Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary cis-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5' long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Psi) is located within an area which includes the 5' LTR and the first 350 bp of gag. Transduction efficiencies of vectors prepared without FIV vif and orf2 accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV rev-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of rev expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.     

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.     

Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter

The fat-1 gene was isolated from roundworm Caenorhabditis elegans, and built into pIRES2-EGFP expression vectors driven by cytomegalovirus (CMV) promoter or cytomegalovirus enhancer and chickenβ-actin (CAG) promoter. Both CMV- and CAG-driven expression vectors were transfected to sheep fetal fibroblast cells. Positive transfected cells were used as donors for somatic cell nuclear transfer (SCNT) and the cloned embryos were transferred into the oviducts of synchronized recipient sheep. Two lambs derived from CMV vector and three lambs derived from CAG vector developed to term. Although Southern analyses using tissues from the two lambs derived from CMV vectors indicated integration of fat-1 gene into the genome, fat-1 mRNAs were not detected by RT-PCR. However, there was fat-1 expression (detected by RT-PCR) in tissues from transgenic lambs driven by CAG vectors. To investigate potential mechanisms involved in the two transgene models, methylation state of the vector promoters were examined. In CMV-driven transgenics, CMV promoters had almost no methylation in transfected cells and the resultant cloned embryos, whereas high methylations were detected in tissues and organs in transgenic lambs. In the CAG-driven transgenics, there were almost no methylations in transgenic cells and transgenic cloned embryos, and cloned lambs expressed fat-1 mRNA (detected by RT-PCR). Moreover, although SV40 promoters which drove neo/kan marker gene in CMV vectors were highly methylated in tissues from transgenic lambs, they were without methylation in cells and embryos. Therefore, we concluded that highly methylated CMV promoters induced the silence of fat-1 transgene expression in sheep. Furthermore, CAG promoter, but not CMV promoter was suitable for generation of fat-1 transgenic sheep.     

A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction

Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 x 10(8) and 1.1 x 10(13) vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 x 10(9) to 3.0 x 10(11) vg/mouse. Vector doses above 3.0 x 10(11) vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 x 10(12) vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 x 10(12) vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.     

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.     

Improved hepatic gene transfer by using an adeno-associated virus serotype 5 vector

Adeno-associated viral (AAV) vectors have been shown to direct stable gene transfer and expression in hepatocytes, which makes them attractive tools for treatment of inherited disorders such as hemophilia B. While substantial levels of coagulation factor IX (F.IX) have been achieved using AAV serotype 2 vectors, use of a serotype 5 vector further improves transduction efficiency and levels of F.IX transgene expression by 3- to 10-fold. In addition, the AAV-5 vector transduces a higher proportion of hepatocytes ( approximately 15%). The subpopulations of hepatocytes transduced with either vector widely overlap, with the AAV-5 vector transducing additional hepatocytes and showing a wider area of transgene expression throughout the liver parenchyma.     

Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle

BACKGROUND: The overlapping approach was developed recently to expand the adeno-associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co-infected cells after homologous recombination. Despite the promising proof-of-principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate-limiting factors including AAV serotype and the transgene sequence. METHODS: To study serotype effect, we delivered AAV-2, -5 and -6 overlapping vectors (5 x 10(8) vg particles of the upstream and the downstream vectors, respectively) and 5 x 10(8) vg particles of the intact gene vector to the tibialis anterior muscles of 7-week-old C57Bl/6 mice, respectively. To determine the effect of transgene sequence, we compared LacZ and alkaline phosphatase (AP) overlapping vectors. Transduction efficiency was quantified 6 weeks later by scoring the percentage of transgene-positive myofibers. RESULTS: AAV-2 overlapping vectors barely resulted in detectable transduction. Transduction efficiency was significantly improved in AAV-5 and AAV-6. The highest level was achieved in AAV-6 that reached 42% and 96% of that of the intact gene vector for the LacZ gene and the AP gene, respectively. Surprisingly, AAV-6 overlapping vector resulted in higher transduction than did AAV-2 and AAV-5 intact gene vectors. CONCLUSIONS: Our findings suggest that AAV serotype and the transgene sequence play critical roles in the overlapping approach. AAV-6 holds great promise for overlapping vector-mediated muscle gene therapy.     

