Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice.

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.


Introduction
A human artificial chromosome (HAC)-based technology developed over the past two decades, represents a technology of engineering, a megabase-sized human artificial vector possessing the main features of a native chromosome, stable episomal maintenance in mammalian cells, high cloning capacity allowing accommodation of the megabase-size genomic loci insertions, and native gene expression [1,2]. In this regard, HACs have become the attractive vectors for gene therapy applications when the provision

Preparation of Metaphase Spreads
The metaphase spread was prepared as previously described [20,21,25]. Exponentially growing HAC-carrying CHO or iPSCs were treated for 4 or 12 h at 37 • C with 0.1 µg/mL Colcemid (Sigma-Aldrich) in a 5% CO 2 incubator. The cells were collected and incubated in hypotonic 0.56% KCl solution for 20 min. Then, the cells were fixed by a solution of methanol/acetic acid (3:1, v/v), washed, and stored in the fixative solution at −20 • C. The cell suspension was placed dropwise on the microscope glass slides (Superfrost; Thermo Scientific, Darmstadt, Germany), air-dried, and kept overnight at RT on air.

Southern-Blot Analysis
Southern-blot analysis with the use of a 32 P-labelled DNA probe was performed as described previously [20,21]. The genomic DNA (5 × 10 5 cells per sample) was cut by SpeI in an agarose plug. The digested CHEF DNA (CHEF Mapper, Bio-Rad Laboratories, Hercules, CA, USA) was separated on an agarose gel (5-250 kb range, 16 h run), transferred onto a nylon Amersham Hybond-N+ membrane (Thermo Fisher Scientific, Waltham, MA, USA), and hybridized with an alphoid tetO -HAC specific 201-bp YAC/BAC DNA probe. The DNA probe was PCR-amplified from the genomic DNA with the use of the 32 P-labeled dNTPs, and the 5 -GGGCAATTTGTCACAGGG-3 and 5 -ATCCACTTATCCACGGGGAT-3 primers. The blot membrane was pre-hybridized at 65 • C for 2 h in Church's buffer (7% SDS and 0.5 M Na-phosphate buffer) supplemented with 100 µg/mL salmon sperm DNA. The membrane was hybridized with heat-denatured 201-bp YAC/BAC DNA probe at 65 • C overnight. The blot was washed twice with 0.05% SDS, 2xSSC at RT for 10 min, and washed Cells 2020, 9, 879 5 of 17 thrice by two times each in 0.05% SDS, 2 × SSC at 60 • C for 5 min, with a reduced concentration of SSC in each subsequent wash (0.5 × SSC, 0.25 × SSC). The blot exposure was at −80 • C for 24-72 h.

Analysis of Mitotic Stability of the FVIII-alphoid tetO -HAC in iPSCs
The iPSCs containing the therapeutic HAC were cultivated in media supplemented with blasticidin (4 µg/mL) for 20 days. Then, the cells were grown without blasticidin for another 15 days. At day 0 and 15 after blasticidin withdrawal, the rate of EGFP-positive cells was assessed by flow cytometry, and the rate of HAC loss on the metaphase spreads was counted manually under microscopy 30 metaphase spreads were examined per each cell line. The daily loss rate of the HAC was calculated as previously described [36,37] applying the formula N 15 = N 0 × (1 − R) 15 where N 15 is the percent of metaphase spreads containing the HAC or EGFP-positive (for FACS assaying) cells after 15 days of blasticidin withdrawal, N 0 is the percent of metaphase spreads containing the HAC or EGFP-positive cells at day 0 of the blasticidin withdrawal, and R is the daily loss of the HAC.

Murine iPSCs Generation
The primary skin fibroblasts were obtained from the tail tip of FVIII Y/- [38], as previously described [25]. In brief, tissue was homogenized with a scalpel and treated with a solution of 1 mg/mL collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) in PBS at 37 • C for 15 min. After washing, the tissue was incubated for 20 min with a 0.05% trypsin/EDTA solution (Thermo Fisher Scientific, Waltham, MA, USA). The cells were cultured in 6-cm plates with MEF medium supplemented with fungizone (Invitrogen) at 37 • C, 5% CО 2 . The cells from 3-4 passages were used for reprogramming. The fibroblasts were seeded in gelatin-coated six-well plates with a cell density of 2 × 10 5 cells per well. On the next day, 600 µL of the MEF medium and 100 µL of each virus supernatant tetO-FUW-OSKM and FUW-M2rtTA (2.5 × 10 6 TU/mL) were added. After 6-8 h of incubation, 800 µL of fresh MEF medium was added to the cells. Cells were routinely cultured in mouse embryonic stem cells (MES) medium containing 5 µg/mL doxycycline (Sigma-Aldrich, St. Louis, MO, USA). The medium was replaced every other day; on day 4, the cells were trypsinized and cultured in 10-cm dishes pre-seeded with a mitomycin-inactivated mouse embryonic fibroblast. The iPSC clones were collected and expanded in the MES media without doxycycline.

Teratoma Formation and Histological Analysis
Teratoma analysis was performed as described previously [25,33]. Cells were trypsinized, washed with PBS, and then 10 6 cells were injected subcutaneously in the hindlimb of NUDE mice. After 3-4 weeks, mice were euthanized, and teratomas were removed and fixed in 4% paraformaldehyde diluted in PBS overnight at 4 • C. Teratomas were cut by pieces with a 5 mm diameter, and dehydrated in a series of ethanol concentrations: 70-80-96%, then with isobutanol and twice in xylene (each step was performed for 1 h at RT). The dehydrated pieces of tissue were incubated once in a solution of 50% paraffin: 50% xylene, and twice in 100% paraffin for 1 h at 56 • C. Paraffin pieces were then cut into 5 µm slices using a microtome Leica RM2235 (Leica Biosystems, Wetzlar, Germany). The paraffin slices were dried at 37 • C overnight on the microscope slides, then washed twice in xylene for 5 min at RT, and rehydrated in a series of ethanol: 96-80-70% for 3 min and in distilled water for 1 min. Next, sections were stained in hematoxylin for 5 min and washed in excess water for 10 min, then stained in eosin for 5 min and washed again in distilled water for 1 min. Sections were dehydrated in a series of ethanol: 70-80-96% for 3 min. Finally, the stained teratoma sections were incubated twice in xylene, mounted with Canadian balsam with a coverslip, and dried at 37 • C overnight. Teratoma histological sections were analyzed using the EVOS Cell Imaging Systems (Thermo Fisher Scientific, Waltham, MA, USA).
2.14. Fluorescence-Activated Cell Sorting (FACS) Assay of iPSCs qFACS assay was performed as described previously [20]. The alphoid tetO -HAC-FVIII-EGFP iPSCs were grown in MES medium in 6-cm dishes, trypsinized, washed with DMEM/F12, and resuspended in 1 mL of DMEM/F12. Cell sorting of the EGFP-positive and EGFP-negative hiPSCs was done using flow cytofluorimeter EPIX XL (Beckman Coulter, Pasadena, CA, USA). The sorted cells were collected in PBS, centrifuged, and seeded on six-cm plates covered with 0.1% gelatin.

Immunocytochemistry
The cells were grown in 48-well plates, washed with PBS, and fixed in 4% paraformaldehyde in PBS for 10 min [33,40]. The fixed cells were washed with PBS, permeabilized by 0,1% Triton X-100 solution in PBS for 15 min, and incubated with 3% bovine serum albumin (BSA) in PBS for 1 h at RT. The cells were incubated with mouse anti-Oct4 (1:500) (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-Nanog (1:1000) (Bethyl Laboratories, Montgomery, TX, USA). Antibodies were diluted in PBS solution with 0.1% Tween 20, 3% BSA overnight at 4 • C. After washing the cells with 0.1% Tween 20 in PBS, they were incubated with secondary antibodies in dilution of 1:1000: anti-mouse-Alexa647 and anti-rabbit-Cy3 conjugated (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at RT. The cells were washed and stained with DAPI in PBS (1:5000), then covered by PBS/sodium azide, and proceeded to an analysis by the EVOS Cell Imaging Systems (Thermo Fisher Scientific, Waltham, MA, USA).

Ethical Statement
All animal procedures were performed according to the guidelines for the humane use of laboratory animals, with standards corresponding to those prescribed by the American Physiological Society. The animal procedures were performed at the Institute of Cytology strictly in agreement with the animal protection legislation acts of the Russian Federation and were approved by the Animal Welfare Assurance of the Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia, received by Office of Laboratory Animal Welfare, NIH/OD/OER, Bethesda, Maryland, USA, the Assurance Identification number-F18-00380 (period of validity 12.10.2017-31.10.2022).

FVIII Targeting Vector Design
To introduce the FVIII gene into the alphoid tetO -HAC, we constructed a vector featuring a loxP site upstream of the 3 part of the HPRT gene for Cre recombinase-mediated integration ( Figure 1). Such integration leads to the restoration of the HPRT gene and acquisition of HAT resistance in HPRT-deficient hamster CHO cells, allowing clonal selection. The vector contains the FVIII gene under the control of the EF1α promoter and the EGFP gene under the CAG promoter. These two genes were inserted in a "head-to-head" orientation and were separated by the cHS4 insulator to minimize interference of the two strong promoters [41]. Besides, the FVIII and the EGFP genes have been flanked by the tRNA insulators to prevent their silencing by HAC centrochromatin [19] (Figure 1). The origin of replication and an ampicillin resistance element were also needed to maintain the targeting vector in bacterial cells. To introduce the FVIII gene into the alphoid tetO -HAC, we constructed a vector featuring a loxP site upstream of the 3′ part of the HPRT gene for Cre recombinase-mediated integration ( Figure 1). Such integration leads to the restoration of the HPRT gene and acquisition of HAT resistance in HPRT-deficient hamster CHO cells, allowing clonal selection. The vector contains the FVIII gene under the control of the EF1α promoter and the EGFP gene under the CAG promoter. These two genes were inserted in a "head-to-head" orientation and were separated by the cHS4 insulator to minimize interference of the two strong promoters [41]. Besides, the FVIII and the EGFP genes have been flanked by the tRNA insulators to prevent their silencing by HAC centrochromatin [19] ( Figure  1). The origin of replication and an ampicillin resistance element were also needed to maintain the targeting vector in bacterial cells. Figure 1. Scheme of construction of the alphoid tetO -HAC-factor VIII vector by Cre-recombinase mediated insertion of the therapeutic human clotting factor VIII DNA. After successful Cre-loxPmediated recombination of the donor vector with the HAC, the HPRT gene was reconstructed, allowing positive selection on the HAT medium. tetO-a tet operator site replacing the CENP-B box sequence. Bsr-the blasticidin resistance gene. White arrows with yellow stripe-alphoid monomers with the CENP-B box sequence. White arrows with a red stripe-alphoid monomers with tetO replacing the CENP-B box sequence. 5′HPRT-5′ and 3′HPRT-3′, according parts of the HPRT gene. The loxP sites are indicated by black arrowheads. The tRNA insulator indicated by a blue rectangle. cHS4-the insulator indicated by a light green rectangle. The FVIII cDNA -indicated by a red arrow. The EF1-alpha promotor (EF1-)-indicated by a yellow arrowhead. CAG promoter-indicated by a brown arrowhead. The EGFP gene-indicated by a green arrow). The ampicillin resistance element (Amp R )-indicated by a grey arrow. The origin of replication (Ori)-indicated by a purple arrowhead.

Recombinase-Mediated Insertion of the Targeting Vector into Alphoid tetO -HAC
The targeting vector was loaded into the HAC in HPRT-deficient CHO cells via Cre/loxrecombination ( Figure 1). Following the growth in the HAT medium, 15 HPRT-positive CHO clones Figure 1. Scheme of construction of the alphoid tetO -HAC-factor VIII vector by Cre-recombinase mediated insertion of the therapeutic human clotting factor VIII DNA. After successful Cre-loxP-mediated recombination of the donor vector with the HAC, the HPRT gene was reconstructed, allowing positive selection on the HAT medium. tetO-a tet operator site replacing the CENP-B box sequence. Bsr-the blasticidin resistance gene. White arrows with yellow stripe-alphoid monomers with the CENP-B box sequence. White arrows with a red stripe-alphoid monomers with tetO replacing the CENP-B box sequence. 5 HPRT-5 and 3 HPRT-3 , according parts of the HPRT gene. The loxP sites are indicated by black arrowheads. The tRNA insulator indicated by a blue rectangle. cHS4-the insulator indicated by a light green rectangle. The FVIII cDNA -indicated by a red arrow. The EF1-alpha promotor (EF1-α)-indicated by a yellow arrowhead. CAG promoter-indicated by a brown arrowhead. The EGFP gene-indicated by a green arrow). The ampicillin resistance element (Amp R )-indicated by a grey arrow. The origin of replication (Ori)-indicated by a purple arrowhead.

Recombinase-Mediated Insertion of the Targeting Vector into Alphoid tetO -HAC
The targeting vector was loaded into the HAC in HPRT-deficient CHO cells via Cre/lox-recombination ( Figure 1). Following the growth in the HAT medium, 15 HPRT-positive CHO clones were selected. PCR analysis revealed that all of them had the FVIII gene with the EF1α promoter. Twelve clones (Figure 2a) were devoid of the spontaneous Cre-recombinase gene insertions in their genome and, as confirmed by Western blot analysis, expressed the FVIII protein (Figure 2b). Four out of these 12 FVIII-expressing clones were next assessed by FISH analysis, which confirmed that all of them contained an autonomous HAC not integrated into the host chromosomes. One of these clones (clone #2) was used as a HAC donor for further experiments (Figure 2c,d).
Cells 2020, 9, x FOR PEER REVIEW 8 of 17 Twelve clones (Figure 2a) were devoid of the spontaneous Cre-recombinase gene insertions in their genome and, as confirmed by Western blot analysis, expressed the FVIII protein ( Figure 2b). Four out of these 12 FVIII-expressing clones were next assessed by FISH analysis, which confirmed that all of them contained an autonomous HAC not integrated into the host chromosomes. One of these clones (clone #2) was used as a HAC donor for further experiments (Figure 2c,d).

iPSC Generation from Fibroblasts of FVIII-deficient Mice
Originally, we had selected several recessive genetic disorders for disease modeling: hemophilia A, dysferlinopathy, and X-linked severe combined immunodeficiency (X-SCID), which are characterized by loss-of-function of the FVIII, dysferlin, and a gamma chain of the IL2 receptor, correspondingly. We have derived iPSCs for each of these mutants. However, in this study, we focused entirely on the hemophilia A gene therapy model. For the derivation of murine iPSCs, adult tail-tip fibroblasts from the FVIII Y/-mice [38] were infected with lentiviruses carrying the doxycyclineinducible polycistronic constructs encoding Oct4, Sox2, Klf4, and c-Myc reprogramming factors and rtTA activator [30,34]. After 16-20 days of reprogramming, 8-10 separate iPSC clones for each mutant were isolated and expanded. The clones were selected based on morphology, growth capacity, and uniform expression of the endogenous Oct4 and Nanog pluripotency markers (Figure 3a). The

iPSC Generation from Fibroblasts of FVIII-Deficient Mice
Originally, we had selected several recessive genetic disorders for disease modeling: hemophilia A, dysferlinopathy, and X-linked severe combined immunodeficiency (X-SCID), which are characterized by loss-of-function of the FVIII, dysferlin, and a gamma chain of the IL2 receptor, correspondingly. We have derived iPSCs for each of these mutants. However, in this study, we focused entirely on the hemophilia A gene therapy model. For the derivation of murine iPSCs, adult tail-tip fibroblasts from Cells 2020, 9, 879 9 of 17 the FVIII Y/mice [38] were infected with lentiviruses carrying the doxycycline-inducible polycistronic constructs encoding Oct4, Sox2, Klf4, and c-Myc reprogramming factors and rtTA activator [30,34]. After 16-20 days of reprogramming, 8-10 separate iPSC clones for each mutant were isolated and expanded. The clones were selected based on morphology, growth capacity, and uniform expression of the endogenous Oct4 and Nanog pluripotency markers (Figure 3a). The pluripotency of selected iPSC clones was confirmed based on their ability to develop teratomas containing three embryonic germ layers (Figure 3b). Two independent FVIII Y/-iPSC clones were used as HAC recipients.
Cells 2020, 9, x FOR PEER REVIEW 9 of 17 containing three embryonic germ layers (Figure 3b). Two independent FVIII Y/-iPSC clones were used as HAC recipients.

MMCT of the FVIII-Therapeutic HAC from CHO Cells in Mouse FVIII Y/-iPSCs
To transfer the assembled FVIII-alphoid tetO -HAC from CHO cells to iPSCs derived from FVIII Y/mouse (clone 2), we used a recently described retro-MMCT method [35] with some modifications [20,25]. The microcells were frozen and kept at −80 °C before fusion with FVIII Y/-iPSCs. Following the MMCT procedure, FVIII Y/-alphoid tetO -HAC-EF1-FVIII bearing cells were grown for one week in the medium containing blasticidin (2 g/mL). Blasticidin was withdrawn, which were then picked up and maintained without blasticidin. At the same time, we also obtained several alphoid tetO -HAC-CMV-FVIII-containing FVIII Y/-iPSC clones by the use of both conventional and retro-MMCT methods. These clones have the episomal HAC vector with FVIII transgene construct driven by the CMV promoter. However, even with early passages, none of these clones showed any FVIII protein expression, as seen by Western blot assay (not shown), indicating that the CMV promoter is not sufficient enough to drive transgene expression in this particular setting. In this regard, we selected only two FVIII Y/-alphoid tetO -HAC-EF1-FVIII (1 and 2) clones obtained by retro-MMCT for further analysis.

MMCT of the FVIII-Therapeutic HAC from CHO Cells in Mouse FVIII Y/-iPSCs
To transfer the assembled FVIII-alphoid tetO -HAC from CHO cells to iPSCs derived from FVIII Y/mouse (clone 2), we used a recently described retro-MMCT method [35] with some modifications [20,25]. The microcells were frozen and kept at −80 • C before fusion with FVIII Y/-iPSCs. Following the MMCT procedure, FVIII Y/alphoid tetO -HAC-EF1α-FVIII bearing cells were grown for one week in the medium containing blasticidin (2 µg/mL). Blasticidin was withdrawn, which were then picked up and maintained without blasticidin. At the same time, we also obtained several alphoid tetO -HAC-CMV-FVIII-containing FVIII Y/-iPSC clones by the use of both conventional and retro-MMCT methods. These clones have the episomal HAC vector with FVIII transgene construct driven by the CMV promoter. However, even with early passages, none of these clones showed any FVIII protein expression, as seen by Western blot assay (not shown), indicating that the CMV promoter is not sufficient enough to drive transgene expression in this particular setting. In this regard, we selected only two FVIII Y/alphoid tetO -HAC-EF1α-FVIII (1 and 2) clones obtained by retro-MMCT for further analysis.

Characterization of iPSC Clones Bearing the Alphoid tetO -HAC-EF1α-FVIII
Two FVIII Y/alphoid tetO -HAC-EF1α-FVIII clones obtained by retro-MMCT were positive for EGFP expression and, according to their expression of the pluripotency markers Oct4 and Nanog and ability to form teratomas containing all three embryonic germ layers, retained pluripotent properties (Figure 4a,b). The FISH analysis of both FVIII Y/alphoid tetO -HAC-FVIII clones confirmed that the HAC was not integrated into the host genome ( Figure 4c) and was maintained as an episomal unit during cell division. We assayed FVIII protein expression in these clones following their cultivation in the standard medium without blasticidin for five and 10 passages. The levels of FVIII protein expression were relatively stable in EGFP-positive cells, selected by flow cytometry during the selected time points ( Figure 5). The clones expressed the FVIII protein at a significantly lower level compared to the donor CHO cells. The derived FVIII Y/-iPSCs cells carrying the alphoid tetO -HAC-EF1α-FVIII will be further characterized and proceeded to the differentiation protocols to treat the hemophilia disorder in mice.
Cells 2020, 9, x FOR PEER REVIEW 10 of 17 ability to form teratomas containing all three embryonic germ layers, retained pluripotent properties (Figure 4a,b). The FISH analysis of both FVIII Y/-alphoid tetO -HAC-FVIII clones confirmed that the HAC was not integrated into the host genome ( Figure 4c) and was maintained as an episomal unit during cell division. We assayed FVIII protein expression in these clones following their cultivation in the standard medium without blasticidin for five and 10 passages. The levels of FVIII protein expression were relatively stable in EGFP-positive cells, selected by flow cytometry during the selected time points ( Figure 5). The clones expressed the FVIII protein at a significantly lower level compared to the donor CHO cells. The derived FVIII Y/-iPSCs cells carrying the alphoid tetO -HAC-EF1α-FVIII will be further characterized and proceeded to the differentiation protocols to treat the hemophilia disorder in mice.  probe (green) and the telomere PNA-TRITS probe (red). The alphoid tetO -HAC-FVIII is indicated by an arrow. Scale bar-10 μm, color-coded markers are indicated on the panels. To analyze whether or not the alphoid tetO -HAC-FVIII vectors remain structurally intact during the course of MMCT from hamster CHO cells to FVIII Y/-iPSCs, Southern blot hybridization was performed with the alphoid tetO -HAC containing genomic DNA of iPSC clones, digested by SpeI endonuclease (Figure 6). This nuclease cuts the input RCA/SAT43 vector-only once, having no recognition site in the alphoid DNA array of the alphoid tetO -HAC [17]. The original alphoid tetO -HAC carries 47 copies of the RCA/SAT43 vector utilized for synthetic alphoid DNA array assembly and propagation [16]. The enzyme digested genomic DNA was separated and hybridized with the probe specific to the tetO-alphoid DNA (see Section 2.5 for details). As seen from the Southern blot, a unique pattern of multiple identical bands of different sizes was observed after SpeI cut of the CHO and iPSCs DNAs of different clones, including the construct with CMV promoter (Figure 6). This indicates the absence of any detectable changes in the HAC structure (both for CMV and EF1 promoter constructs) after its MMCT mediated transfer into FVIII Y/-iPSCs.  To analyze whether or not the alphoid tetO -HAC-FVIII vectors remain structurally intact during the course of MMCT from hamster CHO cells to FVIII Y/-iPSCs, Southern blot hybridization was performed with the alphoid tetO -HAC containing genomic DNA of iPSC clones, digested by SpeI endonuclease (Figure 6). This nuclease cuts the input RCA/SAT43 vector-only once, having no recognition site in the alphoid DNA array of the alphoid tetO -HAC [17]. The original alphoid tetO -HAC carries 47 copies of the RCA/SAT43 vector utilized for synthetic alphoid DNA array assembly and propagation [16]. The enzyme digested genomic DNA was separated and hybridized with the probe specific to the tetO-alphoid DNA (see Section 2.5 for details). As seen from the Southern blot, a unique pattern of multiple identical bands of different sizes was observed after SpeI cut of the CHO and iPSCs DNAs of different clones, including the construct with CMV promoter (Figure 6). This indicates the absence of any detectable changes in the HAC structure (both for CMV and EF1α promoter constructs) after its MMCT mediated transfer into FVIII Y/-iPSCs. To analyze whether or not the alphoid tetO -HAC-FVIII