Mesenchymal stem/stromal cells engineered to express the protease inhibitor alpha-1 antitrypsin for the treatment of inflammatory lung diseases

Chronic inflammatory diseases are a growing global health problem. Chronic obstructive pulmonary disease (COPD) represents a major cause of morbidity and mortality, and is currently the third leading cause of death worldwide. COPD is a devastating progressive lung disease characterized by airflow limitation, chronic bronchitis and bronchiolitis, emphysema, and small airway remodeling. There are no curative options for COPD to date, thus there is a desperate need for novel approaches and treatments. To this end, the goal of this thesis work was to develop and evaluate a novel cell product composed of mesenchymal stem/stromal cells (MSCs) genetically modified to express the protease inhibitor alpha-1 antitrypsin (AAT), thus combining the potent immunomodulatory and cytoprotective properties of MSCs, and of AAT. A major focus was the development of an optimized expression construct/vector system for MSC engineering. Eight versions of gamma-retroviral-based vectors were designed and tested for optimal transgenic expression of human AAT and puromycin-N-acetyltransferase (pac) for antibiotic selection. The constructs differed in the gene promoter used for driving AAT expression, and in the relative position of the expression cassettes within the vector backbone. The vectors were then compared with regards to viral titers, transduction efficacy of primary human MSCs, AAT expression in the supernatant of transduced cells, and cell yield after antibiotic selection. The best results for all parameters were obtained when AAT was expressed from the human eukaryotic translation elongation factor 1 alpha 1 short (EFS) promoter, and the pac gene was linked via an internal ribosome entry site (IRES) sequence. This lead candidate expression cassette was then transferred to a lentiviral backbone to assess if transduction and AAT expression from MSCs could be further increased by using this viral system. While values were higher with the gamma-retroviral vector at low multiplicities of infection (MOIs), transduction reached a plateau and no further increase was possible even when higher MOIs were used. Transduction with the lentiviral construct, however, followed a clear dose-response, reaching transduction efficacies >80% at the highest MOI tested. In vitro functionality of the protease inhibitor AAT expressed from MSCs was confirmed using a neutrophil elastase inhibition assay. A process for large scale transduction and expansion of gene-modified MSCs was subsequently developed. To evaluate the potential therapeutic benefit of AAT-MSCs in vivo, a mouse model of elastase-induced emphysema was used. Treatment of the mice with AAT-MSCs resulted in significant improvement of pulmonary function parameters, while only a slight functional amelioration was observed after treatment with non-modified MSCs. Histopathologic examination of AAT-MSC-treated mice revealed a significant decrease of airspace enlargement, indicating regeneration of pulmonary tissue. Again, the effect was smaller after treatment with non-modified MSCs, demonstrating a more than additive effect of combining AAT and MSCs. In conclusion, the present study provides the first in vivo proof of concept for the treatment of emphysematous COPD using engineered AAT-MSCs.Chronisch entzündliche Erkrankungen sind heutzutage ein globales, im Steigen begriffenes Gesundheitsproblem. Ein wichtiges Beispiel ist die chronisch obstruktive Lungenerkrankung (COPD), eine verheerende, fortschreitende Erkrankung der Lunge mit hoher Morbidität und Mortalität, gegenwärtig die dritthäufigste Todesursache weltweit. Symptome der COPD sind Atemnot, eine chronische Entzündung der Bronchien und Bronchiolen, die Entwicklung eines Emphysems und ein Umbau der engen Atemwege. Da bislang eine Heilung der COPD nicht möglich ist, besteht ein dringender Bedarf an innovativen Therapieansätzen. Wir entwickelten ein neuartiges Zellprodukt, basierend auf mesenchymalen Stamm-/Stromazellen (MSCs), die genetisch modifiziert wurden, sodass sie den Protease-Inhibitor Alpha-1 Antitrypsin (AAT) exprimieren. Diese Kombination ermöglicht es, die immunmodulatorischen und zytoprotektiven Eigenschaften der MSCs mit jenen von AAT in einem Produkt zu vereinen. Dabei wurde in der vorliegenden Arbeit ein Fokus auf die Entwicklung eines optimierten Expressionskonstruktes gelegt. Es wurden acht gamma-retrovirale Vektoren für die Expression von AAT und Puromycin-N-Acetyltransferase (pac), das eine Antibiotikaselektion genmodifizierter Zellen erlaubt, konzipiert und kloniert. Diese Konstrukte unterscheiden sich sowohl im Promoter, der für die AAT-Expression verwendet wird, als auch in der relativen Anordnung der Expressionskassetten im Plasmid. Die Auswahl des besten Konstrukts wurde anhand eines Vergleichs der Vektoren hinsichtlich der erzielten viralen Titer, der Transduktionseffizienz von primären humanen MSCs, des AAT-Gehalts im Zellkulturüberstand von transduzierten MSCs sowie der Zellausbeute nach Antibiotikaselektion durchgeführt. Die jeweils besten Ergebnisse wurden erzielt, wenn AAT von der kurzen Form des Promoters des humanen eukaryotischen Translationselongationsfaktors 1 alpha 1 (EFS) exprimiert wurde, und das pac Gen durch eine interne ribosomale Eintrittsstelle- (IRES)-Sequenz verknüpft war. Diese favorisierte Expressionskassette wurde in ein lentivirales Plasmid transferiert, um zu untersuchen, ob Transduktion und AAT-Expression in einem solchen System weiter verbessert werden können. Es zeigte sich, dass mittels des gamma-retroviralen Vektors größere Transduktionseffizienzen und AAT-Mengen bei niedriger Multiplizität der Infektion (MOI) erzielt werden konnten, aber bei höheren MOIs keine zusätzliche Verbesserung zu erreichen war. Im Gegensatz dazu folgten Transduktion und AAT-Expression mittels des lentiviralen Vektors einer eindeutigen Dosis-Wirkung, die bei hohen MOIs in Transduktionseffizienzen von >80% resultierte. Ein Neutrophile Elastase Inhibitionstest bestätigte die in vitro Funktionalität von MSC-sezerniertem AAT als Proteaseinhibitor, bevor Transduktions- und Expansionsprozesse zur Herstellung von AAT-MSCs im Großformat etabliert wurden. Um die therapeutische Wirkung von AAT-MSCs in vivo zu untersuchen, wurde ein Mausmodell eines Elastase-induzierten Emphysems durchgeführt. Es zeigte sich, dass die Behandlung von Mäusen mit AAT-MSCs zu einer signifikanten Verbesserung der Lungenfunktion der Tiere führte, während nicht-modifizierte MSCs nur eine geringfügige Wiederherstellung der pulmonalen Funktion bewirkten. Die histopathologische Analyse ergab, dass die Überblähung der Lunge durch die Applikation von AAT-MSCs signifikant vermindert werden konnte, was auf eine Regeneration des Lungengewebes hindeutet. Auch in dieser Auswertung fiel der durch die Behandlung mit nicht-modifizierten MSCs erzielte Effekt geringer aus, was eine additive Wirkung von AAT und MSCs nahelegt. Zusammenfassend kann festgestellt werden, dass die vorliegende Arbeit einen ersten Nachweis des Wirkkonzepts von AAT-MSCs für die Behandlung von entzündlichen Lungenerkrankungen wie emphysematöser COPD erbringen konnte

