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Division of Endocrinology, Department of Medicine, Johns Hopkins University School of Medicine, 1830 East Monument Street #333, Baltimore, Maryland 21287, USA 1 Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA
(Correspondence should be addressed to R Salvatori; Email: salvator{at}jhmi.edu)
* (A Sagazio is on leave from Department of Endocrinology and Metabolism, University G D'Annunzio, Chieti 66100, Italy) 
(Present address of Z Wang is at Department of Surgery, The Brooklyn Hospital Center, 121 Dekalb Avenue, Brooklyn, New York 11201, USA)
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Viral vectors provide more long-term effect than traditional drug therapy and represent an alternative efficient mechanism of delivery of proteins. Gene therapy approach to GHD animals has been studied using recombinant adenovirus vectors encoding murine or rat GH and single-stranded adeno-associated viral vectors (ssAAV; Hahn et al. 1996, Marmary et al. 1999, Rivera et al. 1999). The recombinant adenovirus used in the past often elicited a potent immune response, sometimes requiring administration of anti-inflammatory drugs to virus-treated animals (Adesanya et al. 1996), although with more modern vectors this problem is reduced (Alba et al. 2005b). In addition, the conventional AAV vectors tend to have delayed and less effective gene transfer and expression (Wang et al. 2003) because AAV DNA is packaged and delivered as a single-stranded genome, which is transcriptionally inactive until converted into double-stranded (ds) template. Recent research has focused on preparation of AAV vectors that can package a self-complementary dsDNA obtaining rapid, strong, and stable expression in animals (dsAAV; Berns & Hauswirth 1979, Berns & Giraurd 1995, Samulski et al. 1999). This overcomes the rate-limiting step of conversion of single into dsDNA. Non-pathogenicity, non-toxicity and lack of immune response, robust infectivity, and long-term gene transfer identify the new AAV vectors approach. AAV vectors are derived from the replication-defective parvovirus (Berns & Hauswirth 1979, Berns & Giraurd 1995, Samulski et al. 1999). The vectors carrying a foreign gene can infect both dividing and non-dividing cells in vitro and in vivo, obtaining a long-term transgenic expression minimizing the toxicity and cellular immune response (Ferrari et al. 1996, Afione et al. 1999, Bangari & Mittal 2006), resulting in robust and long-term delivery to many tissues, including muscle (Wang et al. 2005), liver (Song et al. 2001), and pancreas (McClane et al. 1997, Ayuso et al. 2004). These dsAAV vectors have successfully delivered gene therapy in animal models of diabetes (McClane et al. 1997, Ayuso et al. 2004, Rehman et al. 2005, Wang et al. 2006), muscular dystrophy (Zhu et al. 2005), hemophilia (Arruda et al. 2005), loss of corneal endothelium (Lai et al. 2005), and cerebral ischemia (Tsai et al. 2002), with possible important future applications in humans.
We have investigated whether such a new delivery system could be a tool to administer GH to GHD animals. We show that normalization of longitudinal growth can be achieved in GHD mice using a single injection of a dsAAV expressing mouse GH cDNA.
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Materials and Methods |
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The AAV8-CMV-green fluorescent protein (GFP) vector (dsAAV8-CMV-GFP) was described earlier (Xiao et al. 1998, Gao et al. 2002). Full-length mouse cDNA was amplified by PCR from pituitary cDNA of a C57BL6 mouse using primers 5'-TTGGGGTCGAGGAAAACAGGTA-3' and 5'-GATGCATCTTAATTTTATTAGAGC-3', and inserted in pCR2.1 TA plasmid (Invitrogen). The dsAAV8-CMV-GH plasmid was generated by replacing the GFP gene of the dsAAV8-CMV-GFP (Wang et al. 2005) using BamHI/NotI restriction sites. The recombinant viral stocks were produced by the adenovirus-free, triple plasmid cotransfection method (Xiao et al. 1998). The AAV particles were subsequently purified by two rounds of CsCl gradient ultracentrifugation. The titer of viral genome particle number was determined by quantitative DNA dot bolt method (Snyder et al. 1996).
Animals and vector administration
We used homozygous GHRHKO male mice and heterozygous male carriers as normal controls to guarantee similar genetic background (mixed C57BL6/129SV; Alba & Salvatori 2004). Following guidance from previous studies (McClane et al. 1997, Song et al. 2001, Tsai et al. 2002, Ayuso et al. 2004, Arruda et al. 2005, Lai et al. 2005, Rehman et al. 2005, Zhu et al. 2005, Wang et al. 2006), dsAAV8-CMV-GH viruses were injected intraperitoneally at the 10th day of age. Treatment groups are described in Table 1. Low dose (LD) groups (12 GHRHKO pups per group) received 1x1011 particles of dsAAV8-CMV-GH viral particles; High dose (HD) group (6 GHRHKO pups) received 1x1011 particles of dsAAV8-CMV-GH at the 10th and 11th days of life (high dose); GFP group (6 GHRHKO pups) received 1x1011 particles of dsAAV8-CMV-GFP; and placebo groups (Plac) (12 GHRHKO pups per group) received placebo injections. Control groups (Ctl) consisted of 12 pups heterozygous (HTZ) for GHRHKO gene. HD, GFP, and one half (six mice) of LD, Plac, and Ctl groups were killed at the 6th week of age. The other half of LD, Plac, and Ctl groups were allowed to reach the 24th week of age (long-term groups).
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Auxological data
Starting from the first week of life, all pups were examined weekly for 5 weeks (LD, HD, GFP, Plac, and Ctl groups) or for 23 weeks (LD, Plac, and Ctl long-term groups) by measuring total body weight (TBW) using a daily-calibrated electronic balance (Scout Pro Balance; Ohaus Corp SP601, Pine Brook, NJ, USA) and body length (nose-to-anus distance, N-A), using an electronic digital caliper (Fisher brand Traceable Digital Caliper; Fisher Scientific, Hampton, NH, USA). Two days before the end of the study, animals were anesthetized using Avertin (tribromoethanol; Sigma–Aldrich) and 
200 µl blood were collected with heparinized capillaries by retro-orbital bleed for GH and IGF-I measurements. Following the same method at the 10th, 14th, 18th, 22nd weeks of life, we collected blood from four mice of LD and Ctl long-term groups for IGF-I measurements. Serum was stored at –20 °C until the day of assay. Mice were killed by halothane overdose. After killing, we harvested heart, liver, spleen, right kidney, right testis, and right gastrocnemius muscle. Organs were weighed using an Ohaus Adventurer Pro Analytical Balance (AV264 Ohaus, Pine Brook, NJ, USA). Femur and tibia length were measured using an electronic caliper after dissection of the surrounding tissues and careful disarticulation of the bones. Right femur length was considered as the maximal distance between the head of the great trochanter and the distal condyles, while right tibia length as the maximal distance between proximal condyles and malleolus. To determine the effects of the GH expression on body composition, animals were skinned, the perirenal and epidydimal fat pads were pooled (visceral fat, VF), while the sum of fat pads from the interscapular and axillary region, thighs, and inguinal region was considered s.c. fat (SF). Lean mass (LM) was measured by weighing animals deprived of tail, skin, adipose tissue, and internal organs. LM, VF, and SF weights of each animal were normalized to TBW by calculating the percentage as follows: (weight (g)/TBW (g))x100.
Detection of GH RNA expression
Total RNA was isolated using TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer's recommendations. We used 25/50 mg of tissue/mice from liver, heart, right gastrocnemius muscle, spleen, right kidney, and right testis. Total RNA was quantified spectrophotometrically at 260 nm (DU 640 Spectrophotometer; Beckman-Coulter, Fullerton, CA, USA). One microgram of total RNA was used to generate cDNA using reverse transcriptase (Moloney murine leukemia virus (M-MLV) Reverse Transcriptase; Promega). Control tubes were used without reverse transcriptase (reverse transcriptase negative). GH cDNA was amplified by PCR using primers described above, expected to generate an 859 bp band corresponding to the full-length mouse GH cDNA.
Serum hormones measurements
We measured serum GH and IGF-I at the 6th week of age in LD, HD, GFP, Plac, and Ctl groups. In addition, IGF-I serum levels were measured at the 10th, 14th, 18th, and 22nd weeks of age in four mice from LD and Ctl, and at the 24th week of age in long-term LD, Plac, and Ctl groups. GH serum levels were measured at the 24th week in four mice from LD, Plac, and Ctl groups.
Serum GH was measured by RIA (Rat GH RIA, RGH-45HK; Linco-Millipore, Billerica, MA, USA). The standard curve of the assay performed in accordance with the manufacturer's provided samples. Each sample was assayed in duplicate.
Serum IGF-I was measured using mouse/rat IGF-I kit (DSL-2900; DSL Webster, TX, USA), after acid–ethanol extraction, following the manufacturer's recommendations. The assay included quality controls provided by the manufacturer. The standard curve of the assay performed in accordance with the manufacturer's provided samples. Each sample was assayed in duplicate.
Fluorescence analysis
Tissues were fixed in 4% paraformaldyde (PFA, pH 7.3), washed in 0.2 M PO4 buffer, and conserved –20 °C in Tissue-Teck OCT Compound. Transversal sections (7 µm) were cut from frozen tissue. All tissues were placed on glass slides and covered by 25 µl Vectashield Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA) containing propidium iodine for direct evaluation of GFP expression under the fluorescence microscope.
Statistical analysis
Data were analyzed by ANOVA using the SPSS statistical package (SPSS Inc., Chicago, IL, USA), with post hoc analysis using Bonferroni's method. Data were considered statistically significant at P<0.05.
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Results |
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As shown in Fig. 1A and B, all groups of GHRHKO mice injected with dsAAV8-CMV-GH virus reached significant increase in the final N-A length (6 and 24 weeks). At the 6th week of age N-A length of HD group was significantly higher than LD and Ctl groups. Weight normalized in all virus-treated groups at the 6th and 24th weeks of age (Fig. 2A and B). As shown in Fig. 3A–C, both low- and high-dose-treated groups at the 6th and 24th weeks of age normalized in femoral and tibial lengths.
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GH mRNA was detected by RT-PCR only in liver, skeletal, and heart muscle of virus-injected animals both at the 6th (Fig. 5A) and 24th weeks (Fig. 5B), but not in spleen, kidney, and testis.
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As shown in Fig. 6A, at 6 weeks of age GH levels of low- and high-dose groups were significantly higher than levels of HTZ control mice. Serum GH levels of control mice were higher than placebo- and GFP-treated animals. At 24 weeks of age, serum GH levels of virus-treated mice were higher than placebo but not different from HTZ mice (Ctl; Fig. 6B).
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As shown in Fig. 7A, at 6 weeks of age, serum IGF-I levels were significantly higher in the high-dose-treated group compared with heterozygous mice, but no statistically significant difference was observed between low dose and HTZ controls. Serum IGF-I levels were significantly higher in both groups treated with dsAAV8-GH virus than in placebo-treated GHRHKO mice and mice treated dsAAV8-CMV-GFP. There was no statistical difference between serum IGF-I levels in the low-dose-treated group and HTZ mice at the 10th, 14th, 18th, 22nd, and 24th weeks of age, and they were both higher than in placebo-treated GHRHKO mice at week 24 (Fig. 7B).
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Microscopy analysis showed that in GHRHKO mice injected with dsAAV8-CMV-GFP, GFP was expressed in liver, skeletal, and (to a lesser extent) heart muscle, while no detectable expression was observed in spleen and kidney in agreement with the result of GH mRNA in mice injected with dsAAV8-CMV-GH (Fig. 8A). No fluorescence was observed in tissues from HTZ mice (Fig. 8B).
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Discussion |
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The detection of normal levels of serum GH and IGF-I at 24 weeks of life (22.5 weeks after the injection of the virus) confirmed previous studies showing that this viral vector has stable and long-term expression limited to the liver, skeletal, and cardiac muscle (McClane et al. 1997, Song et al. 2001, Tsai et al. 2002, Ayuso et al. 2004, Arruda et al. 2005, Lai et al. 2005, Rehman et al. 2005, Zhu et al. 2005, Wang et al. 2006). This is confirmed by both detection of GH mRNA and fluorescence studies in mice injected with GFP-expressing vector. The pattern of expression (as shown by GH mRNA) was similar at 6 and 24 weeks, suggesting stable and long-term gene expression after the initial infection. Our results are similar to previous works that has used single-stranded adenoviral vector that caused expression of recombinant GH up to 7 weeks (Hahn et al. 1996). In that paper, however, expression beyond 7 weeks was not assessed, and tissue expression of the virus was not examined. Another GH delivery system using a single-stranded adeno-associated virus has been shown to last up to 10 months after a single injection, but it is used in nude mice unable to mount an immune response (Rivera et al. 1999). Therefore, our results show that in GHD immunocompetent mice a species-specific GH cDNA continue to exert biological activity up to 6 months from a single injection.
Serum levels of GH at 6 weeks in the low-dose group were higher than at 24 weeks, showing that the degree of GH expression reduces over time. This reduction did not reflect a parallel decrease in serum IGF-I. This is not completely surprising. Serum IGF-I reflects mostly liver IGF-I production, and in the past we have noted a frequent discrepancy between the effects of GH or GHRH on growth and serum IGF-I, which seems to be a less than optimal index of GH effect in mice (Alba et al. 2005a, Fintini et al. 2005a,b). Indeed, in mice with liver-specific ablation of the IGF-I gene, somatic growth is essentially normal despite markedly reduced serum IGF-I (Yakar et al. 1999). Nevertheless, the fact that serum IGF-I levels at 24 weeks are significantly higher in virus-treated than in placebo-treated GHRHKO mice shows that the expressed GH is still functional, and that no resistance to its effect developed over time. This is not surprising, as we have used a species-specific GH cDNA, and previous reports describing the development of anti-GH antibodies in GH-treated GHD rodents used exogenous GH from different species (Groesbeck & Parlow 1987). As we do not have a group that was allowed to survive longer than 6 months, we cannot determine whether eventually the transgene expression would disappear. However, long-term gene expression over a year mediated by AAV vectors in experimental animals has been well documented in different animal species using various transgenes (Rivera et al. 2005). The positive effect of the dsAAV virus on growth was not accompanied by any readily evident side effect, despite the fact that GH expression was not physiologically regulated. Obviously, the use of a universal promoter is a limitation to any possible clinical application of this approach, due to the well-known long-term risks associated with excessive and unregulated GH secretion as seen in patients with GH-secreting pituitary adenomas (Melmed 2006). In addition, cardiac expression and possible long-term effects of locally produced GH on the heart muscle is also a concern (Lombardi et al. 1997). Therefore, future studies are being designed using either inducible promoters (that may be regulated at will), similarly to the data published by Rivera et al. (1999), or promoters that are specific for a given tissue (e.g. muscle) which may allow significant systemic delivery despite limited local expression. Finally, the occurrence of circulating anti-AAV antibodies has been reported in rodents (Peden et al. 2004) and liver enzyme increase has been reported in human hemophilic subjects treated with intrahepatic AAV vector expressing factor IX (Manno et al. 2006). All these factors may limit future clinical use of this approach.
While the applicability of the findings of this work is very far from any conceivable clinical application in humans, the advantages of new dsAAV vectors offer a good starting point for the development of novel regulated viral gene delivery systems for GH administration.
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Acknowledgements |
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References |
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Received in final form 12 September 2007
Accepted 17 October 2007
Made available online as an Accepted Preprint 17 October 2007
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