Clinical Gene Therapy Protocols Research Articles

Analysis of hepatic lentiviral vector transduction; implications for preclinical studies and clinical gene therapy protocols.

Lentiviral vector-transduced T-cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the host genetic variation effects on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge-gap, we characterized hepatic gene delivery by lentiviral vectors across the Collaborative Cross (CC) mouse genetic reference population. For 24 weeks, we periodically measured hepatic luciferase expression from lentiviral vectors in 41 CC mouse strains. Hepatic and splenic vector copy numbers were determined. We report that CC mouse strains showed highly diverse outcomes following lentiviral gene delivery. For the first time, moderate correlation between mouse strain-specific sleeping patterns and transduction efficiency was observed. We associated two quantitative trait loci (QTLs) with intra-strain variations in transduction phenotypes, which mechanistically relates to the phenomenon of metastable epialleles. An additional QTL was associated with the kinetics of hepatic transgene expression. Genes comprised in the above QTLs are potential targets to personalize gene therapy protocols. Importantly, we identified two mouse strains that open new directions in characterizing continuous viral vector silencing and HIV latency. Our findings suggest that wide-range patient-specific outcomes of viral vector-based gene therapy should be expected. Thus, novel escalating dose-based clinical protocols should be considered.

UM171 Enhances Fitness and Engraftment of Gene Modified Hematopoietic Stem Cells from Sickle Cells Disease Patients

Sickle cell disease (SCD) is one of the most common genetic blood diseases. Autologous hematopoietic stem cell gene therapy using lentiviral vectors is an effective therapeutic strategy for SCD. However, the occurrence of post gene therapy myelodysplastic syndrome and acute myeloid leukemia not related to vector insertion has suggested an underlying genetic risk or acquisition of damage during ex vivo culture or from proliferative stress associated with reconstitution. Here, we aimed to improve the transduction efficiency, stem cell fitness and reconstitution of SCD CD34+ cells transduced with a lentiviral vector (LV) containing BCL11A shmiR currently in clinical trials (Esrick et al. NEJM, 2021, NCT03282656), to minimize culture-induced stress and reduce genomic damage during ex vivo culture. UM171, an HSC self-renewal pyrimidoindole derivative agonist, has been shown to expand HSCs and enhance multilineage blood cell reconstitution in mice. We examined the effect of addition of UM171 during ex vivo transduction on HSCs from SCD patients. Culture of SCD CD34+ HSC in HSC expansion conditions with UM171 increased the proportion of CD34+CD133-CD45RA-CD90+ long term HSCs (LT-HSCs), from 1.1% to 4.1% (N = 3, P < 0.001), and the absolute number of LT-HSCs increased 4-fold (N = 3, P < 0.001). Treatment with UM171 also significantly enhanced the transduction efficiency of BCL11A shmiR containing lentiviral vector (LV-BCL11A) in SCD CD34+ cells from 0.7 vector copies/diploid genome (VCN) to 1.0 VCN (N = 5, P < 0.001), and significantly decreased apoptosis of stem cells from 22.5% to 14.3% (N = 3, P < 0.001), and DNA damage as measured by γ-H2AX from 10.6% to 6.6% (N = 3, P < 0.001). Given these results, we assessed the engraftment capability and clonality of SCD CD34+ transduced in the presence or absence of UM171 in NBSGW mice. UM171 increased the human CD45 engraftment of mice measured in BM at 16 weeks post-transplantation from 32.5% to 69.4% in hCD45+ BM cells (N = 6, P < 0.01) and enhanced the engraftment of transduced in SCD CD34+-derived CD45+ cells from 11.2% to 18.2% in gene-marked BM cells (N = 6, P < 0.01), while maintaining multilineage differentiation in this model. Barcode analysis revealed the engraftment of 500-1800 unique barcodes with a high degree of clonal diversity and a trend towards increased polyclonal engrafting populations in transplanted mice. To assess the effect of UM171 more rigorously on functionally defined HSCs, we performed a competitive transplantation assay using CD34+ cells transduced in the presence vs absence of UM171. Analysis of BM obtained from the mice at 16 weeks post-transplantation showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions, 62.6% in gene-marked erythroid cells of UM171 treated cells compared with 37.4% of control cells (N = 9, P < 0.001), and UM171 maintains LV-BCL11A induced γ-globin expression in erythroid cells. In summary, a short-term exposure of SCD CD34+ PB cells to UM171 enhances stem cell fitness. Implementing these findings in clinical gene therapy protocols may improve the efficacy and sustainability of gene therapy and generate new opportunities in the field of gene editing.

Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

Efficient ExVivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

SummaryEx vivo gene therapy based on CD34+ hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+ cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38− cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing.

Incremental Innovation of Ex Vivo Hematopoietic Stem Cell Engineering to Expand Clinical Gene Therapy Applications

Transplantation of genetically engineered, autologous hematopoietic stem and progenitor cells (HSPC) is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases, avoiding the risks of graft versus host disease and prolonged immunosuppression. Most clinical gene therapy protocols are based on CD34+ HSPC engineered during >2 days of ex vivo culture. By xenotransplanting mobilized peripheral blood (mPB) CD34+ HSPC, which were lentivirally (LV) marked with different fluorescent proteins according to CD38/CD90 expression levels allowing quantitative assessment of the contribution of CD38/CD90 subpopulations to hematopoietic reconstitution (n=48 NSG mice, 3 experiments), we identified 2 distinct waves of reconstitution: (1) short term repopulation (up to 2 months) mostly driven by CD34+CD38intCD90+/- cells and (2) long-term repopulation driven by CD34+CD38-CD90+ (70%) and CD34+CD38-CD90- cells (30%). Notably, an intermediate wave extending from 2 to 4 months driven by CD34+CD38low cells was selectively eliminated by prolonged ex vivo culture and could be rescued when culture time was reduced to 1 day. We therefore developed a novel LV transduction protocol able to provide curative levels of gene transfer during a single day of ex vivo culture. Stimulating CD34+ cells or CD34+CD38- cells with Prostaglandin E2 (PGE2) increased gene transfer with VSVg-pseudotyped LVs by 1.5-2 fold acting on early steps of transduction, an effect that was further potentiated by the late-acting compound Cyclosporin A. Using large-scale vector preparations for gene therapy of mucopolysaccharidosis type 1, chronic granulomatous disease or beta-thalassemia, we show by in vitro and xenotransplantation assays that a 1-day PGE2 protocol achieved similar transduction efficiencies into BM or MPB HSPC from healthy donors and patients as our 62h benchmark protocol. PGE2 treatment did not result in toxicity or skewed multi-lineage differentiation. However, shortening ex vivo culture increased engraftment levels in the NSG mouse model. To entirely avoid culturing progenitor cells, we explored the feasibility to limit ex vivo manipulation to HSC-enriched CD34+CD38- cells that may be co-transplanted with unmanipulated CD34+ progenitor cells devoid of long-term engraftment potential. This could further improve hematopoietic reconstitution, increase safety by reducing the LV integration load infused into the patient and downscale ex vivo manipulation making the process more efficient and economically sustainable. To this end, we optimized a sequential bead-based, GMP-compatible selection procedure to separate mPB into a CD34+CD38- stem and CD38+ progenitor cell fraction. We reached high purity (87+/-6.6% CD34+) and recovery of CD34+CD38- cells (37.3+/-8.7%), making their isolation clinically viable. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells into NSG mice resulted in rapid engraftment followed by near-complete replacement of untransduced short-term repopulating progenitors by gene-marked HSPC deriving from CD34+CD38- cells after the 3rd month post-transplant. Finally, we explored ex vivo expansion of mPB CD34+CD38- cells with arylhydrocarbon receptor antagonists and/or pyrimido-indole-derivatives. These cells expanded 3-10 fold in a 7-14 d time-window, far less than seen for total CD34+ cells, thereby facilitating culture handling and reducing cost. Unlike CD34+ cells, expanded mPB CD34+CD38- cells largely maintained their SCID-repopulating potential providing proof-of-concept for the expansion of gene-modified HSC. This clinically applicable platform will improve the efficacy, safety and sustainability of ex vivo gene addition and open up new opportunities in the field of gene editing. DisclosuresCiceri:MolMed SpA: Consultancy.

Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates

Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols.—Mattar, C. N., Wong, A. M. S., Hoefer, K., Alonso-Ferrero, M. E., Buckley, S. M. K., Howe, S. J., Cooper, J. D., Waddington, S. N., Chan, J. K. Y., Rahim, A. A. Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.

Development of dual-activity vectors by co-envelopment of adenovirus and SiRNA in artificial lipid bilayers.

Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.

An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols.

Refocus the Recombinant DNA Advisory Committee

The Recombinant DNA Advisory Committee, formed in 1974, was originally charged with advising on potential risks of recombinant technology and took on review of clinical gene therapy protocols in the late 1980s. But as gene therapy has made headway with safety, it's time to shift the committee's mission toward broader ethical and technical issues linked to the field.

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: A comparison between ex vivo and in vivo data

Purpose: Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis.Materials and methods: A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression.Results: We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo.Conclusions: However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.

