Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes

Chronic non-healing wounds such as diabetic ulcers or burns represent a devastating health problem with significant clinical, physical and social implications. The healing can be frustrating and painful for patients. The difficult healing process requires advanced therapeutic strategies such as the use of primary human keratinocytes (HK) as autologous transplants, which may be considered for clinical use. To improve engraftment or to introduce therapeutic genes into primary HK, efficient and safe vectors are required. One of the most promising vector systems today is based on the adeno-associated virus (AAV), a member of the parvovirus family. Recombinant AAV (rAAV) vectors possess a number of attractive properties including low immunogenicity, high stability and the potential to integrate site-specifically without known side-effects. Unfortunately, cell entry into primary HK of rAAV2 is barely detectable and consequentially, HK are poor targets of rAAV2-mediated transductions. As demonstrated in this thesis, primary HK do not express AAV2´s primary receptor heparan sulphate proteoglycan (HSPG), the presence of which, however, is required for binding to AAV2´s internalization receptors. Cell surface targeting allows re-directing the viral vector tropism towards a novel receptor mediating thereby transduction of cells in absence of AAV’s natural receptors. These AAV capsid mutants have displayed improved transduction efficiency in wild-type-AAV non-permissive cells and have provided the opportunity of rAAV-mediated, cell-type-specific gene transfer. As documented in this study, new rAAV vectors were developed as promising tools for modifying primary HK. Using an AAV peptide display library that displayed 7mer peptides of random sequence at capsid position 587; three AAV peptide insertion mutants differing in sequence of inserted ligand (Kera1, Kera2 and Kera3) were selected and subsequently analyzed. To select rAAV targeting vectors with a re-directed tropism, the library was optimized by depleting mutants capable of binding to HSPG prior to selection by heparin affinity chromatography. Furthermore, the selection was performed on primary HK obtained from different donors to target a common receptor and the selection pressure was continuously increased by decreasing the vector genomes per cell ratio to select for the fittest variant. The thereby developed rAAV targeting vectors Kera1 (RGDTATL), Kera2 (PRGDLAP) and Kera3 (RGDQQSL) showed a remarkable change in tropism, transducing primary HK with high efficiency and specificity even in mixed cultures of target and non-target cells. In this study, a novel microarray based bioinformatic approach (comparative gene analysis (CGA)), was used for the identification of the receptor that targeted the mutant that showed the most striking change in tropism, Kera2. Briefly, in cooperation with Giovanni Di Pasquale (NCI/NIH, Bethesda, USA), a screening of the NIH cell line panel was performed, pointing towards the involvement of beta8 integrin subunit for cell transduction by Kera2. Beta8 is unique as it is solely described as heterodimer with alpha V and the integrin αVβ8 could be detected on cell surface of primary human keratinocytes. By blocking experiments with blocking αV- or αVβ8-antibodies experimental evidence was provided that the integrin αVβ8 serves as receptor for Kera2. Finally, this study has shown that the targeting vectors Kera1, Kera2 and Kera3 transduced airlifted differentiated keratinocytes in organotypic 3D cultures. In summary, the three rAAV targeting vectors Kera1, Kera2 and Kera3, selected from an optimized library and using a novel selection strategy, are excellent candidates for successful application in clinical use

Emerging contaminant exposure to aquatic systems in the Southern African Development Community

The growing production and use of chemicals and the resultant increase in environmental exposure is of particular concern in developing countries where there is rapid industrialization and population growth but limited information on the occurrence of emerging contaminants. Advances in analytical techniques now allow for the monitoring of emerging contaminants at very low concentrations with the potential to cause harmful ecotoxicological effects. Therefore, we provide the first critical assessment of the current state of knowledge about chemical exposure in waters of the Southern African Developmental Community (SADC). We achieved this through a comprehensive literature review and the creation of a database of chemical monitoring data. Of the 59 articles reviewed, most (n = 36; 61.0%) were from South Africa, and the rest were from Botswana (n = 6; 10.2%), Zimbabwe (n = 6; 10.2%), Malawi (n = 3; 5.1%), Mozambique (n = 3; 5.1%), Zambia (n = 2; 3.4%), Angola (n = 1; 1.7%), Madagascar (n = 1; 1.7%), and Tanzania (n = 1; 1.7%). No publications were found from the remaining seven SADC countries. Emerging contaminants have only been studied in South Africa and Botswana. The antiretroviral drug ritonavir (64.52 µg/L) was detected at the highest average concentration, and ibuprofen (17 times) was detected most frequently. Despite being the primary water source in the region, groundwater was understudied (only 13 studies). High emerging contaminant concentrations in surface waters indicate the presence of secondary sources of pollution such as sewage leakage. We identify research gaps and propose actions to assess and reduce chemical pollution to enable the SADC to address the Sustainable Development Goals, particularly Goal 3.9, to reduce the deaths and illnesses from hazardous chemicals and contamination. Environ Toxicol Chem 2022;41:382–395

Antibiotics and antibiotic resistance in agroecosystems : State of the science

We propose a simple causal model depicting relationships involved in dissemination of antibiotics and antibiotic resistance in agroecosystems and potential effects on human health, functioning of natural ecosystems, and agricultural productivity. Available evidence for each causal link is briefly summarized, and key knowledge gaps are highlighted. A lack of quantitative estimates of human exposure to environmental bacteria, in general, and antibiotic-resistant bacteria, specifically, is a significant data gap hindering the assessment of effects on human health. The contribution of horizontal gene transfer to resistance in the environment and conditions that might foster the horizontal transfer of antibiotic resistance genes into human pathogens also need further research. Existing research has focused heavily on human health effects, with relatively little known about the effects of antibiotics and antibiotic resistance on natural and agricultural ecosystems. The proposed causal model is used to elucidate gaps in knowledge that must be addressed by the research community and may provide a useful starting point for the design and analysis of future research efforts

Emerging contaminant exposure to aquatic systems in the Southern African Developmental Community

The growing production and use of chemicals and the resultant increase in environmental exposure is of particular concern in developing countries where there is rapid industrialization and population growth but limited information on the occurrence of emerging contaminants. Advances in analytical techniques now allow for the monitoring of emerging contaminants at very low concentrations with the potential to cause harmful ecotoxicological effects. Therefore, we provide the first critical assessment of the current state of knowledge about chemical exposure in waters of the Southern African Developmental Community (SADC). We achieved this through a comprehensive literature review and the creation of a database of chemical monitoring data. Of the 59 articles reviewed, most (n = 36; 61.0%) were from South Africa, and the rest were from Botswana (n = 6; 10.2%), Zimbabwe (n = 6; 10.2%), Malawi (n = 3; 5.1%), Mozambique (n = 3; 5.1%), Zambia (n = 2; 3.4%), Angola (n = 1; 1.7%), Madagascar (n = 1; 1.7%), and Tanzania (n = 1; 1.7%). No publications were found from the remaining seven SADC countries. Emerging contaminants have only been studied in South Africa and Botswana. The antiretroviral drug ritonavir (64.52 µg/L) was detected at the highest average concentration, and ibuprofen (17 times) was detected most frequently. Despite being the primary water source in the region, groundwater was understudied (only 13 studies). High emerging contaminant concentrations in surface waters indicate the presence of secondary sources of pollution such as sewage leakage. We identify research gaps and propose actions to assess and reduce chemical pollution to enable the SADC to address the Sustainable Development Goals, particularly Goal 3.9, to reduce the deaths and illnesses from hazardous chemicals and contamination

Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction

Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lenti-viral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying alpha v beta 8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy

Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy