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Stem Cells 2004;22:324-333 www.StemCells.com
© 2004 AlphaMed Press

Transfer and Stable Transgene Expression of a Mammalian Artificial Chromosome into Bone Marrow-Derived Human Mesenchymal Stem Cells

S. Vanderbyl, G. N. MacDonald, S. Sidhu, L. Gung, A. Telenius, C. Perez, E. Perkins

Chromos Molecular Systems Inc., Burnaby, British Columbia, Canada

Key Words. Mammalian artificial chromosomes • ACEs • Gene therapy • Cell therapy • Mesenchymal stem cells

Sandra Vanderbyl, MSc, Chromos Molecular Systems Inc., 8081 Lougheed Highway, Burnaby, British Columbia, Canada V5A 1W9. Telephone: 604-415-7100; Fax: 604-415-7151; e-mail: svanderbyl{at}chromos.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammalian artificial chromosomes (ACEs) transferred to autologous adult stem cells (SCs) provide a novel strategy for the ex vivo gene therapy of a variety of clinical indications. Unlike retroviral vectors, ACEs are stably maintained, autonomous, and nonintegrating. In this report we assessed the delivery efficiency of ACEs and evaluated the subsequent differentiation potential of ACE-transfected bone marrow-derived human mesenchymal stem cells (hMSCs). For this, an ACE carrying multiple copies of the red fluorescent protein (RFP) reporter gene was transferred under optimized conditions into hMSCs using standard cationic transfection reagents. RFP expression was detectable in 11% of the cells 4–5 days post-transfection. The RFP-expressing hMSCs were enriched by high-speed flow cytometry and maintained their potential to differentiate along adipogenic or osteogenic lineages. Fluorescent in situ hybridization and fluorescent microscopy demonstrated that the ACEs were stably maintained as single chromosomes and expressed the RFP transgenes in both differentiated cultures. These findings demonstrate the potential utility of ACEs for human adult SC ex vivo gene therapy.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recent events in gene therapy applications have driven the development of vectors that maintain stable expression of therapeutic genes without the risk of cell transformation or the stimulation of the host immune system [1]. However despite advances, current eukaryotic viral vector systems remain problematic. For example, integration into the host chromosome by retroviral-based vectors may lead to variegated gene expression, insertional mutagenesis, and oncogenesis [2–4]. Similar concerns have also been raised regarding the integration of adeno-associated virus vectors and their association with chromosomal deletions and other rearrangements that are frequently located on human chromosome 19 [5]. Furthermore, it has been demonstrated that retroviruses preferentially integrate near transcription start regions thus raising the likelihood of insertional mutagenesis with retrovirus-based vectors [6]. Transient expression of therapeutic products is also a concern. Most notably, using nonintegrating vector systems such as adenoviral-based vectors may elicit a host immunological response to the vector, including death [7].

Artificial chromosomes (ACEs) are being investigated as an alternative to current eukaryotic viral vector systems. Hadlaczky and colleagues developed a unique methodology to construct mammalian ACEs through the induction of large scale amplifications of "satellite" DNA sequences in pericentromeric heterochromatin [8, 9]. De novo centromeres and dicentric chromosomes were formed upon the integration of exogenous DNA sequences into the specific regions of acrocentric chromosomes—those containing both pericentromeric heterochromatin and the tandemly repeated ribosomal genes (rDNA). Ensuing breakage during mitosis generated new chromosomes ranging in size from 10 to 360 megabases [10–12]. Considering that these ACEs, termed satellite DNA-based artificial chromosomes (SATACs), can be manipulated or engineered to contain a variety of exogenous sequences, they present a unique opportunity for a variety of applications, including gene therapy [13, 14]. SATACs have recently been engineered to contain multiple recombination acceptor sites in order to facilitate the insertion of desired transgenes onto the ACEs. These engineered SATACs are commonly referred to as Platform ACEs or ACEs.