Mesenchymal stem/stromal cells engineered to express the protease inhibitor alpha-1 antitrypsin for the treatment of inflammatory lung diseases

Chronic inflammatory diseases are a growing global health problem. Chronic obstructive pulmonary disease (COPD) represents a major cause of morbidity and mortality, and is currently the third leading cause of death worldwide. COPD is a devastating progressive lung disease characterized by airflow limitation, chronic bronchitis and bronchiolitis, emphysema, and small airway remodeling. There are no curative options for COPD to date, thus there is a desperate need for novel approaches and treatments. To this end, the goal of this thesis work was to develop and evaluate a novel cell product composed of mesenchymal stem/stromal cells (MSCs) genetically modified to express the protease inhibitor alpha-1 antitrypsin (AAT), thus combining the potent immunomodulatory and cytoprotective properties of MSCs, and of AAT. A major focus was the development of an optimized expression construct/vector system for MSC engineering. Eight versions of gamma-retroviral-based vectors were designed and tested for optimal transgenic expression of human AAT and puromycin-N-acetyltransferase (pac) for antibiotic selection. The constructs differed in the gene promoter used for driving AAT expression, and in the relative position of the expression cassettes within the vector backbone. The vectors were then compared with regards to viral titers, transduction efficacy of primary human MSCs, AAT expression in the supernatant of transduced cells, and cell yield after antibiotic selection. The best results for all parameters were obtained when AAT was expressed from the human eukaryotic translation elongation factor 1 alpha 1 short (EFS) promoter, and the pac gene was linked via an internal ribosome entry site (IRES) sequence. This lead candidate expression cassette was then transferred to a lentiviral backbone to assess if transduction and AAT expression from MSCs could be further increased by using this viral system. While values were higher with the gamma-retroviral vector at low multiplicities of infection (MOIs), transduction reached a plateau and no further increase was possible even when higher MOIs were used. Transduction with the lentiviral construct, however, followed a clear dose-response, reaching transduction efficacies >80% at the highest MOI tested. In vitro functionality of the protease inhibitor AAT expressed from MSCs was confirmed using a neutrophil elastase inhibition assay. A process for large scale transduction and expansion of gene-modified MSCs was subsequently developed. To evaluate the potential therapeutic benefit of AAT-MSCs in vivo, a mouse model of elastase-induced emphysema was used. Treatment of the mice with AAT-MSCs resulted in significant improvement of pulmonary function parameters, while only a slight functional amelioration was observed after treatment with non-modified MSCs. Histopathologic examination of AAT-MSC-treated mice revealed a significant decrease of airspace enlargement, indicating regeneration of pulmonary tissue. Again, the effect was smaller after treatment with non-modified MSCs, demonstrating a more than additive effect of combining AAT and MSCs. In conclusion, the present study provides the first in vivo proof of concept for the treatment of emphysematous COPD using engineered AAT-MSCs.Chronisch entzündliche Erkrankungen sind heutzutage ein globales, im Steigen begriffenes Gesundheitsproblem. Ein wichtiges Beispiel ist die chronisch obstruktive Lungenerkrankung (COPD), eine verheerende, fortschreitende Erkrankung der Lunge mit hoher Morbidität und Mortalität, gegenwärtig die dritthäufigste Todesursache weltweit. Symptome der COPD sind Atemnot, eine chronische Entzündung der Bronchien und Bronchiolen, die Entwicklung eines Emphysems und ein Umbau der engen Atemwege. Da bislang eine Heilung der COPD nicht möglich ist, besteht ein dringender Bedarf an innovativen Therapieansätzen. Wir entwickelten ein neuartiges Zellprodukt, basierend auf mesenchymalen Stamm-/Stromazellen (MSCs), die genetisch modifiziert wurden, sodass sie den Protease-Inhibitor Alpha-1 Antitrypsin (AAT) exprimieren. Diese Kombination ermöglicht es, die immunmodulatorischen und zytoprotektiven Eigenschaften der MSCs mit jenen von AAT in einem Produkt zu vereinen. Dabei wurde in der vorliegenden Arbeit ein Fokus auf die Entwicklung eines optimierten Expressionskonstruktes gelegt. Es wurden acht gamma-retrovirale Vektoren für die Expression von AAT und Puromycin-N-Acetyltransferase (pac), das eine Antibiotikaselektion genmodifizierter Zellen erlaubt, konzipiert und kloniert. Diese Konstrukte unterscheiden sich sowohl im Promoter, der für die AAT-Expression verwendet wird, als auch in der relativen Anordnung der Expressionskassetten im Plasmid. Die Auswahl des besten Konstrukts wurde anhand eines Vergleichs der Vektoren hinsichtlich der erzielten viralen Titer, der Transduktionseffizienz von primären humanen MSCs, des AAT-Gehalts im Zellkulturüberstand von transduzierten MSCs sowie der Zellausbeute nach Antibiotikaselektion durchgeführt. Die jeweils besten Ergebnisse wurden erzielt, wenn AAT von der kurzen Form des Promoters des humanen eukaryotischen Translationselongationsfaktors 1 alpha 1 (EFS) exprimiert wurde, und das pac Gen durch eine interne ribosomale Eintrittsstelle- (IRES)-Sequenz verknüpft war. Diese favorisierte Expressionskassette wurde in ein lentivirales Plasmid transferiert, um zu untersuchen, ob Transduktion und AAT-Expression in einem solchen System weiter verbessert werden können. Es zeigte sich, dass mittels des gamma-retroviralen Vektors größere Transduktionseffizienzen und AAT-Mengen bei niedriger Multiplizität der Infektion (MOI) erzielt werden konnten, aber bei höheren MOIs keine zusätzliche Verbesserung zu erreichen war. Im Gegensatz dazu folgten Transduktion und AAT-Expression mittels des lentiviralen Vektors einer eindeutigen Dosis-Wirkung, die bei hohen MOIs in Transduktionseffizienzen von >80% resultierte. Ein Neutrophile Elastase Inhibitionstest bestätigte die in vitro Funktionalität von MSC-sezerniertem AAT als Proteaseinhibitor, bevor Transduktions- und Expansionsprozesse zur Herstellung von AAT-MSCs im Großformat etabliert wurden. Um die therapeutische Wirkung von AAT-MSCs in vivo zu untersuchen, wurde ein Mausmodell eines Elastase-induzierten Emphysems durchgeführt. Es zeigte sich, dass die Behandlung von Mäusen mit AAT-MSCs zu einer signifikanten Verbesserung der Lungenfunktion der Tiere führte, während nicht-modifizierte MSCs nur eine geringfügige Wiederherstellung der pulmonalen Funktion bewirkten. Die histopathologische Analyse ergab, dass die Überblähung der Lunge durch die Applikation von AAT-MSCs signifikant vermindert werden konnte, was auf eine Regeneration des Lungengewebes hindeutet. Auch in dieser Auswertung fiel der durch die Behandlung mit nicht-modifizierten MSCs erzielte Effekt geringer aus, was eine additive Wirkung von AAT und MSCs nahelegt. Zusammenfassend kann festgestellt werden, dass die vorliegende Arbeit einen ersten Nachweis des Wirkkonzepts von AAT-MSCs für die Behandlung von entzündlichen Lungenerkrankungen wie emphysematöser COPD erbringen konnte

Criteria for vaccine introduction: results of a DELPHI discussion among international immunisation experts on a stepwise decision-making procedure

Background: Based on a model of a stepwise approach for decision-making on vaccine introduction, this study aimed to reveal unpublished decision aids, to assess cut-off limits or thresholds for vaccine introduction that have already been used, and to discuss the comprehensiveness and feasibility of our suggested model. Methods: Forty international immunisation experts were invited to a DELPHI discussion, 14 finally participated. Experts received a questionnaire and were asked for comments on other experts’ opinions and specification of their previously given answers in the second DELPHI round. We did not intend to develop a consensus document. Results: Though most of the DELPHI participants were not aware of decision aids other than the five that had been used for the development of our model, the international discussion revealed four additional national documents that define decision-making criteria. Except for one example with a cost-utility ratio, no defined thresholds or cut-off limits have been used in vaccine introduction decisions so far. The majority of experts believe that a stepwise approach could enhance the feasibility of decision aids. The experts agreed that the influence of each single criterion of our model should be at least “important” for decision-making. The most often mentioned possible negative consequence that could arise from a rigid stepwise procedure, was a delay of the vaccine introduction process. Conclusions: The suggested stepwise procedure provides a systematic and evidence-based standardised way to support public health immunisation policy decisions. A framework could be a common starting point

