TGF‐β gene transfer and overexpression via rAAV vectors stimulates chondrogenic events in human bone marrow aspirates

Genetic modification of marrow concentrates may provide convenient approaches to enhance the chondrogenic differentiation processes and improve the repair capacities in sites of cartilage defects following administration in the lesions. Here, we provided clinically adapted recombinant adeno‐associated virus (rAAV) vectors to human bone marrow aspirates to promote the expression of the potent transforming growth factor beta (TGF‐β) as a means to regulate the biological and chondrogenic activities in the samples in vitro. Successful TGF‐β gene transfer and expression via rAAV was reached relative to control (lacZ) treatment (from 511.1 to 16.1 pg rhTGF‐β/mg total proteins after 21 days), allowing to durably enhance the levels of cell proliferation, matrix synthesis, and chondrogenic differentiation. Strikingly, in the conditions applied here, application of the candidate TGF‐β vector was also capable of reducing the hypertrophic and osteogenic differentiation processes in the aspirates, showing the potential benefits of using this particular vector to directly modify marrow concentrates to generate single‐step, effective approaches that aim at improving articular cartilage repair in vivo.     

The IGF‐I regulatory system and its impact on skeletal and energy homeostasis

Insulin‐like growth factor (IGF)‐I is important in the acquisition and maintenance of both soft and hard tissues. Skeletal remodeling requires energy and recent work has demonstrated that bone can influence insulin sensitivity and thereby regulate metabolic processes. New insights from mouse models into the role of IGF‐binding proteins (IGFBPs) as more than mere depots for the IGFs has reignited investigations into the metabolic targets influenced by the IGF regulatory system and the pathways that link bone to adipose tissue. Although there remains continued uncertainty about the relative balance between the effects of circulating versus tissue IGF‐I actions, the role of the IGFBPs has been redefined both as modulators of IGF‐I action and as independent signaling factors. This review highlights several recent findings that shed new light on the physiologic role of the IGF regulatory system and its influence on skeletal and fat metabolism. J. Cell. Biochem. 111: 14–19, 2010. © 2010 Wiley‐Liss, Inc.     

IGF‐I regulates the age‐dependent signaling peptide humanin

Aging is influenced by endocrine pathways including the growth hormone/insulin‐like growth factor‐1 (GH/IGF) axis. Mitochondrial function has also been linked to the aging process, but the relevant mitochondrial signals mediating the effects of mitochondria are poorly understood. Humanin is a novel signaling peptide that acts as a potent regulator of cellular stress responses and protects from a variety of in vitro and in vivo toxic and metabolic insults. The circulating levels of humanin decline with age in mice and humans. Here, we demonstrate a negative correlation between the activity of the GH‐IGF axis and the levels of humanin, as well as a positive correlation between humanin and lifespan in mouse models with altered GH/IGF‐I axis. Long‐lived, GH‐deficient Ames mice displayed elevated humanin levels, while short‐lived GH‐transgenic mice have reduced humanin levels. Furthermore, treatment with GH or IGF‐I reduced circulating humanin levels in both mice and human subjects. Our results indicate that GH and IGF are potent regulators of humanin levels and that humanin levels correlate with lifespan in mice. This suggests that humanin represents a circulating mitochondrial signal that participates in modulating the aging process, adding a coordinated mitochondrial element to the endocrine regulation of aging.     

Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system

BACKGROUND: The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined. METHODS: The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale. RESULTS: An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter). CONCLUSION: These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization.     

Suppression of IGF‐I signals in neural stem cells enhances neurogenesis and olfactory function during aging

Downregulation of insulin-like growth factor (IGF) pathways prolongs lifespan in various species, including mammals. Still, the cellular mechanisms by which IGF signaling controls the aging trajectory of individual organs are largely unknown. Here, we asked whether suppression of IGF-I receptor (IGF-1R) in adult stem cells preserves long-term cell replacement, and whether this may prevent age-related functional decline in a regenerating tissue. Using neurogenesis as a paradigm, we showed that conditional knockout of IGF-1R specifically in adult neural stem cells (NSC) maintained youthful characteristics of olfactory bulb neurogenesis within an aging brain. We found that blocking IGF-I signaling in neural precursors increased cumulative neuroblast production and enhanced neuronal integration into the olfactory bulb. This in turn resulted in neuro-anatomical changes that improved olfactory function. Interestingly, mutants also displayed long-term alterations in energy metabolism, possibly related to IGF-1R deletion in NSCs throughout lifespan. We explored Akt and ERK signaling cascades and revealed differential regulation downstream of IGF-1R, with Akt phosphorylation preferentially decreased in IGF-1R^−/− NSCs within the niche, and ERK pathway downregulated in differentiated neurons of the OB. These challenging experimental results were sustained by data from mathematical modeling, predicting that diminished stimulation of growth is indeed optimal for tissue aging. Thus, inhibiting growth and longevity gene IGF-1R in adult NSCs induced a gain-of-function phenotype during aging, marked by optimized management of cell renewal, and enhanced olfactory sensory function.     

Longevity effect of IGF‐1R+/− mutation depends on genetic background‐specific receptor activation

Growth hormone (GH) and insulin-like growth factor (IGF) signaling regulates lifespan in mice. The modulating effects of genetic background gained much attention because it was shown that life-prolonging effects in Snell dwarf and GH receptor knockout vary between mouse strains. We previously reported that heterozygous IGF-1R inactivation (IGF-1R^+/−) extends lifespan in female mice on 129/SvPas background, but it remained unclear whether this mutation produces a similar effect in other genetic backgrounds and which molecules possibly modify this effect. Here, we measured the life-prolonging effect of IGF-1R^+/− mutation in C57BL/6J background and investigated the role of insulin/IGF signaling molecules in strain-dependent differences. We found significant lifespan extension in female IGF-1R^+/− mutants on C57BL/6J background, but the effect was smaller than in 129/SvPas, suggesting strain-specific penetrance of longevity phenotypes. Comparing GH/IGF pathways between wild-type 129/SvPas and C57BL/6J mice, we found that circulating IGF-I and activation of IGF-1R, IRS-1, and IRS-2 were markedly elevated in 129/SvPas, while activation of IGF pathways was constitutively low in spontaneously long-lived C57BL/6J mice. Importantly, we demonstrated that loss of one IGF-1R allele diminished the level of activated IGF-1R and IRS more profoundly and triggered stronger endocrine feedback in 129/SvPas background than in C57BL/6J. We also revealed that acute oxidative stress entails robust IGF-1R pathway activation, which could account for the fact that IGF-1R^+/− stress resistance phenotypes are fully penetrant in both backgrounds. Together, these results provide a possible explanation why IGF-1R^+/− was less efficient in extending lifespan in C57BL/6J compared with 129/SvPas.