ACEs are safe, stable systems, and many numerous reviews have addressed their utility. In addition to ease of loading genes onto Platform ACEs, they can be isolated and stably transferred. This further expands their utility. As part of the overall ACE System, multiple transgenes (cDNAs or larger genomic encoding regions) can be efficiently targeted onto the Platform ACE through site-specific DNA recombination [15]. Platform ACEs and transgene-loaded ACEs can be isolated by flow cytometry to high purities (>95%) and yields (>1,000,000 ACEs/hr) [16] and can be transferred in vitro into a variety of mammalian cell lines and primary cells by cationic lipids and dendrimers [17, 18]. ACEs can also be used to generate transgenic mice through microinjection of purified ACEs into fertilized oocytes [19]. Stability is exemplified by the fact the resulting transgenic mice were shown to stably maintain the ACE through four generations of the germline [20].

Human mesenchymal stem cells (hMSCs) have emerged as a promising strategy for ex vivo gene therapy [21–24]. Improvements in isolation, in vitro cultivation of hMSCs [25–27], and in vivo studies on differentiation into functional cells of multiple tissue types [28] make them attractive delivery vehicles for autologous cell-mediated gene therapy. Characteristics that make them appealing cell hosts include ease of purification from clinical bone marrow (BM) aspirates (frequency of 1/105) and the ability to culture and expand hMSCs in vitro without apparent loss of differentiation potential into multiple cell types [26]. Additionally MSCs have been shown to confer immune privilege by not presenting alloantigens and by suppressing T-cell lymphocyte proliferation [29–31]. These features may enable a cellular therapy scenario whereby hMSCs can be derived from a "universal" donor (irrespective of their major histocompatability complex haplotype), expanded, and prepared as an "off-the-shelf" reagent.

hMSCs are being evaluated as cellular delivery vehicles for therapeutic genes for a variety of clinical indications. This list includes the replacement of genes to correct acquired or inherited disorders of bone, muscle, or cartilage by transfer of genes encoding bone morphogenic proteins -2, -4, -9 [21, 32–40], the transfer of genes encoding the blood coagulation factors VIII and IX for the treatment of hemophilia [41–47], human growth hormone [46, 48], insulin-like growth factor 1 [49], and interleukin-3 [50]. This list also includes: erythropoietin [51], {alpha}-l-iduronidase for treatment of mucopolysaccharidosis type I [52], pro{alpha}2 collagen (I) for treatment of osteogenesis imperfecta [53], sox9 [54] to enhance chondrogenesis, and interferon to treat tumors [55]. In several of these studies retroviral-based vectors were utilized, thereby increasing the risk of inducing insertional mutagenesis. The risk of insertional mutagenesis can be reduced prior to patient treatment by physical screening of selected recombinants for vector integration into genetically safe regions; however, this approach is problematic when transducing committed stem cells (SCs) with limited cell doublings. Furthermore, the integrated vectors often result in pronounced transgene silencing leading to short-term therapeutic gain. Theoretically, the ACE System would overcome these issues as ACEs are nonintegrating and can be engineered to optimize long-term transgene expression.

Cell therapy regimes are restricted by the ability to target host cells, the expression and stability of the inserted constructs, the delivery efficiency of vectors, and safety. When considering the application of ACE-based cell therapies in the clinic, a limitation that requires further investigation is the delivery efficiency of protein-DNA complexes. When compared to viral vectors, which can reach transduction efficiencies of 100%, the transfer efficiency of the ACEs is at least an order of magnitude lower [18]. Therefore, strategies that facilitate the enrichment of cells harboring a transferred ACE must be simultaneously developed. To this end, the use of human cell surface or drug selectable marker genes is being investigated as a means to enrich for the ACE-containing cells within a population.

In addition to the enrichment and ex vivo delivery strategies for ACE-containing cells, we are currently initiating animal studies to investigate the potential for application of ACE-based cell therapies in disease models along with the relative safety and tolerability of these modified cells. With respect to the latter, current data suggest that ACEs are mitotically and meiotically stable, nontumorigenic, and nonmutagenic, as transgenic mice containing an ACE were phenotypically normal, as were their offspring through four generations [19, 20]. Although these observations will need to be substantiated in the context of cellular therapies, our current plans include these and additional cellular assays to determine cell fate, stability of the ACE, duration of transgene expression, and immune response. Current animal studies focus on the utility of ACE-modified MSCs in a small animal model of disease that, if successful, will represent the first demonstration of a therapeutic benefit using a chromosome-based ex vivo cell therapy.

In this work we evaluated the ability of BM-derived hMSCs to be genetically modified with ACEs expressing the red fluorescent protein (RFP). An intact ACE chromosome was introduced into the hMSCs, and reporter gene expression was detected in the transfected hMSCs. hMSCs containing the ACEs maintained their multipotential capacity as evidenced by their ability to be induced to differentiate along osteogenic and adipogenic lineages. Furthermore, RFP expression was detected in the differentiated cells suggesting that the ACE chromosomal environment is well suited for transgene expression in differentiating SC populations. To our knowledge this is the first report of the stable expression of a transgene introduced into human adult SCs by mammalian ACE. This finding demonstrates the potential utility of the ACE technology for human adult SC ex vivo gene therapy and provides a novel approach for a broad range of tissue engineering applications.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of RFP-ACEs
For this study, an ACE expressing the RFP (RFP-ACE) was utilized. The construction and targeted integration of DNA encoding the RFP gene on an ACE1 platform has been described previously [15]. For bulk production of chromosomes prior to transfection, RFP-ACEs were isolated from a murine LMTK host production cell line and purified by flow cytometry as described previously [16].

Transfection of hMSCs with RFP-ACEs
After the harvesting and purifying of the RFP-ACEs from the production cell line, RFP-ACEs were transferred into recipient hMSCs using commercially available transfection reagents. A purified culture of hMSCs (CD105+ CD166+ CD29+ CD44+ CD14– CD34– CD45–) was purchased from a commercial source and expanded for no more than three passages in defined medium according to the manufacturer’s protocol (Mesenchymal Stem Cell Growth Media [MSCGM]; Cambrex Bio Science; Walkersville, MD; http://www.cambrex.com). ACE transfection procedures have been previously described [17, 18]. Briefly, approximately 17–48 hours prior to transfection exponentially growing cells were trypsinized and plated at 200,000 cells per well into six-well dishes. One million RFP-ACEs (in a volume of 800 µl) were combined with either 5–8 µl of SuperfectTM (Qiagen) or 8–12 µl of LipofectAMINETM PLUSTM (Invitrogen; Burlington, ON, Canada; http://www.invitrogen.com). The RFP-ACEs transfection mixtures were allowed to complex for 10–15 minutes at room temperature and then applied to the hMSCs (~5 x 105 cells/well) in 6-well dishes and incubated for 3–5 hours at 37°C in a 5% CO2 incubator. Recipient cells were incubated for 24 hours at 37°C in a 5% CO2 incubator and then passaged into 10-cm dishes.

FISH Analysis of ACE-transfected hMSCs
In order to cytogenically assess the ACE-transfected hMSC population, fluorescent in situ hybridization (FISH) analysis was performed on selected transfected samples and scored for the presence and integrity of the ACE, 3–10 days post-transfection. The heterogeneous population (transfected and nontransfected cells) was blocked in mitosis by colchicine treatment and analyzed by FISH for the presence of intact ACEs [56]. Cell preparations were hybridized with digoxigenin-labeled (DIG-Nick Translation Mix; Roche Applied Science; Mannheim, Germany; http://www.roche-applied-science.com) mouse major satellite-DNA probes to detect ACE-specific DNA sequences. A minimum of 50 interphase nuclei or metaphase spreads was scored for each condition comparing duplicate experiments. Statistical analysis included standard error of mean and Student’s t-tests (p-value 0.05) to compare the frequency of RFP-ACEs per transfected hMSC to the frequency of RFP-expressing hMSCs.

Enrichment of RFP-Expressing hMSCs
Prior to differentiation studies, enrichment of the RFP-expressing population was performed in order to increase the genetically modified population available for analysis. The enrichment steps were as follows. A single-cell suspension was prepared in defined growth media. The transfected hMSCs were initially enriched from an 11% to a 35% RFP-expressing population by using a FACS Vantage flow cytometer (Becton Dickinson; San Jose, CA; http://www.bd.com) 488nM excitation and 585/42 band pass filter for optimal detection of red fluorescence. Three sequential rounds of high speed flow sorting and expansion generated a population of stable transformants (>95% RFP+) for further evaluation. Expansion and enrichment of the RFP-expressing hMSCs were limited to 13–15 doublings to minimize the onset of senescence.

Differentiation of hMSCs
To test the ability of the transfected hMSCs to differentiate, the enriched RFP-expressing MSC population was induced to adipocytes and osteocytes. The hMSCs were differentiated in defined adipocyte and osteocyte induction media according to manufacturer’s instructions (Cambrex Bio Science). Osteocyte differentiation media consist of osteogenic basal media supplemented with L-glutamine and the induction reagents dexamethasone and ascorbate; while adipocyte differentiation media consists of adipogenic basal media supplement with L-glutamine, 3-isobutyl-1-methyl-xanthine, dexamethasone, indomethacin, and human recombinant insulin. Incubations were performed at 37°C in 5% CO2 atmosphere.

Adipogenic Induction   The hMSCs were plated at approximately 2 x 105 cells/well in 6-well dishes and fed with MSCGM until the cells reached confluence (5–13 days). At this point the hMSC populations were exposed to three adipogenic induction/maintenance cycles. Each cycle consisted of incubating the hMSCs with supplemented adipogenesis induction media for 3 days, followed by a 1–3 day incubation in adipogenic maintenance media. Non-induced control hMSCs were fed on the same schedule but with only the adipogenic maintenance media. After three cycles of induction/maintenance culturing, the cells were incubated for a maximum of 1 week in maintenance media, rinsed with phosphate buffered saline (PBS), fixed with 10% buffered formalin, and stained with Oil Red "O." The frequency of differentiation was scored as the number of cells that contained red-stained lipid vacuoles.

Osteogenic Induction   The hMSCs were seeded at approximately 3–10 x 104 cells/well in six-well plates in MSCGM. Cultures were fed with osteogenesis induction medium every 3–4 days for 2–3 weeks. Non-induced control cells were grown in parallel in normal growth media. Osteocyte differentiation was determined by harvesting cells in calcium-free PBS and assaying calcium deposition using a commercial kit (Sigma Diagnositics Kit, Procedure 587-A; Sigma-Aldrich; Oakville, ON, Canada; http://www.sigmaaldrich.com).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analyses
In addition to morphological changes, i.e., the formation of lipid vacuoles (adipogenesis) or calcium deposits (osteogenesis), the differentiation of hMSCs expressing RFP was assessed by monitoring the expression of lineage specific gene transcripts. For this, total RNA from RFP expressing populations was extracted from all cell samples using the RNAqueousTM-4PCR kit (Ambion; Austin, TX; http://www.ambion.com). First strand cDNA synthesis was performed on 50 ng of total cellular RNA using the SuperscriptTM First Strand Synthesis System for RT-PCR (Invitrogen). Amplification primer pairs for ß-actin, lipoprotein lipase (LPL), PPAR{gamma}2 (peroxisome proliferator activated receptor gamma) and osteopontin were chosen. Amplimer DNA sequences were: ß-actin (predicted PCR product of 515 bp), sense: 5'-GCACTCTTCCAGCCTTC CTTCC-3', antisense: 5'-TCACCTTCACCGTTCCAGTT TTT-3'; PPAR{gamma}2 (predicted PCR product of 352 bp), sense: 5'-GCTGTTATGGGTGAAACTCTG-3', antisense: 5'-ATA AGGTGGAGATGCAGGCTC -3'; osteopontin (predicted PCR product of 330 bp), sense: 5'-CTAGGCATCACCTG TGCCATACC-3', antisense: 5'-CAGTGACCAGTTCATC AGATTCATC-3'; low-density lipotrotein (LDL) (predicted PCR product of 276 bp) sense: 5'-GAGATTTCTCT GTATGGCACC-3', antisense: 5'-CTGCAAATGAGACA CTTTCTC-3'. PCR was performed using a PTC-200 Peltier Thermal Cycler (MJ Research; Scarborough, ON, Canada; http://www.mjr.com) with an initial denaturation of 1 minute at 95°C, followed by 35 cycles consisting of 30 seconds at 95°C, 30 seconds at 50°C, 1 min at 72°C, and a final extension of 10 minutes at 72°C. PCR products were fractionated on a 1% agarose gel, stained with ethidium bromide and photographed under ultraviolet light.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ACE-Transgene Expression in hMSCs
As a first step pursuant to the use of ACEs in a gene therapy context, we tested the ability to introduce flow-sorted and concentrated RFP-ACEs into a purified hMSC population. The RFP-ACEs were transfected into limited passaged hMSCs using commercially available transfection reagents. After transfer of the RFP-ACEs, the cells were passaged in 10-cm dishes, and the level of transgene expression (RFP fluorescence) was monitored by flow cytometry. From 4 to 5 days post-transfection, RFP expression was detected in 11 ± 4% (n = 3; scoring a minimum of 10,000 cells) of the total hMSC population. Intact RFP-ACEs, as determined by FISH analysis, were detected in 5 ± 3% (n = 2; scoring a minimum of 50 metaphase chromosome spreads) of total hMSC population. A representative example of FISH analysis of the RFP-ACEs-modified hMSCs can be seen in Figure 1AGo. Since the frequency of intact delivered RFP-ACEs/transfected hMSC, as determined by FISH analyses, was not statistically different from the observed frequency of RFP-expressing hMSCs in the total cell population that was transfected (p-value 0.05), this suggested that in general the transfection of the RFP-ACEs into the hMSCs resulted in the delivery of an intact chromosome.



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  		Figure 1. FISH analyses. A) Untransfected control hMSCs (i) and ACE-modified hMSCs (ii) were incubated with colchicine, fixed to microscope slides, and hybridized to digoxigenin-labeled mouse major satellite DNA (red signal). Note that the ACE is intact and is maintained as an autonomous chromosome. B) ACE-modified hMSCs were induced to differentiate into either osteocytes (i) or adipocytes (ii) analysis done with interphase cells. Cells were fixed and hybridized to digoxigenin-labeled mouse major satellite DNA (red signal).

 
ACE-Transgene Expression During hMSC Differentiation
We evaluated whether the hMSCs carrying the RFP-ACEs still maintained their potential to differentiate into unique cell lineages. To do so, we elected to monitor reporter gene expression in the hMSC population as the cells differentiated along adipogenic and osteogenic lineages. These two pathways were chosen because the differentiated cells were visually distinctive, based on microscopic examination, and simple biochemical identification assays were available—staining lipids with Oil Red "O" or measuring insoluble calcium deposition to identify adipocytes or osteocytes, respectively. Initially differentiation was monitored in heterogeneous cell populations prior to enrichment of RFP-ACEs containing hMSCs (population with 11% RFP-expressing hMSCs). In this case, the RFP-ACE-transfected hMSCs appear to differentiate at a rate comparable to the untransfected controls. Greater than 95% of the cells had differentiated 2–3 weeks post-induction into either adipogenic or osteogenic lineages (visual inspection, data not shown). Furthermore, during differentiation RFP expression was maintained (visual inspection). FISH analyses were performed on terminally differentiated cultures 2–3 weeks post-induction in order to evaluate the integrity of the transferred RFP-ACEs during differentiation (Fig. 1BGo). The frequencies of RFP-ACEs were scored as 25% ± 7% (n = 2) in adipocytes and 6% ± 4% (n = 2) in osteocytes. These preliminary experiments indicate that the hMSCs stably maintained RFP-ACEs and concomitant RFP expression after osteogenic and adipogenic differentiation (Fig. 2AGo, 2BGo).



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  		Figure 2. A) RFP-expressing adipocytes at 2 weeks post induction in the adipocyte differentiation pathway. Panel (i): Phase microscopic view from a representative field. Panel (ii): RFP-expressing cells from the identical microscopic field as depicted in panel (i). B) RFP-expressing osteocytes at 2 weeks post induction in the osteocyte differentiation pathway. Panel (i): Phase microscopic view from a representative field. Panel (ii): RFP-expressing cells from the identical microscopic field as depicted in panel (i).

 
RT-PCR analyses of selected gene markers (PPAR{gamma}2, LDL, osteopontin) from induced and non-induced cultures were performed to further confirm the differentiated phenotype of the mesenchymal cultures containing the RFP-ACEs. Previous reports have utilized the expression of PPAR{gamma}2 and LDL as hallmarks of adipogenic differentiation and osteopontin as a biomarker for osteogenic differentiation [57, 58].

In order to detect transgene expression and monitor cytogenetic events during differentiation, the RFP-expressing hMSCs were enriched by flow cytometry 3–5 days post-transfection, and plated into three sets of wells. One sample (non-induced control) was maintained under hMSC culturing conditions, whereas parallel cultures were induced to differentiate into osteogenic and adipogenic lineages. Samples of induced cells were harvested at various times during the 3-week induction period. The cultures were analyzed by RT-PCR to measure the induced expression of lineage specific marker genes and by FISH. RT-PCRs for ß-actin transcripts were conducted as measures of RNA integrity for all culturing conditions since ß-actin expression is ubiquitous for both undifferentiated and differentiated hMSCs (top panels Fig. 3Go, lanes C-F). Only hMSCs induced to the adipogenic lineage expressed LDL or PPAR{gamma}2 mRNA (Summary RT-PCR results [Table 1Go], representative LDL profile Fig. 3Go bottom panels I-M). RT-PCR detected expression of the LDL and PPAR{gamma}2 genes in the genetically modified adipocyte cultures as early as 1 week post induction. Parallel osteogenic-induced cultures were tested for the presence of adipogenic lineage marker genes LDL or PPAR{gamma}2. For all of the osteogenic-induced cultures, expression of LDL or PPAR{gamma}2 genes was not detected (data not shown). Similar results showing lineage specificity were found when assessing osteogenic-induced cultures for expression of the osteogenic lineage marker, osteopontin, 13 days post-induction (Table 1Go). Overall, the RFP-ACE-modified hMSCs continued to express the RFP transgene and to maintain the integrity of the RFP-ACE after in vitro differentiation.



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  		Figure 3: RT-PCR analyses using the ubiquitous cell lineage marker ß-actin (row B-F) and the specific adipose marker LPL (row I-M) from adipocyte mRNA. A ß-actin PCR product was found in all samples except for the negative PCR control. Top row: 515-bp ß-actin products. A) 100-bp ladder; B) negative control (no RNA); C) transfected hMSC (undifferentiated); D) transfected, 7-days post adipocyte induction; E) transfected, 13-days post adipocyte induction; F) nontransfected, 13-days post adipocyte induction; G) 100-bp ladder. Bottom row: 276-bp LPL products; H) 100-bp ladder; I) negative control (no RNA); J) transfected hMSC (undifferentiated); K) transfected, 7-days post adipocyte induction; L) transfected, 13-days post adipocyte induction; M) nontransfected, 13-days post adipocyte induction; N) 100-bp ladder. For summary of RT-PCR analyses refer to Table 1Go.

 

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  		Table 1. Summary of RT-PCR of hMSCs cells
 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
With the greater knowledge of SC function as well as the increased demand for treatment of degenerative musculoskeletal diseases, the emergence of SC-based therapeutic applications is becoming a cornerstone for regenerative medicine. Moreover, the enhanced proliferative and multipotential differentiation properties of SCs make them ideal delivery candidates for cell-based therapeutics. In particular, MSCs or progenitor MSCs are an attractive source of biological material for tissue engineering. In addition to their in vivo and in vitro ability to develop into particular mesenchymal tissue including bone, fat, cartilage, and BM stroma, the ease of isolation and expansion of these adherent regenerative cell populations makes subsequent genetic modifications possible prior to therapeutic delivery. Of particular importance for autologous cell-based therapies is the ability to maintain MSCs in an undifferentiated state in vitro during the introduction of exogenous genetic material. MSCs and progenitor cells also maintain the potential to develop into myocytes, tenocytes, skeletal muscle, cardiac muscle, and smooth muscle thus expanding the uses of these cells for muscle regeneration, tendon repair, and cardiac cell engineering (recently reviewed) [59, 60]. The isolation of multipotent adult progenitor cells that can be passaged indefinitely without loss of differentiation potential and the subsequent demonstration of their intrinsic in vitro pluripotency greatly expands the therapeutic potential of adult-derived MSCs [61].

While the great majority of strategies employed for the genetic modification of MSCs have focused around viral-based or plasmid-based vector systems, these vector systems are constrained by their requirement for insertion into the host genome (thereby increasing the likelihood of insertional mutagenesis) or their limited carrying capacity for introducing exogenous genetic material. The development of mammalian site-specific integration systems derived from bacteriophage integrases or engineered transposases can greatly reduce the potential for insertional mutagenesis [62–70], however, these systems are limited in the number of gene copies that can be introduced into the genome.

Recently, novel ACE technology has been developed that consists of A) a Platform ACE that contains greater than 50 sites for targeted integration; B) a Targeting Vector that "shuttles" the transgene to the Platform ACE, and C) an ACE Integrase which catalyzes the site-specific recombination between the Targeting Vectors and the Platform ACE and results in multiple transgene "loading" (manuscript in preparation). This ACE System has numerous advantages over current systems that include genetic control and flexibility combined with the potential for increased safety. Enhanced genetic control is enabled by the loading of a broad size range of DNA sequences encoding complete gene sequences, including introns, enhancers, locus control regions, tissue specific promoters, insulators, and matrix attachment regions. Flexibility is increased by loading multiple gene copies to increase gene expression and by loading other sequences such as genes encoding small interfering RNAs [71–75] to "knock-down" the expression of endogenous disease genes and "knock-in" the corrected versions. Loading suicide genes, such as the herpes simplex virus thymidine kinase gene, as a means to rationally terminate gene therapies can augment safety.

The ability to engineer hMSCs with Platform ACEs greatly expands the therapeutic potential of both the MSC cell delivery system and the chromosome-based system. In addition, ACEs are stable, nonintegrating, allow for long-term expression, have a large carrying capacity, and are easily transferred. Notwithstanding that ACE-based technology is a relatively new technology that requires further characterization, our work to date demonstrates that this technology provides a viable strategy to complement current viral or DNA vectors in gene therapy models. For that reason, we investigated the ability to introduce ACEs into hMSCs.

The work presented here represents the first step towards the prospective use of ACEs in an ex vivo gene therapy application. To our knowledge this is the first report of the introduction of mammalian ACEs into human adult SC BM-derived hMSCs. More importantly, the introduction of Platform ACEs into SCs provides a potent implement applicable ab initio to a variety of cell-based therapeutics, i.e., the ability to tractably engineer a variety of genetic factors into a cell that can form a myriad of tissue types. This potential will be particularly invaluable in the treatment of complex genetic disease traits. Currently we are testing the efficacy of ACE-engineered hMSCs to express a therapeutic protein product in a small animal model. Towards that end, we have recently engineered hMSCs to express a human therapeutic protein product contained on the Platform ACE. The resulting engineered hMSCs will be transplanted into non-obese diabetic/severe combined immunodeficient mice, and the production of the therapeutic protein will be monitored in this in vivo small animal system. Long-term stability of the ACE can be better investigated in an in vivo setting. Our goal is to demonstrate that ACEs transferred to hMSCs will provide the flexibility, safety, and long-term transgene expression imperative for viable ex vivo cell-based human gene therapy.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chromos Molecular Systems, Inc. was responsible for all material and financial support for this research. We would like to thank Diane Monteith and Tom Stodola, for purifying ACEs and technical support; Harry Ledebur and Joseph Zendegui for critical discussions; and Shamila Gorjian for manuscript preparation.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 9, 2003; accepted for publication November 16, 2003.



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