End of life care for frail older patients in family practice (ELFOP) – protocol of a longitudinal qualitative study on needs, appropriateness and utilisation of services

BACKGROUND: Frail elderly people represent a major patient group in family practice. Little is known about the patients’ needs, and how their needs evolve over time with increasing frailty towards the end of life. This study will address end-of-life care needs, service utilisation, and experiences of frail elderly patients and their informal caregivers, with regard to family practice. This paper aims to introduce the research protocol. METHODS/DESIGN: The study uses a multiple perspective approach qualitative design. The first study part consists of serial six-monthly in-depth interviews with 30 community-dwelling elderly patients (≥70 years) with moderate to severe frailty and their key informal caregivers, over a period of 18 months. Additionally, semi-structured interviews with the patients’ family physician will be conducted. The serial interviews will be analysed with grounded theory and narrative approaches. Special attention will be paid to the comparison of distinct views of the patients’, informal caregivers’, and family physicians’ as well as on chronological aspects. In the second study part, five focus groups with experts in family medicine, geriatrics, palliative medicine, and nursing will be conducted. Finally, the implications for family practice and health care policy will be discussed in an expert workshop. DISCUSSION: To our knowledge, this is the first prospective, longitudinal qualitative study on the needs of elderly patients with advanced frailty towards the end of life in German family practice, which integrates the perspectives of patients, informal caregivers, family physicians and other health professionals. The study will contribute to the understanding of the clinical, psychosocial and information needs of patients and their caregivers, and of respective changes of experiences and needs along the illness/frailty trajectory including the last phase of life. It will provide an empirical basis for improving patient-centred care for this increasingly relevant target group

Skeletal variation in bird domestication: limb proportions and sternum in chicken, with comparisons to mallard ducks and Muscovy ducks.

Background Domestication, including selective breeding, can lead to morphological changes of biomechanical relevance. In birds, limb proportions and sternum characteristics are of great importance and have been studied in the past for their relation with flight, terrestrial locomotion and animal welfare. In this work we studied the effects of domestication and breed formation in limb proportions and sternum characteristics in chicken (Gallus gallus), mallard ducks (Anas plathyrhynchos) and Muscovy ducks (Cairina moschata). Methods First, we quantified the proportional length of three long bones of the forelimb (humerus, radius, and carpometacarpus) and the hind limb (femur, tibiotarsus, and tarsometatarsus) in domestic chickens, mallard ducks, and Muscovy ducks and their wild counterparts. For this, we took linear measurements of these bones and compared their proportions in the wild vs. the domestic group in each species. In chicken, these comparisons could also be conducted among different breeds. We then evaluated the proportional differences in the context of static and ontogenetic allometry. Further, we compared discrete sternum characteristics in red jungle fowl and chicken breeds. In total, we examined limb bones of 287 specimens and keel bones of 63 specimens. Results We found a lack of significant change in the proportions of limb bones of chicken and Muscovy duck due to domestication, but significant differences in the case of mallard ducks. Variation of evolvability, allometric scaling, and heterochrony may serve to describe some of the patterns of change we report. Flight capacity loss in mallard ducks resulting from domestication may have a relation with the difference in limb proportions. The lack of variation in proportions that could distinguish domestic from wild forms of chicken and Muscovy ducks may reflect no selection for flight capacity during the domestication process in these groups. In chicken, some of the differences identified in the traits discussed are breed-dependent. The study of the sternum revealed that the condition of crooked keel was not unique to domestic chicken, that some sternal characteristics were more frequent in certain chicken breeds than in others, and that overall there were no keel characteristics that are unique for certain chicken breeds. Despite some similar morphological changes identified across species, this study highlights the lack of universal patterns in domestication and breed formation

Algorithms to identify COPD in health systems with and without access to ICD coding: a systematic review

