Gene Therapy Approaches for Disc Regeneration




Survival of luciferase-expressing stem cells following implantation into the intervertebral disc region. Stem cells expressing the luciferase gene were implanted in a caudal disc space in the rat. At each time point listed, luciferin was injected into the implantation site. Images and quantified data were generated using the BLI system. Arrows indicate luciferase-expressing cells in the disc region. RLU relative luminescence unit



Bioluminescence imaging can be used to monitor cell survival over time both in vitro and in vivo. To apply this method, luciferase-expressing cells are required. Following gene delivery and cell culture or implantation, the luciferase substrate luciferin is added to cells in culture or injected into the implantation area in the animal. In vitro and in vivo imaging is performed using a cooled charge-coupled device (CCCD), which can detect light (photons) emitted from tissue following luciferin degradation by luciferase (Sheyn et al. 2011).

Reference

Sheyn D, Kallai I et al (2011) Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Mol Pharm 8:1592–1601




24.3 Delivery and Activity of Anti-inflammatory/Anti-degeneration Genes


As discussed in other chapters of the book, during disc degeneration there is a progressive loss of water, proteoglycans, and collagen II from the matrix of the nucleus pulposus. Although the events that lead to these degenerative changes have not been clearly defined, there is overwhelming evidence to indicate that inflammatory mediators are elevated during degeneration. Kang et al. (1997) showed that cells obtained from normal, nondegenerate human discs and cultured for 72 h with interleukin (IL)-1β were biologically responsive and increased their production of matrix metalloproteinases, nitric oxide, IL-6, and prostaglandin E2. The effect was significantly higher in normal, nondegenerate discs, in which spontaneous synthesis of these mediators is low. These agents are inhibitors of proteoglycan synthesis, but IL-1 is likely to have a direct effect on proteoglycan breakdown through activation of members of the metalloproteinase (MMP) family of enzymes.

Aside from the interleukins, Seguin et al. (2005) studied the in vitro effect of TNF-α on nucleus pulposus tissue from bovine caudal spines. These studies reported decreased proteoglycan and collagen gene expression and protein synthesis and activation of aggrecanase-mediated proteoglycan degradation, involving MMPs, and ADAMTS. Since MMP activity is normally inhibited within the matrix by tissue inhibitors of MMPs (TIMPs) (Nagase and Woessner 1999), it is reasonable that members of this family were used to explore whether candidate genes would stop or slow the disc degradation process.


24.3.1 Anti-inflammatory/Anti-degeneration Gene Delivery into Animal and Human Cells: In Vitro and In Vivo Studies


In 2003, Wallach et al. (2003 b) isolated cells from degenerate intervertebral disc obtained from patients undergoing elective surgical procedures and infected the cells with an adenoviral-tissue inhibitor of metalloproteinase-1 (Adeno-TIMP-1) construct. Gene delivery of TIMP-1 increased proteoglycan synthesis in a dose–response manner. Disc cells treated with Adeno-TIMP-1 demonstrated an optimal response at an MOI of 100; cell pellets treated with Adeno-TIMP-1 at 100 MOI were consistently larger than controls and pellets treated with other virus concentrations. Successful delivery of the anticatabolic gene TIMP1 resulted in increased levels of proteoglycan in three-dimensional cultures of degenerate disc cells. It is likely that TIMP-1 had no direct effect on proteoglycan synthesis, but rather inhibited proteoglycan degradation, thus promoting its accumulation in the culture. In addition, these results may reflect a secondary effect, that is, inhibition of aggrecanase-1 (ADAMTS-4), which is also inhibited by the TIMP family, as reported elsewhere (Hashimoto et al. 2001).

In a more recent study, Liu et al. (2010) investigated the effect of combined connective tissue growth factor (CTGF) and TIMP1 expression mediated by AAV. These researchers isolated rhesus monkey lumbar disc nucleus pulposus cells; cultured and transduced the cells with rAAV2-CTGF-IRES-TIMP-1 (a vector that elicits both CTGF and TIMP1 expression), rAAV2-CTGF, or rAAV2-TIMP-1 at an MOI of 106; and then measured the expression of collagen II and proteoglycan using reverse transcription polymerase chain reaction (RT-PCR) analysis and Western blotting. By comparing transduced with nontransduced cells, these researchers showed that CTGF induced the synthesis of collagen II and proteoglycan, whereas TIMP1 enhanced expression of proteoglycan, but had no effect on collagen. Expression of both genes in the lumbar disc nucleus pulposus cells significantly increased the synthesis of proteoglycan and collagen II. In this particular case, single gene transduction of CTGF or TIMP1 increased proteoglycan synthesis (Lee et al. 2001). Overexpression of CTGF caused an increase in collagen II protein synthesis. Combined transduction of both CTGF and TIMP1 significantly promoted the expression of proteoglycan and collagen II to levels greater than that achieved using each of the individual genes. This finding is of considerable importance as it supports the concept that one approach to preventing degradation is by promoting inhibition of catabolic processes. It thus serves as a promising avenue of research for the treatment of degenerative disc disease via gene therapy.

IL-1 is an inflammatory mediator implicated in the ­degeneration of intervertebral discs, especially the nucleus ­pulposus matrix (Elfervig et al. 2001; Shen et al. 2003; Jimbo et al. 2005; Le Maitre et al. 2005). In 2006, Le Maitre et al. targeted the IL-1 receptor using an IL1 receptor antagonist (IL1Ra) gene that was transferred ex vivo to the intervertebral disc. Normal and degenerate human disc cells, growing in a monolayer or in alginate beads, were infected with an adenoviral vector carrying the IL1Ra gene (Adeno-IL-1Ra). Protein production was measured, and the ability of the infected cells to inhibit the effects of IL-1 was assessed. In addition, normal and degenerate intervertebral disc cells infected with Adeno-IL-1Ra were injected into degenerate disc tissue explants, and IL-1Ra production in these discs was evaluated. Nucleus pulposus cells and annulus fibrosus cells infected with Adeno-IL-1Ra produced elevated levels of IL-1Ra for prolonged time periods, and the infected cells were unaffected by IL-1. IL-1Ra protein expression was increased and maintained for 2 weeks when infected cells were injected into disc explants. In a further study by the same group (Le Maitre et al. 2007), Adeno-IL-1Ra was introduced into degenerate disc explants directly or via genetically engineered nucleus pulposus cells. This resulted in cessation of degenerate intervertebral disc matrix, degradation; in situ zymographic and immunohistochemical investigations showed that there was downregulation of MMP-1, MMP-3, MMP-7, and MMP-13 expression as well as ADAMTS4 expression. Single injections of disc cells engineered to overexpress IL-1Ra significantly inhibited degradation enzyme expression for 2 weeks. These studies indicated that since IL-1 is a key cytokine that promotes matrix degradation, direct delivery of IL-1Ra or direct delivery by gene therapy provides a powerful new way to prevent or inhibit disc matrix degradation.

In 2005 Glasson and colleagues reported on the prevention of cartilage degradation in a murine model of osteoarthritis by deletion of the ADAMTS5 “a disintegrin and metalloproteinase with thrombospondin motifs” gene (Glasson et al. 2005). To pursue the goal of looking for additional candidate genes that would stop or slow the disc degeneration process, the effect of ADAMTS5 was investigated by Seki using siRNA oligonucleotide in a rabbit annulus needle-puncture model (Seki et al. 2009). The efficiency of the siRNA constructs was first evaluated by growing rabbit NP cells in culture and transfecting them with the siRNA oligonucleotide specific to ADAMTS5. Compared with control, the ADAMTS5 siRNA-transfected cells displayed an approximately 75 % decrease in levels of ADAMTS5 mRNA. For the in vivo portion of the study, the nucleus pulposus was punctured, and 7 days later the ADAMTS5 siRNA oligonucleotide was injected into the rabbit nucleus. The early timing of the injection was to ascertain the impact of ADAMTS5 siRNA during the acute phase of disc degeneration. Eight weeks after the siRNA oligonucleotide injection, the rabbit spines were examined ex vivo by using MRI. The T2 signal intensity was stronger in ADAMTS5 siRNA-injected nucleus pulposi than in the control siRNA-injected nucleus. Histological studies supported the MRI results, showing partial maintenance of nucleus pulposus tissues in the ADAMTS5 siRNA-injected group, compared with the control group.


24.3.2 Fas-Fas Ligand (L) and the Intervertebral Disc as an Immune-Privileged Tissue


Since the concept of immune privilege is of high relevance in the biology of the intervertebral disc, the concept is discussed further in the accompanying box. Early in 1995, Griffith et al. (1995) published a paper showing that Fas-FasL interactions appear to be an important mechanism for the maintenance of immune privilege. That publication had as its focus the compartmentalization of tissues of the eye. In a related publication, Takada and his group reported that FasL exists in the intervertebral disc and confers immune privilege (Takada et al. 2002). In their study, Takada et al. (2002) used immunohistochemical and RT-PCR analysis to ascertain if FasL was present in human and rat disc specimens. They reported that nucleus pulposus cells of human and rat origin exhibited intense positive reactions for FasL. Outer annulus fibrosus cells and notochordal cells exhibited weak immunostaining. The results of RT-PCR confirmed the existence of FasL in the rat disc cells, but analysis was not performed on human tissues.

Results of investigations reported by Han et al. (2009) showed that FasL-induced apoptosis of rat nucleus pulposus cells was sharply reduced by Fas siRNA, suggesting that it is feasible to suppress nucleus cell death using an RNAi approach. The authors recognized that this was successful in short-term in vitro assays, but for research purposes and especially as a therapeutic strategy a much longer period of inhibition may be required. Because nucleus pulposus cells reportedly express FasL constitutively and since FasL is recognized as a marker gene, a study led by Suzuki et al. (2009) investigated the applicability of RNAi technology to downregulate the expression of this endogenous gene in rat intervertebral discs in vivo. As mentioned earlier in this chapter, the Suzuki group previously used RNAi in cultured nucleus pulposus cells in vitro, targeting two exogenous reporter luciferase plasmids, firefly and Renilla (Kakutani et al. 2006). For the endogenous FasL gene study, siRNAs that target rat FasL was injected into coccygeal intervertebral discs. After the injection, therapeutic ultrasound was applied to the skin surface covering the injected discs. There was significant inhibition (53 %) of endogenous FasL gene expression compared with the control group, which was transfected with nonspecific siRNA, at both 4 and 20 weeks posttransfection. One important conclusion from this study is that there is long-term downregulation, which is mediated by siRNA for the endogenous gene Fas in discs in vivo. Besides the ­techniques used for Fas-FasL genetic manipulation, it is important to take into consideration the actual debate on the paradoxical role of FasL. Reports have indicated that FasL is involved in the formation of the intervertebral disc and its immune privilege, and also that the expression of FasL is decreased in degenerate discs, but does not vary with age (Kaneyama et al. 2008). On the other hand, FasL has also been found to have a close relationship with disc cell apoptosis and is seen as another factor involved in disc degeneration (Han et al. 2009).


24.3.3 Box 24.2 Immune Privilege


Immune privilege (IP) is the ability of certain organs to suppress the immune response to foreign bodies or organisms within their specific boundaries. IP was first described by Sir Peter Medawar in 1948, based on the prolonged survival of an allogenic tissue graft that had been placed in the anterior chamber of the eye (Hori et al. 2010). Additional sites of IP include the brain, pregnant uterus, and testis. Recent studies have led to the identification of more tissues with IP-like characteristics (i.e., tissues that adopt IP mechanisms and hence avoid a destructive immune response) such as the gut, skin, and lung (Arck et al. 2008). The oral mucosa displays a similar tolerance: it rarely exhibits acute inflammatory or allergic reactions to high levels of bacterial colonization or frequent contacts with allergens (Novak et al. 2008). Although IP was first thought to be a passive process, it soon became evident that many active immunoregulatory processes take place in maintenance of IP (Hong and Van Kaer 1999; Arck et al. 2008; Novak et al. 2008; Hori et al. 2010). This phenomenon is believed to be an evolutionary protective mechanism designed to prevent a destructive inflammatory immune response in critical parts of the body (Hong and Van Kaer 1999; Arck et al. 2008). The potential clinical use of IP is clear. From allogenic cornea replacement (Hori et al. 2010) to implantation of xenogenic pancreatic islets performed in both rodents and large-animal models (Gores et al. 2003), the possibility of exploiting sites of IP to avoid the immune response to implanted tissue is attractive. In addition, the inherent IP properties of stem cells overcome the host immune rejection response, as in the case of ex vivo expanded hematopoietic stem cells, which when used in allogenic transplantation in a mouse model surmounts the major histocompatibility complex barrier (Zheng et al. 2011). Translated to humans, this strategy can significantly enhance the availability of implanted tissues and organs as well as promote novel therapies.

