Recombinant adeno-associated viruses (AAVs) have emerged as a widely used gene delivery platform for both basic research and human gene therapy. To ensure and improve the safety profile of AAV vectors, substantial efforts have been dedicated to the vector production process development using suspension HEK293 cells. Here, we studied and compared two downstream purification methods, iodixanol gradient ultracentrifugation versus immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column). We tested multiple vector batches that were separately produced (including AAV5, AAV8, and AAV9 serotypes). To account for batch-to-batch variability, each batch was halved for subsequent purification by either iodixanol gradient centrifugation or affinity chromatography. In parallel, purified vectors were characterized, and transduction was compared both in vitro and in vivo in mice (using multiple transgenes: Gaussia luciferase, eGFP, and human factor IX). Each purification method was found to have its own advantages and disadvantages regarding purity, viral genome (vg) recovery, and relative empty particle content. Differences in transduction efficiency were found to reflect batch-to-batch variability rather than disparities between the two purification methods, which were similarly capable of yielding potent AAV vectors.

Anh K. Lam

Patrick L. Mulcrone

Dylan Frabutt

Junping Zhang

Matthew Chrzanowski

Sreevani Arisa

Maite Munoz

Xin Li

Moanaro Biswas

David Markusic

Roland W. Herzog

Weidong Xiao

Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity Chromatography

Liver cirrhosis is the final stage of chronic liver disease and can be life-threatening. Despite extensive studies on its treatment, a standard therapy is yet to be developed. Considering the complex mechanism of fibrogenic and fibrolytic processes in liver cirrhosis, combined therapy may have clinically significant effects on cirrhotic livers. In this study, we used thioacetamide (TAA) administration and matrix metalloproteinase-13 (MMP13) gene delivery to induce extracellular matrix generation and degradation in rats. The aim of this study was to determine whether hydrodynamics-based gene delivery of MMP13 to cirrhotic liver has regressive and suppressive effects on fibrogenesis. MMP13-encoding plasmids were hydrodynamically delivered to TAA-induced cirrhotic livers, and intravascular pressure was monitored. Therapeutic effect with and without continuous TAA exposure was assessed 8 weeks after the gene delivery. Test results indicated successful gene delivery and gene expression in the cirrhotic livers. Furthermore, microscopic imaging showed that MMP13 delivery resulted in significant degradation of fibrotic areas. Quantitative analysis of hydroxyproline content supported the microscopic findings. These results suggest that transgene delivery of MMP13 can be a promising candidate to treat liver fibrosis and that hydrodynamics-based gene delivery can be a good option for delivery of MMP13 to cirrhotic livers.

Takeshi Yokoo

Kenya Kamimura

Ryosuke Nozawa

Moeno Sugita

Osamu Shibata

Yuji Kobayashi

Hiroyuki Abe

Hiromi Miura

Masato Ohtsuka

Shuji Terai

Therapeutic Effect of Hydrodynamics-Based Delivery of Matrix Metalloproteinase-13 Gene on Thioacetamide-Induced Liver Fibrosis in Rats

Mucopolysaccharidosis type I (MPS I), an inherited lysosomal storage disorder characterized by deficiency of α-L-iduronidase (IDUA) activity, causes multisystemic pathology due to sequelae of accumulated heparan and dermatan sulfates (HS and DS), the substrates of IDUA. Current treatments, though life-prolonging, inadequately address skeletal dysplasia and do not forestall progressive and painful degenerative joint disease. Previous studies demonstrated that intra-articular enzyme replacement cleared cellular lysosomal storage and reduced joint inflammation. Three nontolerized MPS I canines were studied to assess safety, efficacy, and durability of IDUA gene replacement therapy delivered via intra-articular injection. After baseline joint tissue biopsies, the right shoulder and stifle of each animal were injected in the intra-articular space with AAV9-IDUA and contralateral joints with AAV9-eGFP. Animals received either 5E11 or 5E12 vector genomes/joint. Necropsy was performed at 2- or 52-week postinjection. All animals tolerated injections without adverse effects. At two weeks, supraphysiologic IDUA enzyme activity was measured in AAV9-IDUA-treated but not AAV9-eGFP-treated synovium, with corresponding normalization of HS content and synoviocyte morphology. The AAV9-IDUA-treated cartilage had normal physiologic levels of IDUA enzyme, reduced but not normalized HS and DS levels compared to untreated MPS I cartilage, and healthy chondrocyte morphology. Liver IDUA transgene and IDUA enzyme activity were identified, as was serum IDUA activity which was 40% of wild-type serum enzyme activity. At 52-week postinjection, AAV9-IDUA-treated synovium and cartilage IDUA enzyme activity declined in both animals, corresponding to high tissue HS and DS levels and severe lysosomal storage. Liver and serum IDUA activity levels were undetectable. A dose-dependent serum anti-IDUA antibody response was observed which, together with loss of transgene with age, likely contributed to decline in tissue enzyme activity and treatment efficacy. Our study demonstrates successful proof-of-concept for intra-articular gene replacement therapy as a treatment for MPS-related joint dysplasia. Our observations suggest the possibility of multimodal gene replacement therapy to address multiple refractory manifestations of MPS I. Subsequent studies, in conjunction with immune tolerization and functional assessments of joint pathology, will investigate this possibility.

Raymond Yu-Jeang Wang

Shih-Hsin Kan

Haoyue Zhang

Jodi D. Smith

Afshin Aminian

Elizabeth Snella

Jackie K. Jens

Sarah P. Young

Patricia I. Dickson

N. Matthew Ellinwood

Intra-Articular AAV9 α-L-Iduronidase Gene Replacement in the Canine Model of Mucopolysaccharidosis Type I

The study of an organism’s genome, often known as “genomics,” has advanced quickly, producing a wealth of publicly accessible genetic data. Despite how valuable the genome is; proteins essentially control most aspects of cell function. Proteomics, or the comprehensive study of proteins, has emerged as an important technology for disease characterization, diagnosis, prognosis, drug development, and therapy. Proteomics technologies are now used to support the diagnosis and treatment of both infectious and noninfectious diseases. Nevertheless, it is more difficult to describe a proteomic profile since a single gene product may result in a number of unique proteins, and proteins have a wider range of chemical configurations. The proteome profiles of a particular organism, tissue, or cell are impacted by a variety of environmental factors, including those triggered by infectious agents. This review intends to highlight the applications of proteomics in the study of disease diagnosis and treatment. In this review, the different technologies used in proteomics studies, like two-dimensional gel electrophoresis, mass spectrometry, and protein microarray as well as biomarker discovery and drug target identification using proteomics, have also been focused on.

Fanuel Bizuayehu Yihunie

Mequanint Addisu Belete

Gizachew Fentahun

Solomon Getachew

Teshager Dubie

Diagnostic and Therapeutic Application of Proteomics in Infectious Disease

Mendelian genetics contribute largely to the development of hypertension; therefore, the identification of genetic variants related to blood pressure (BP) regulation remains crucial and may reveal new therapeutic drug targets. The purpose of the present study was to screen the captive-bred Vervet colony for salt-sensitive sequence variants or single nucleotide polymorphisms (SNPs) in the selected Renin-Angiotensin-Aldosterone System (RAAS) genes associated with salt sensitivity. Blood samples were collected from 16 captive-bred Vervet monkeys for genotyping and gene expression analysis. The impact of the identified sequence variants was determined using online prediction tools. Sanger sequencing analysis revealed 21 sequence variants in AGT, CYP3A5, GRK4, and SCL4A5, of which 19 were novel and two were previously reported in humans. All novel variants were either predicted to be polymorphic, disease-causing, or possibly damaging by prediction tools. Furthermore, the mRNA expression for AGT was significantly higher in the normal BP group (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.42943pt" id="M1" height="10.2124pt" version="1.1" viewBox="-0.0498162 -6.78297 7.83752 10.2124" width="7.83752pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-113" d="M570 304C570 398 525 448 414 448C385 448 343 445 312 434L329 511L321 518C297 504 262 482 244 460L233 411C195 397 159 381 128 358L135 332C160 347 189 360 224 373L111 -147C97 -210 84 -218 17 -231L13 -257L254 -247L259 -218L233 -216C183 -212 177 -202 189 -142L218 -1C238 -10 266 -12 283 -12C351 3 429 48 483 105C543 168 570 242 570 304ZM482 289C482 161 380 33 304 33C278 33 248 51 233 69L303 396C326 400 352 403 369 403C428 403 482 380 482 289Z"/></g></svg> value = 0.02), and a similar trend was observed for CYP3A5 and GRK4, whereas SCL4A5 was higher in the hypertensive group. The identified salt-sensitive variants specifically in GRK4 may be suggestive to be the attributing factor of the elevated BP levels in these captive-bred Vervet monkeys. Therefore, RAAS variants could be considered as a biomarker to identify the potential risk of developing hypertension in both humans and nonhuman primates.

Sanele Khoza

Meenu Ghai

Chesa G. Chauke

Zandisiwe E. Magwebu

The Association between Genetic Variants and Gene Expression in RAAS Genes Using Captive-Bred Vervet Monkeys (Chlorocebus aethiops)

Immunotherapy for cancer treatment is growing at an unprecedented rate since the inception of chimeric antigen receptor T (CAR-T) cells. However, the efficacy of CAR-T cells against solid tumors is hampered by various issues, including “on-target, off-tumor toxicities,” T cell exhaustion, and immunosuppressive tumor microenvironment. To overcome these limitations, recent advances focus on optimizing CAR-T cells using vaccines to develop more effective cell immunotherapies. Here, we summarize the most recent studies on how vaccine-based CAR-T therapies are advancing the response of cancer immunotherapy as well as the current state of their clinical and preclinical development. Finally, we share perspectives on how future studies can incorporate other strategies to augment the antitumor response of vaccine-assisted CAR-T cell therapy.

Emmanuel Owusu Ansah

Andy Baah

Emmanuel Boateng Agyenim

Vaccine Boosting CAR-T Cell Therapy: Current and Future Strategies

About this journal

Advances in Cell and Gene Therapy publishes original, high-quality, peer-reviewed articles covering basic and clinical research relating to advances in cell, gene, and immune therapies and their use in the treatment of a range of diseases, including cancer, genetic and immune diseases, and others.

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Chief Editor, Carol H. Miao, is Professor of Pediatrics at University of Washington and&nbsp;Principal Investigator in the&nbsp;Center for Immunity and Immunotherapies at&nbsp;Seattle Children's Research Institute.&nbsp;

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Advances in Cell and Gene Therapy publishes original, high-quality, peer-reviewed articles covering basic and clinical research relating to advances in cell, gene, and immune therapies and their use in the treatment of a range of diseases, including cancer, genetic and immune diseases, and others.

Advances in Cell and Gene Therapy