Neutralizing antibody evasion and transduction with purified extracellular vesicle-enveloped AAV vectors.

AUTHORS

Dr. Ming Cheng, Ms. Laura Dietz, Dr. Yi Gong, Dr. Florian Eichler, Ms. Josette Nammour, Miss Carrie Ng, Dr. Dirk Grimm, and Dr. Casey A Maguire

ABSTRACT

Adeno-associated virus (AAV) is classified as a non-enveloped DNA virus. However, several years ago we discovered that in media of packaging cells producing recombinant AAV vectors, AAV capsids can associate with the interior and surface of extracellular vesicles (EVs), sometimes referred to as exosomes. Since then we and others have demonstrated that exosome-enveloped AAV, exo-AAV, can enhance transduction in vivo as well as evade neutralizing antibodies. While promising, these data were generated with differential centrifugation to pellet the exo-AAV. This method results in a heterogeneous mixture of exo-AAV, co-precipitating proteins, as well as free AAV capsids. To define the properties of exo-AAV more accurately, here we used a density gradient method to purify exo-AAV. We next performed head-to-head comparisons of standard AAV1, differential centrifuged exo-AAV1, and gradient purified exo-AAV1 for antibody evasion and transgene expression in the murine brain. We found purified exo-AAV1 to be more resistant to neutralizing antibodies than the other AAV preparations. Direct intracranial injection of purified exo-AAV1 into mice resulted in robust transduction, which transduced a larger area of brain than standard AAV1. We also identified the recently described membrane-associated accessory protein (MAAP) by mass spectrometry of purified exo-AAV1 preparations. Finally, we used a scalable method, size-exclusion chromatography to isolate exo-AAV1, and demonstrated functional transduction in cultured cells and increased antibody resistance. Together, these data suggest that higher purity exo-AAV will have beneficial characteristics for gene delivery and also may lead to mechanistic insights into the incorporation of AAV into EVs.

Regional Gene Therapy with Transduced Human Cells: The Influence of “Cell Dose” on Bone Repair

AUTHORS

Hansel Ihn, Hyunwoo Kang, Brenda Iglesias, Osamu Sugiyama, Amy Tang, Roger Hollis, Sofia Bougioukli, Tautis Skorka, Sanghyun Park, Donald Longjohn, Daniel A. Oakes, Donald B. Kohn, and Jay R. Lieberman

ABSTRACT

Regional gene therapy using a lentiviral vector containing the BMP-2 complementary DNA (cDNA) has been shown to heal critical-sized bone defects in rodent models. An appropriate “cellular dose” needs to be defined for eventual translation into human trials. The purpose of this study was to evaluate bone defect healing potential and quality using three different doses of transduced human bone marrow cells (HBMCs). HBMCs were transduced with a lentiviral vector containing either BMP-2 or green fluorescent protein (GFP). All cells were loaded onto compression-resistant matrices and implanted in the bone defect of athymic rats. Treatment groups included femoral defects that were treated with a low-dose (1 × 106 cells), standard-dose (5 × 106 cells), and high-dose (1.5 × 107 cells) HBMCs transduced with lentiviral vector containing BMP-2 cDNA. The three control groups were bone defects treated with HBMCs that were either nontransduced or transduced with vector containing GFP. All animals were sacrificed at 12 weeks. The bone formed in each defect was evaluated with plain radiographs, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. Bone defects treated with higher doses of BMP-2-producing cells were more likely to have healed (6/14 of the low-dose group; 12/14 of the standard-dose group; 14/14 of the high-dose group; χ2(2) = 15.501, p < 0.001). None of the bone defects in the control groups had healed. Bone defects treated with high dose and standard dose of BMP-2-producing cells consistently outperformed those treated with a low dose in terms of bone formation, as assessed by microCT and histomorphometry, and biomechanical parameters. However, statistical significance was only seen between defects treated with high dose and low dose. Larger doses of BMP-2-producing cells were associated with a higher likelihood of forming heterotopic ossification. Femurs treated with a standard- and high-dose BMP-2-producing cells demonstrated similar healing and biomechanical properties. Increased doses of BMP-2 delivered through higher cell doses have the potential to heal large bone defects. Adapting regional gene therapy for use in humans will require a balance between promoting bone repair and limiting heterotopic ossification.

Adipogenic Differentiation Alters Properties of Vascularized Tissue-Engineered Skeletal Muscle

AUTHORS

Francisca M. Acosta, Kennedy K. Howland, Katerina Stojkova, Elizabeth Hernandez, Eric M. Brey, and Christopher R. Rathbone

ABSTRACT

Advances in the engineering of comprehensive skeletal muscle models in vitro will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes.

Deletion of Glut1 in early postnatal cartilage reprograms chondrocytes toward enhanced glutamine oxidation

AUTHORS

Cuicui Wang, Jun Ying, Xiangfeng Niu, Xiaofei Li, Gary J. Patti, Jie Shen & Regis J. O’Keefe

ABSTRACT

Glucose metabolism is fundamental for the functions of all tissues, including cartilage. Despite the emerging evidence related to glucose metabolism in the regulation of prenatal cartilage development, little is known about the role of glucose metabolism and its biochemical basis in postnatal cartilage growth and homeostasis. We show here that genetic deletion of the glucose transporter Glut1 in postnatal cartilage impairs cell proliferation and matrix production in growth plate (GPs) but paradoxically increases cartilage remnants in the metaphysis, resulting in shortening of long bones. On the other hand, articular cartilage (AC) with Glut1 deficiency presents diminished cellularity and loss of proteoglycans, which ultimately progress to cartilage fibrosis. Moreover, predisposition to Glut1 deficiency severely exacerbates injury-induced osteoarthritis. Regardless of the disparities in glucose metabolism between GP and AC chondrocytes under normal conditions, both types of chondrocytes demonstrate metabolic plasticity to enhance glutamine utilization and oxidation in the absence of glucose availability. However, uncontrolled glutamine flux causes collagen overmodification, thus affecting extracellular matrix remodeling in both cartilage compartments. These results uncover the pivotal and distinct roles of Glut1-mediated glucose metabolism in two of the postnatal cartilage compartments and link some cartilage abnormalities to altered glucose/glutamine metabolism.

Morin attenuates osteoclast formation and function by suppressing the NF-κB, MAPK and calcium signalling pathways

AUTHORS

Yifeng Shi, Lin Ye, Shiwei Shen, Tianchen Qian, Youjin Pan, Yuhan Jiang, Jinghao Lin, Chen Liu, Yaosen Wu, Xiangyang Wang, Jiake Xu, Haiming Jin

ABSTRACT

Morin is a natural compound isolated from moraceae family members and has been reported to possess a range of pharmacological activities. However, the effects of morin on bone-associated disorders and the potential mechanism remain unknown. In this study, we investigated the anti-osteoclastogenic effect of morin in vitro and the potential therapeutic effects on ovariectomy (OVX)-induced osteoporosis in vivo. In vitro, by using a bone marrow macrophage-derived osteoclast culture system, we determined that morin attenuated receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation via the inhibition of the mitogen-activated protein kinase (MAPK), NF-κB and calcium pathways. In addition, the subsequent expression of nuclear factor of activated T cells c1 (NFATc1) and c-fos was significantly suppressed by morin. In addition, NFATc1 downregulation led to the reduced expression of osteoclastogenesis-related marker genes, such as V-ATPase-d2 and Integrin β3. In vivo, results provided that morin could effectively attenuate OVX-induced bone loss in C57BL/6 mice. In conclusion, our results demonstrated that morin suppressed RANKL-induced osteoclastogenesis via the NF-κB, MAPK and calcium pathways, in addition, its function of preventing OVX-induced bone loss in vivo, which suggested that morin may be a potential therapeutic agent for postmenopausal osteoporosis treatment.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling

AUTHORS

Yu Shi, Xueyang Liao, James Y.Long, Lutian Yao, Jianquan Chen, Bei Yin, Feng Lou, Guangxu He, Ling Ye, Ling Qin, Fanxin Long

ABSTRACT

Teriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.