References

Arck PC, Gilhar A et al (2008) The alchemy of immune privilege explored from a neuroimmunological perspective. Curr Opin Pharmacol 8:480–489

Gores PF, Hayes DH et al (2003) Long-term survival of intratesticular porcine islets in nonimmunosuppressed beagles Transplantation 75:613–618

Hong, SL, Van Kaer L (1999) Immune privilege: keeping an eye on natural killer T cells. J Exp Med 190:1197–1200

Hori J, Vega JL et al (2010) Review of ocular immune privilege in the year 2010: modifying the immune privilege of the eye. Ocul Immunol Inflamm 18:325–333

Novak N, Haberstok J et al (2008) The immune privilege of the oral mucosa. Trends Mol Med 14:191–198

Zheng J, Umikawa M et al (2011) Ex vivo expanded hematopoietic stem cells overcome the MHC barrier in allogeneic transplantation. Cell Stem Cell 9:119–130


24.4 Gene Targeting of Growth and Transcription Factors


To maintain the health and integrity of the intervertebral disc, both in the nucleus pulposus and annulus fibrosus, there should be a balance among systems that regulate the synthesis, breakdown, and accumulation of macromolecules in the matrix. When reviewing work published to date on the gene therapy approach to treat disc degeneration, four main intracellular regulators were found: BMPs and TGF-β as morphogens and LMP-1 and Sox-9. Growth factors are polypeptides that bind to cell membranes and regulate matrix production and regeneration of various cell types in paracrine and autocrine pathways (Masuda and An 2004) (see also Chap.​ 25). In normal conditions, intervertebral disc cells express and secrete anabolic growth factors to maintain the balance between normal matrix synthesis and degradation. Included in this group of molecules is insulin-like growth factor (IGF), transforming growth factor-beta (TGF-β), and members of the BMP family (Thompson et al. 1991; Osada et al. 1996; Cui et al. 2008). Another group of targets includes transcriptional factors that are “master regulators” of the chondrogenic and osteogenic phenotype. Sox-9 is one such factor that has received study for cartilage and intervertebral disc basic and preclinical research; retroviral expression of Sox-9 can also efficiently induce ASCs differentiation into chondrocyte-like cells. Thus, Sox-9 is a candidate gene for use as a possible therapeutic tool for the treatment of degenerative disc diseases (Yang et al. 2011). LIM mineralization protein-1 (LMP-1) is another regulatory protein that positively regulates BMP secretion (Boden et al. 1998). The activities of both anabolic growth factors and selected transcription factors have been used to promote regeneration through augmentation of matrix production thereby impacting disc height and function (Zhang et al. 2006).


24.4.1 In Vitro Gene Delivery of Growth and Transcription Factors


Most experiments conducted in this field are still performed in vitro. Research is focused on finding the set of genes and cells that will elicit the most promising results (Zhang et al. 2006; Bron et al. 2009; Zhang et al. 2009). Most experiments are conducted in 3D cultures known to support chondrogenesis and nucleus pulposus-like differentiation (Risbud et al. 2004). Another established method for in vitro differentiation is a whole-disc culture model, which mimics the in vivo environment (Zhang et al. 2008).

Originally identified by their ability to induce the formation of bone and cartilage, BMPs are regarded today as key signaling proteins responsible for organization of tissue architecture throughout the entire body. Today, 20 proteins are known to be associated with this group. The Food and Drug Administration has recently allowed the initiation of Investigational New Drug clinical trials on osteogenic protein-1 and growth differentiation factor-5 in the United States (Reddi and Reddi 2009; Zhang et al. 2011). Zhang and colleagues compared the effects of overexpression of recombinant adenoviral vectors expressing various BMPs (BMP-2, BMP-3, BMP-4, BMP-5, BMP-7, BMP-8, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, and BMP-15) on extracellular matrix accumulation by bovine intervertebral disc cells. Of the 12 vectors evaluated by these researchers, BMP-2 and BMP-7 (also known as OP-1) were best at stimulating proteoglycan accumulation in nucleus pulposus cells, whereas BMP-4 and BMP-14 (also known as GDF-5) optimally stimulated collagen production (Zhang et al. 2006, 2009). Similar results were seen using the AAV-BMP-7 vector in canine nucleus pulposus cells (Wang et al. 2011). In another study, researchers injected knee chondrocytes transduced with Adeno-BMP-7 or Adeno-BMP-10 into whole intervertebral disc explants cultured in vitro. Discs treated with chondrocytes expressing BMP-7 demonstrated a 50 % increase in proteoglycan within the nucleus pulposus, whereas discs treated with chondrocytes expressing BMP-10 evidenced no increase in proteoglycan accumulation (Zhang et al. 2008).

