty images were taken randomly for each well under a bright field microscope at 100x magnification.
The number of LacZ positive cells and the total number of cells in each image were recorded with ImageJ.The transduction and integration efficiencies were calculated according to the following formulas: % transduction = 100 x sum of LacZ positive cells from all 20 images / sum of total number of cells from all 20 images; % integration = 100 x sum of LacZ positive cells from all 20 images / (sum of total number of cells from all 20 images x transduction efficiency).

Single cell colonies
Transduced IB3-1 cells were cultured for 12 passages before single cell sorting.Upon 70% confluency, the cells were suspended in ice-cold PBS, pH 7.4 containing 1% FBS.The cells were incubated on ice in the dark with 7-AAD (BioLegend, San Diego, CA) at a concentration of 100 ng per million cells for 10 minutes.Single cells were sorted directly into 96-well plates containing 20% DMEM medium supplemented with 20% FBS (MoFloXDP BRV/UV).Sorted cells were cultured for 2 weeks until assaying.cDNA were measured as the internal control using primers 5' GTT CGA CAG ACA GCC GTG TG 3' (forward primer) and 5' ATG GCG ACA ATG TCC ACT TTG C 3' (reverse primer).

Flow cytometry
Relative hCFTR mRNA expression was calculated as expression = 2 -CT .

CFTR Immunofluorescence
IB3-1 cells were seeded onto collagen-coated round glass cover slips in 6-well plates 3 days prior to staining.At confluency, cells were washed in PBS, pH 7.4, fixed in ice-cold methanol for 10 minutes, and permeabilized with 0.5% Triton-100 in PBS.Cells were then blocked for 1 hour in block solution (5% goat serum, 0.5% BSA, 0.05% Triton-100 in PBS).Cells were incubated with mouse monoclonal antibodies against the human CFTR R-domain MAB1660 and C-terminus MAB25031 (R&D Systems, Minneapolis, MN) at 1:500 dilution overnight at 4°C, washed in PBS with 0.05% Triton-100, and then incubated in dark for 1 hour in 1:750 diluted CF555 goat anti-mouse IgG (H+L) (Biotium, Fremount, CA).After washes in dark, cells on the glass slips were taken out from the 6-well plates, and mounted with one drop of VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlington, ON).

Protein isolation and CFTR Immunoblot
IB3-1 cells were gently washed with ice-cold PBS, pH 7.4 then scraped from the bottom of the plate with a cell scraper in 1 mL of ice-cold PBS and centrifuged at 4°C separation, primary and secondary antibody probing, and chemi-fluorescence detection, was done automatically by the JESS system (ProteinSimple, San Jose, CA) under 5 hours.Results from 24 samples were displayed and analyzed using the Compass analysis software.

Membrane potential assay
Protocol for membrane potential assay was adapted from Ahmadi et al [51].Cells were seeded to black-walled, clear-bottom 96-well plates and cultured until 100% confluency.The blue membrane potential dye (Molecular Devices) was dissolved in a chloride-free buffer (150 mM NMDG-gluconate, 3 mMKCl, 10 mM HEPES, pH 7.35, osmolarity 300 mOsm) and loaded to the cells.The plates were incubated at 37°C with 5% CO2 and humidified air for 30 minutes, then transferred to the i3 multi-well microplate reader (Molecular Devices, San Jose, CA).
Eleven baseline reads were made, followed by addition of 2.5 L forskolin per well.After adding the drug, 31 reads were made.Then, 21 scans were made after the reaction was terminated by addition of 10 M CFTRinh-172.For negative controls, 2.5 microliter of DMSO was added instead of forskolin.

Statistical analysis
Unpaired student's t-test was used for the comparison between means of two groups.A p value less than 0.05 was considered statistically significant.Error bars were shown in standard error of the mean (SEM).Off-target analysis for TALEN integration vector.Top 3 potential off-target sites were provided using TALENoffer (http://galaxy.informatik.uni-halle.de/root?tool_id=TALENoffer). T7E1 assays for IB3-1 cells transduced with 100MOI of HD-Ad-UBClacZ-TALEN vector were RVDs HD-HD-HD-HD-NG-HD-HD-NI-HD-HD-HD-HD-NI-HD-NI-NN-NG and NG-NG-NG-HD-NG-NN-NG-HD-NI-HD-HD-NI-NI-NG-HD-HD-NG recognize a region in the AAVS1site within the human chromosome 19 with a 15-nucleotude spacer.Generation of the UBCLacZ expression cassette was as described in the previously[49].Left and right homology arms (4kb) were amplified from human cell line A549 (ATCC CCL-185), and cloned into the UBCLacZ vector by in-fushion cloning (Clontech).The UBCLacZ expression cassette with homology arms was cloned into an HD-Ad backbone plasmid pC4HSU (NotI/SalI)[50].The PmeI sites on the resulting vectors were switched to PacI sites using infusion cloning.The assembled UBCLacZ plasmid pC4HSU-UBCLacZ-L4-R4 was linearized with NheI followed by blunt end treatment with T4 DNA polymerase (New England Biolabs, Whitby, ON), then digested with NsiI.The DNA fragment for TALEN expression was cleaved and ligated using FspI and Nsilsites.The resulting vectors were denominated as pHD-Ad-UBCLacZ-TALEN.

Figure 2 .
Figure 2. Characterization of site-specific gene integration.A. Identification of the integration

Figure 5 .
Figure 5. Analysis of CFTR mRNA and protein expression, and channel function.A. qPCR

Figure 6 .
Figure 6.Disappearance of TALEN expression and vector genome over time.A. GFP