Lentivirus vector can be readministered to nasal epithelia without blocking immune responses

For many envisioned applications of lentivirus vectors as tools in respiratory biology and therapeutic gene delivery, the efficiency of gene transfer must be improved. We previously demonstrated stable, persistent (>11 months) in vivo expression following a single application of a feline immunodeficiency virus (FIV)-based lentivirus vector (GP64-FIV) to murine nasal epithelia. Here we investigate the efficacy of repeated administration of lentivirus vectors to the airways. Using quantitative bioluminescent imaging, we found that consecutive daily dosing achieved a linear increase in gene expression and greatly increased the number of epithelial cells targeted. Surprisingly, reporter gene expression also increased additively following each of seven doses of FIV delivered over consecutive weeks (1 dose/week), without the development of systemic or local neutralizing antibodies. This approach enhanced expression of both reporter and therapeutic transgenes. Transduction efficiency achieved following a single dose of FIV expressing mouse erythropoietin was insufficient to increase hematocrit, whereas seven consecutive daily doses significantly increased hematocrit. These unexpected results contrast strikingly with findings reported for adenovirus vectors. Prolonged gene expression has been observed in vivo following a single dose of virus vector; however, depending on the application, repeated administration of vector may be necessary to achieve stable, therapeutic gene expression.     

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.     

Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.     

Transgenic embryos and mice produced from low titre lentiviral vectors

Lentiviral vectors are now recognised as an efficient transgene delivery system which can result in greater than 90% of founder animals carrying the transgene. Vector injection into the perivitelline space has emerged as the standard delivery method but is limited by the need for high-titre lentivirus vector preparations. Based on a modified perivitelline injection method we demonstrate that transgenic animals can be generated from low-titre virus vector preparations further simplifying lentiviral transgenesis. Repeat injection of 10⁷ TU/ml vector preparation resulted in 23% of embryos carrying the transgene compare to 1% from a single injection. Embryos exposed to repeat injection of vector developed to blastocyst with the same efficiency as non-injected embryos and produced transgenic mice capable of transmitting the transgene through the germline     

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.     

Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands

The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1alpha receptor (FGFR1alpha), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1alpha-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.     

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.     

In vivo electroporation: a new frontier for gene delivery and embryology

One of the key techniques in developmental biology is introducing transgenes into tissues and analyzing their subsequent effects on morphogenesis and organogenesis. In mammals, the transgenic approach is a way to misexpress foreign genes in various tissues and organs. However, targeting expression to certain tissues is totally dependent on the availability of specific promoters. Hence, it is not an easy task to control transgene expression temporally and spatially during embryogenesis. Further, if the transgene is toxic, embryonic development can be disrupted, resulting in premature death before the desired stages of development. As alternative systems, Xenopus and zebrafish are used frequently. In these vertebrate models, overexpression of genes can be carried out by injecting synthetic RNAs into eggs. However, genetic techniques in these systems are limited only to early development, prohibiting the precise analysis of gene effects on organogenesis in later stages. In contrast, the chick embryo has long served as a powerful and useful model system, holding a unique position in the field of developmental biology. Although trials of transgenic chicks have never been successful, easy accessibility to the developing embryo through a window opened in an eggshell enables performance of a variety of techniques, such as time-lapse cinephotomatography, microsurgical manipulations (including chick/quail chimeras), transplantation of cells and tissues, New's in vitro culture, etc. (Bortier et al., 1996; Douarin et al., 1996; Selleck, 1996). In addition to these experimental advantages, retrovirus-mediated gene delivery, and recently, adenovirus-mediated misexpression have been employed routinely in chick embryos (Leber et al., 1996; Morgan and Fekete, 1996).     

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.     

Efficient site-specific integration of large transgenes by an enhanced herpes simplex virus/adeno-associated virus hybrid amplicon vector

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in approximately 22% of transduced cells; of those cells, approximately 70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.     

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells

BACKGROUND: Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS: A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS: In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS: These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.