GDF-5 (BMP-14) is known for its participation in joint formation and endochondral ossification (Cui et al. 2008). It is also known that a deficiency in GDF-5 leads to abnormalities in intervertebral disc structure (Li et al. 2004). Studies have shown that overexpression of GDF-5 in disc cells using viral (adenoviral vector) and nonviral (nucleofection) methods augments expression of proteoglycan proteins and collagen (Cui et al. 2008; Feng et al. 2009). LMP-1 is an intracellular regulatory molecule known to induce secretion of multiple BMPs from leukocytes and osteoblasts (Boden et al. 1998). Boden and colleagues used this activity to increase proteoglycan production by intervertebral disc cells (Yoon et al. 2004; Kuh et al. 2008). They have shown that transduction of rat disc cells in a 3D culture or in a ­monolayer with Adeno-LMP1 results in significant increases in proteoglycan and aggrecan mRNA levels as well as elevations in BMP-2 and BMP-7 mRNA levels.

A nonviral gene delivery system is a promising alternative that avoids the risks of insertional mutagenesis of retroviruses, immunogenicity of adenoviruses, and acquisition of replication competence (Nishida et al. 2000). Using a gene gun, Matsumoto et al. demonstrated successful transfection of bovine intervertebral disc cells with BMP-7, which led to a higher proteoglycan content (Matsumoto et al. 2001).

In common with the BMPs discussed above, TGF-β is a secreted signaling protein that regulates many aspects of development and tissue homeostasis, including growth, patterning, and cellular differentiation. Three isoforms of TGF-β (1, 2, and 3) act through the same heteromeric receptor complex (Baffi et al. 2004). TGF-β signaling plays a key role in the development and maintenance of cartilage and, in particular, the intervertebral disc (Nishida et al. 1999). Lee and colleagues examined the effect of TGF-β on human disc cells in vitro. Disc cells were transfected with an adenovector expressing TGF-β, after which the cells were grown in 3D pellets. In response to this treatment, the cells increased synthesis of proteoglycan and collagen II (Lee et al. 2001).

Although positive results have been achieved with gene therapy, mainly when using adenoviral vectors, transfer of a single gene using high doses of viral vector can produce hazardous systemic effects, in particular cytotoxicity, and an immune response (Lohr et al. 2001). It is likely that these effects can be mitigated by administration of several vectors so as to decrease the level of a single vector and possibly augment the regenerative potential. Moon and colleagues tested this concept in human intervertebral disc cells. The cells were transduced with different combinations of Adeno-TGF-β1, Adeno-BMP-2, and Adeno-IGF-1, after which the cells were placed in a 3D culture in alginate beads. Cells treated with double and triple gene combinations were found to have higher proteoglycan synthesis rates than cells treated with a single vector, hence allowing the use of less viral ­vector (Moon et al. 2008).

Sox-9 is another transcription factor that plays a key role in the differentiation process. This transcription factor positively controls collagen II synthesis and is a key gene for chondrogenesis, thus suggesting that its expression could be used to promote nucleus pulposus cell survival (Bell et al. 1997; Paul et al. 2003; Yang et al. 2011). After infection with Adeno-Sox-9 vector, cells from a chondroblast line, human disc cells derived from degenerated discs, and bovine nucleus pulposus-derived cells demonstrated elevated collagen II mRNA expression and raised protein levels (Paul et al. 2003; Zhang et al. 2006). In another study, rat ASCs were infected with a retro-Sox-9 vector based on the murine leukemia virus. In a 3D culture system, supplemented with TGF-β, these genetically modified cells demonstrated increased levels of Sox-9 and collagen II. These researchers also cocultured these genetically modified ASCs with nucleus pulposus cells. The coculturing technique has been shown to enhance differentiation toward a nucleus pulposus-like fate since the ASC secrete growth factors into the culture medium. When this was performed, a marked increase in proteoglycan and collagen II production by the nucleus pulposus cells was noted (Yang et al. 2011).


24.4.2 In Vivo Gene Delivery of Growth and Transcription Factors: Animal Models


There are few published studies dealing with gene therapy for intervertebral disc regeneration in vivo. The experiments described in this section are all focused on early stages of development and were performed to evaluate the feasibility and safety of using gene therapy as a tool for disc regeneration. The model used for these studies is based on intradiscal injection of a therapeutic vector into the degenerated intervertebral disc and takes advantage of the avascularity and immune privilege of the disc (Nishida et al. 2008).

Nishida and colleagues evaluated injections of Adeno-TGF-β into rabbit IVDs. The discs were harvested 1 week after virus injection and were subjected to histological analysis and biochemical assays. The injected discs showed extensive increases in TGF-β expression and production, as well as in proteoglycan synthesis (Nishida et al. 1999). In a similar study, Adeno-GDF-5 or Adeno-Luc vector was injected directly into mouse lumbar degenerated intervertebral discs. Gene ­expression of the delivered vector lasted up to 6 weeks. In addition, delivery of the vector succeeded in arresting the decrease in disc height and loss of proteoglycan due to disc degeneration (Liang et al. 2010). Injection of Adeno-LMP-1 into the native lumbar discs of a rabbit resulted in elevations of LMP-1, BMP-2, and BMP-7 mRNA levels (Yoon et al. 2004).

Surprisingly, to date, there is only one paper reporting on degenerated rabbit discs injected with adenoviral vector expressing the Sox9 gene. The Sox-9-treated discs retained their “chondrogenic” appearance, unlike control discs injected with Adeno-GFP vector or control degeneration-induced discs (Paul et al. 2003).


24.5 Use of Gene Therapy for Intervertebral Disc Repair: Theoretical and Practical Consideration


Concomitant with increased human life expectancy, disc degeneration and associated spinal disorders are a major health concern. Due to the limited regenerative capacity of the disc tissues, it is almost impossible to reverse or even stop the process of degeneration, a topic extensively discussed in this book. Thus, there is increasing interest in developing new biological approaches to regenerating the degenerating disc. In this chapter, we have reviewed how this can be achieved through genetic modification of intradiscal gene expression via gene therapy. Other therapeutic approaches to disc regeneration include injection of growth factors, with or without a carrier, and use of mature cells, progenitor cells, and stem cells, with or without scaffolding, topics addressed in other chapters of this book.

Accordingly, we have reviewed studies of intervertebral disc gene therapy performed in the last decade, summarized in Table 24.1. The vast majority of the investigations performed in vitro use reporter genes as proof of concept. Several studies were conducted to deliver reporter genes into disc cells isolated from animal tissues. Those experiments addressed the feasibility and limitations of gene delivery into intervertebral disc-derived cells. LacZ and GFP genes were used in various animal and human cells and were delivered via viral and nonviral methods. Small iRNA technology was also used in vitro in rat and human nucleus pulposus cells to downregulate luciferase activity. Following studies performed on animal cells, the delivery of reporter genes to human disc cells was also pursued. Adenovector harboring the LacZ or luciferase reporter genes transduced 100 % of cultured human intervertebral disc cells, healthy or degenerate. Another in vitro study demonstrated that it is possible to exert exogenous control over gene activity using human cells in vitro. To date, both viral and nonviral vectors have been used to deliver reporter genes into intervertebral discs in vivo. When different doses of both adenovector and AAV ­encoding either GFP or LacZ were injected into rabbit discs, no clinical, biochemical, or histological deficits were noted. However, this was not the case when BMP2 or TGFβ was delivered (Levicoff et al. 2008). It is therefore probable that the safety of gene delivery cannot be assessed using only reporter genes. Cell- or stem cell-mediated gene therapy is another tactic of choice and is viewed as a “more physiological” approach to regulating gene expression. Luciferase-expressing porcine MSCs were implanted into a minipig disc, but massive cell loss followed implantation. siRNA against the exogenous Luciferase gene was used in a rat model in vivo. Because the luciferase gene was silenced for 24 weeks, the siRNA activity was active for a considerable time period (Suzuki et al. 2009).
Mar 25, 2017 | Posted by in ORTHOPEDIC | Comments Off on Gene Therapy Approaches for Disc Regeneration

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