Autologous Transplantation of Lentivector/Acid Ceramidase–Transduced Hematopoietic Cells in Nonhuman Primates

Farber disease is a rare lysosomal storage disorder (LSD) that manifests due to acid ceramidase (AC) deficiencies and ceramide accumulation. We present a preclinical gene therapy study for Farber disease employing a lentiviral vector (LV-huAC/huCD25) in three enzymatically normal nonhuman primates. Autologous, mobilized peripheral blood (PB) cells were transduced and infused into fully myelo-ablated recipients with tracking for at least 1 year. Outcomes were assessed by measuring the AC specific activity, ceramide levels, vector persistence/integration, and safety parameters. We observed no hematological, biochemical, radiological, or pathological abnormalities. Hematological recovery occurred by approximately 3 weeks. Vector persistence was observed in PB and bone marrow (BM) cells by qualitative and quantitative PCR. We did not observe any clonal proliferation of PB and BM cells. Importantly, AC-specific activity was detected above normal levels in PB and BM cells analyzed post-transplantation and in spleens and livers at the endpoint of the study. Decreases of ceramide in PB cells as well as in spleen and liver tissues were seen. We expect that this study will provide a roadmap for implementation of clinical gene therapy protocols targeting hematopoietic cells for Farber disease and other LSDs.

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

Parachuting in the epigenome: the biology of gene vector insertion profiles in the context of clinical trials

Retroviral pre-integration complexes (PICs) provide a most efficient mechanism to integrate foreign DNA into cellular chromatin. This has made retrovirus-based vectors a preferred tool for gene delivery in therapeutic settings where the chromosomal integration of a recombinant expression cassette that encodes a protein of interest can lead to a long-lasting correction of monogenetic diseases (so-called gene addition strategy). However, the efficiency of retroviral gene addition comes at the expense of a lack of precision in the choice of the integration site. Insertional mutagenesis with potential activation of proto-oncogenes as a first hit in a multistep scenario of cancer development thus represents one of the major hurdles to a more widespread exploration of gene-based treatments (Kustikova et al, 2010). To date, four clinical trials were reported to be associated with severe adverse reactions induced by insertional mutagenesis in haematopoietic stem and progenitor cells (HSC/P); two targeting the X-linked form of severe combined immunodeficiency (SCID-X1), one targeting chronic granulomatous disease, and most recently another trial exploring gene therapy for the Wiskott–Aldrich-Syndrome. In contrast, in the SCID caused by mutations in the gene encoding the metabolic enzyme adenosine deaminase (ADA), retroviral gene addition so far has been free of such complications. Furthermore, numerous trials using similar gene vectors to transfer genes into mature T cells have not been complicated by clonal outgrowth. This explains the great interest in a deeper understanding of retroviral vector–host interactions in the therapeutic setting of SCID-ADA.

Radiolabeled Nucleoside Analogues for PET Imaging of HSV1-tk Gene Expression

The HSV1-tk gene has been explored as a reporter and/or suicide gene in molecular imaging of gene expression. Gene therapy with HSV1-tk and the use of this gene as a marker have been applied in patients with various forms of cancer. However, the conditions for clinical gene therapy protocols have yet to be optimized. A method to monitor the activity of HSV-tk in vivo would be extremely useful to optimize clinical gene therapy protocols. Positron emission tomography (PET) with a suitable probe can offer information about both the extent of gene expression and its distribution, provided that an appropriate reporter gene is included in the therapeutic cassette. PET imaging provides higher resolution and sensitivity and allows noninvasive quantification of biological processes. Several radiolabeled pyrimidine (thymidine) and purine (acycloguanosine) derivatives have been developed as reporter probes for imaging of HSV-TK enzyme activity with PET. In this review, information on radiolabeling and PET imaging of HSV1-tk gene expression with various nucleoside analogues is presented.

SIN Retroviral Vectors Expressing COL7A1 Under Human Promoters for Ex Vivo Gene Therapy of Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.

Cellular immune response to cryptic epitopes during therapeutic gene transfer

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8(+) T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8(+) CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.

Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. This unit describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using cationic lipid reagents.

Production of adenoviral vectors and its recovery

The current demands for adenoviral vectors are increasing to satisfy pre-clinical and clinical gene therapy protocols. Consequently, there is a necessity of methodologies to improve production and recovery of intact particles with the minimum effect upon bioactivity. The production of adenoviral vectors in HEK 293 cells and the potential of an alternative aqueous two-phase system (ATPS) composed of PEG 300-phosphate in recovery of adenoviral vectors were investigated. The production of adenoviral vectors was carried out using a 2 L bioreactor equipped with two Rushton impellers. Different parameters including initial cell density, harvesting time and the addition of a buffer (HEPES) were studied in order to improve the production of adenoviral vectors in HEK 293 cells. A yield of 8 × 10 11 infective particles was achieved under the conditions characterized by the addition of Pluronic F-68, inoculation at an initial cell density of 3.5 × 10 5 cells/mL and harvest of infected cells at 48 h post infection (hpi). This material was used for the evaluation of the ATPS recovery processes. It was demonstrated that the chemical components of the ATPS did not have a significant effect upon the infectivity of the adenoviral vectors and a total recovery of approximately 90% was obtained. These findings contribute to the process development for the manufacture of adenoviral vectors and other nanoparticulate bioproducts.