vectors remain structurally intact during the course of MMCT from hamster CHO cells to FVIII Y/-iPSCs, Southern blot hybridization was performed with the alphoid tetO -HAC containing genomic DNA of iPSC clones, digested by SpeI endonuclease (Figure 6). This nuclease cuts the input RCA/SAT43 vector-only once, having no recognition site in the alphoid DNA array of the alphoid tetO -HAC [17]. The original alphoid tetO -HAC carries 47 copies of the RCA/SAT43 vector utilized for synthetic alphoid DNA array assembly and propagation [16]. The enzyme digested genomic DNA was separated and hybridized with the probe specific to the tetO-alphoid DNA (see Section 2.5 for details). As seen from the Southern blot, a unique pattern of multiple identical bands of different sizes was observed after SpeI cut of the CHO and iPSCs DNAs of different clones, including the construct with CMV promoter (Figure 6). This indicates the absence of any detectable changes in the HAC structure (both for CMV and EF1α promoter constructs) after its MMCT mediated transfer into FVIII Y/-iPSCs.  We noticed that after blasticidin withdrawal, some FVIII Y/alphoid tetO -HAC-EF1α-FVIII iPSCs became EGFP-negative, indicating HAC loss during cell division. This loss was previously observed in human iPSCs bearing the alphoid tetO -HAC-EGFP [20]. With this in mind, we compared HAC stability in these two mouse iPSC clones with the previously characterized mouse ESCs bearing the alphoid tetO -HAC-EGFP [25] by quantitative FACS and FISH analyses. In addition, we also checked the HAC mitotic stability in the iPSC-HAC-CMV-FVIII clone by FISH analysis. In agreement with the previous results, the alphoid tetO -HAC-EGFP was mitotically stable, and in our hands, it showed the daily loss rate-0.008 and 0.004 by FISH and FACS, correspondingly (Tables 1 and 2), which is consistent with the HAC stability in human HT1080 cells [16]. Interestingly, the daily loss rate of the alphoid tetO -HAC-EF1α-FVIII in both FVIII Y/-iPSCs clones was around 10 times higher (0.04-0.045 and 0.03-0.06, correspondingly) than that of the alphoid tetO -HAC-EGFP in ESCs (Figure 7 and Table 1). On the other hand, the mitotic stability of alphoid tetO -HAC-CMV-FVIII (daily loss rate-0.006) was comparable to the stability of alphoid tetO -HAC-EGFP (Table 2). In this regard, we assume that mitotic stability does not depend on the transgene. Isolation of more alphoid tetO -HAC-FVIII clones may allow the identification of the clone with the higher mitotic stability that would be suitable for gene therapy application. In addition, it is known that the mitotic stability of the HACs varies in different types of host cells [24,27,42]. We also cannot exclude that the lower mitotic stability can be associated with the method of HAC transfer because similar poor mitotic stability has been observed for HAC-EGFP human iPSC clones derived with the use of retro-MMCT method [20]. The variability in the mitotic stability of circular HACs [16,24,42] may be due to their ability to form dicentrics by sister HACs recombination. The dicentric HACs are very unstable. At present, we develop an isogenic lineal alphoid-HAC vector, that is likely more stable than the circular HAC due to the presence of the telomere sequences. If a linear HAC is stable in iPSCs, we will use it as a gene therapy vector in our future experiments. Further studies should clarify the cause of the low mitotic stability of the alphoid tetO -HAC in certain cells in order to better utilize the HAC for therapeutic applications. The HAC-based therapeutic model for treatment of hemophilia A was developed by Oshimura's group [9,10]. A top-down engineered 21HAC2 vector [43] carrying the CAG promoter-driving FVIII cDNA was constructed and maintained in hamster CHO cells and human MSCs. This HAC showed strong and stable FVIII transgene expression [9]. In another study, it was shown that FVIII Y/-iPSC-derived megakaryocytes/platelets carrying the megakaryocyte-specific platelet factor-4 (PF4) promoter-driven FVIII cDNA within the PF4-FVIII-HAC showed a sufficient level of FVIII secretion into the culture medium [10]. Multiple studies demonstrated that de novo synthetized HACs with different transgenes could be delivered to and functionally expressed in various human and murine cell lines [44][45][46][47], as well as successfully used for generation of transgenic mice [48][49][50][51]. Ito and colleagues reported successful treatment of nonalbumine rats by transplantation of immortalized hepatocytes using de novo HAC with the SV40T antigen [50,51]. The alphoid tetO -HAC featured a conditional centromere, which can be inactivated by the expression of tet-repressor (tetR) fusion proteins [16,17]. The structure of alphoid tetO -HAC has been completely defined, and this HAC is the best characterized among all types of HACs to date [17]. Besides its extensive implementation in various basic chromosome-related studies, this HAC is also being used in terms of its application in gene therapy modeling [15,18,27]. The alphoid tetO -HAC was examined for its capacity to carry two average-sized human genes, von Hippel-Lindau tumor suppressor (VHL) and NBS1 protein, to complement genetic deficiencies of cell lines lacking these genes [15]. Here, we have utilized the alphoid tetO -HAC for the development of gene therapy using the murine hemophilia A model. The FVIII cDNA was successfully inserted into the alphoid tetO -HAC vector, and then the HAC was transferred into mouse iPSCs derived from FVIII Y/mutant fibroblasts. Several features of the newly developed HAC-based therapeutic vector require further investigation to improve its quality towards higher mitotic stability and a stable transgene expression. Our results show, as a proof of principle, usage of the alphoid tetO -HAC vector in the HAC-based gene therapy model. The HAC-based therapeutic model for treatment of hemophilia A was developed by Oshimura's group [9,10]. A top-down engineered 21HAC2 vector [43] carrying the CAG promoter-driving FVIII cDNA was constructed and maintained in hamster CHO cells and human MSCs. This HAC showed strong and stable FVIII transgene expression [9]. In another study, it was shown that FVIII Y/-iPSCderived megakaryocytes/platelets carrying the megakaryocyte-specific platelet factor-4 (PF4) promoter-driven FVIII cDNA within the PF4-FVIII-HAC showed a sufficient level of FVIII secretion into the culture medium [10]. Multiple studies demonstrated that de novo synthetized HACs with different transgenes could be delivered to and functionally expressed in various human and murine cell lines [44][45][46][47], as well as successfully used for generation of transgenic mice [48][49][50][51]. Ito and colleagues reported successful treatment of nonalbumine rats by transplantation of immortalized hepatocytes using de novo HAC with the SV40T antigen [50,51]. The alphoid tetO -HAC featured a conditional centromere, which can be inactivated by the expression of tet-repressor (tetR) fusion proteins [16,17]. The structure of alphoid tetO -HAC has been completely defined, and this HAC is the best characterized among all types of HACs to date [17]. Besides its extensive implementation in various basic chromosome-related studies, this HAC is also being used in terms of its application in gene therapy modeling [15,18,27]. The alphoid tetO -HAC was examined for its capacity to carry two average-sized human genes, von Hippel-Lindau tumor suppressor (VHL) and NBS1 protein, to complement genetic deficiencies of cell lines lacking these genes [15]. Here, we have utilized the alphoid tetO -HAC for the development of gene therapy using the murine hemophilia A model. The FVIII cDNA was successfully inserted into the alphoid tetO -HAC vector, and then the HAC was transferred into mouse iPSCs derived from FVIII Y/-mutant fibroblasts. Several features of the newly developed HAC-based therapeutic vector require further investigation to improve its quality towards higher mitotic stability and a stable transgene expression. Our results show, as a proof of principle, usage of the alphoid tetO -HAC vector in the HAC-based gene therapy model.

Conclusions
The alphoid tetO -HAC represents a de novo synthesized, structurally defined, high capacity episomal vector for fundamental chromosome research and biomedical applications [14,17,[52][53][54]. In this study, we utilized this HAC to develop the FVIII-carrying therapeutic vector, then delivered this vector into mouse FVIII Y/-iPSCs and confirmed the stable expression of the transgene in these cells. Thus, the alphoid tetO -HAC is a suitable and powerful vector for its implementation in the human hemophilia A disease model in the mouse. Nevertheless, there are still several limitations of HAC-based vectors for gene therapy, which include a low efficiency of HAC transfer to different recipient cells, HAC mitotic instability, an insufficient level of transgene expression in some cells, low efficiency of HAC formation, the complex repeated DNA structure of the HACs, and challenges in amplification of the HAC vector DNA outside of the eukaryotic cells [18,26,27,55,56]. Further investigation is needed to improve HAC vectors for their biomedical therapeutic application.