Background Chronic obstructive pulmonary disease (COPD) causes significant morbidity and mortality worldwide. Estimation of incidence, prevalence and disease burden through routine insurance data is challenging because of under-diagnosis and under-treatment, particularly for early stage disease in health care systems where outpatient International Classification of Diseases (ICD) diagnoses are not collected. This poses the question of which criteria are commonly applied to identify COPD patients in claims datasets in the absence of ICD diagnoses, and which information can be used as a substitute. The aim of this systematic review is to summarize previously reported methodological approaches for the identification of COPD patients through routine data and to compile potential criteria for the identification of COPD patients if ICD codes are not available. Methods A systematic literature review was performed in Medline via PubMed and Google Scholar from January 2000 through October 2018, followed by a manual review of the included studies by at least two independent raters. Study characteristics and all identifying criteria used in the studies were systematically extracted from the publications, categorized, and compiled in evidence tables. Results In total, the systematic search yielded 151 publications. After title and abstract screening, 38 publications were included into the systematic assessment. In these studies, the most frequently used (22/38) criteria set to identify COPD patients included ICD codes, hospitalization, and ambulatory visits. Only four out of 38 studies used methods other than ICD coding. In a significant proportion of studies, the age range of the target population (33/38) and hospitalization (30/38) were provided. Ambulatory data were included in 24, physician claims in 22, and pharmaceutical data in 18 studies. Only five studies used spirometry, two used surgery and one used oxygen therapy. Conclusions A variety of different criteria is used for the identification of COPD from routine data. The most promising criteria set in data environments where ambulatory diagnosis codes are lacking is the consideration of additional illness-related information with special attention to pharmacotherapy data. Further health services research should focus on the application of more systematic internal and/or external validation approaches

Skeletal variation in bird domestication: limb proportions and sternum in chicken, with comparisons to mallard ducks and Muscovy ducks

Background Domestication, including selective breeding, can lead to morphological changes of biomechanical relevance. In birds, limb proportions and sternum characteristics are of great importance and have been studied in the past for their relation with flight, terrestrial locomotion and animal welfare. In this work we studied the effects of domestication and breed formation in limb proportions and sternum characteristics in chicken (Gallus gallus), mallard ducks (Anas plathyrhynchos) and Muscovy ducks (Cairina moschata). Methods First, we quantified the proportional length of three long bones of the forelimb (humerus, radius, and carpometacarpus) and the hind limb (femur, tibiotarsus, and tarsometatarsus) in domestic chickens, mallard ducks, and Muscovy ducks and their wild counterparts. For this, we took linear measurements of these bones and compared their proportions in the wild vs. the domestic group in each species. In chicken, these comparisons could also be conducted among different breeds. We then evaluated the proportional differences in the context of static and ontogenetic allometry. Further, we compared discrete sternum characteristics in red jungle fowl and chicken breeds. In total, we examined limb bones of 287 specimens and keel bones of 63 specimens. Results We found a lack of significant change in the proportions of limb bones of chicken and Muscovy duck due to domestication, but significant differences in the case of mallard ducks. Variation of evolvability, allometric scaling, and heterochrony may serve to describe some of the patterns of change we report. Flight capacity loss in mallard ducks resulting from domestication may have a relation with the difference in limb proportions. The lack of variation in proportions that could distinguish domestic from wild forms of chicken and Muscovy ducks may reflect no selection for flight capacity during the domestication process in these groups. In chicken, some of the differences identified in the traits discussed are breed-dependent. The study of the sternum revealed that the condition of crooked keel was not unique to domestic chicken, that some sternal characteristics were more frequent in certain chicken breeds than in others, and that overall there were no keel characteristics that are unique for certain chicken breeds. Despite some similar morphological changes identified across species, this study highlights the lack of universal patterns in domestication and breed formation

Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the \u27plant mosaic disease\u27 eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of \u27smart\u27 functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing \u27systems-on-a-chip\u27

Preclinical Demonstration of Lentiviral Vector-mediated Correction of Immunological and Metabolic Abnormalities in Models of Adenosine Deaminase Deficiency

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)–deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA−/− mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA−/− mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34+ cells transduced with 1–5 × 107 TU/ml had 1–3 vector copies/cell and expressed 1–2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis