ATMP development and pre-GMP environment in academia: a safety net for early cell and gene therapy development and manufacturing.

Advanced therapy medicinal products (ATMP) are medicines for human use that are based on genes, cells or tissues. Over the past years, an increasing number of ATMP entered the market for treatment of cancer, genetic disorders, skeletal defects and metabolic diseases. However, the ATMP production methods often change from the initial concept to commercialization. This change is needed to improve the manufacturing feasibility for scaling up or scaling out. Moreover, the production must adhere to current good manufacturing practices (GMP), and needs to follow a risk-based approach, which often is challenging to implement due to the novelty of the products. Since most of the early ATMP development is done in academia, an environment that is not familiar with regulatory requirements for ATMP production in GMP, the initial manufacturing choice for pre-clinical studies is usually very different from what is required for clinical use. This leads to a lengthy production process optimization, unnecessary repetition of experiments and ultimately waste of funding. This consideration prompted us to provide an intermediate step between early ATMP production in research settings to GMP manufacturing. We built a dedicated facility, and we called this environment 'pre-GMP' to highlight that it is a step toward preparation to GMP manufacturing. This environment supports process development and provides a manufacturing fitness room before transferring to GMP suites. This paper addresses the relevance of pre-GMP, underlining the advantages and the possible disadvantages of this additional framework that may be key in accelerating the pace of ATMP toward clinic.

Immunization with HIV-1 envelope T20-encoding DNA vaccines elicits cross-clade neutralizing antibody responses.

BACKGROUND: Genetic immunization is expected to induce the expression of antigens in a native form. The encoded peptide epitopes are presented on endogenous MHC molecules, mimicking antigen presentation during a viral infection. We have explored the potential of enfuvirtide (T20), a short HIV peptide with antiviral properties, to enhance immune response to HIV antigens. To generate an expression vector, the T20 sequence was cloned into a conventional plasmid, the novel minicircle construct, and a replicon plasmid. In addition, 3 conventional plasmids that express the envelope of HIV-1 subtypes A, B and C and contain T20 in their gp41 sequences were also tested. RESULTS: All combinations induced HIV-specific antibodies and cellular responses. The addition of T20 as a peptide and as an expression cassette in the 3 DNA vectors enhanced antibody responses. The highest anti-HIV-1 Env titers were obtained by the replicon T20 construct. This demonstrates that besides its known antiviral activity, T20 promotes immune responses. We also confirm that the combination of slightly divergent antigens improves immune responses. CONCLUSIONS: The antiretroviral T20 HIV-1 sequence can be used as an immunogen to elicit binding and neutralizing antibodies against HIV-1. These, or similarly modified gp41 genes/peptides, can be used as priming or boosting components for induction of broadly neutralizing anti-HIV antibodies. Future comparative studies will reveal the optimal mode of T20 administration.

HIVIS-DNA or HIVISopt-DNA priming followed by CMDR vaccinia-based boosts induce both humoral and cellular murine immune responses to HIV.

BACKGROUND: In order to develop a more effective prophylactic HIV-1 vaccine it is important optimize the components, improve Envelope glycoprotein immunogenicity as well as to explore prime-boost immunization schedules. It is also valuable to include several HIV-1 subtype antigens representing the world-wide epidemic. METHODS: HIVIS-DNA plasmids which include Env genes of subtypes A, B and C together with Gag subtypes A and B and RTmut/Rev of subtype B were modified as follows: the Envelope sequences were shortened, codon optimized, provided with an FT4 sequence and an immunodominant region mutated. The reverse transcriptase (RT) gene was shortened to contain the most immunogenic N-terminal fragment and fused with an inactivated viral protease vPR gene. HIVISopt-DNA thus contains fewer plasmids but additional PR epitopes compared to the native HIVIS-DNA. DNA components were delivered intradermally to young Balb/c mice once, using a needle-free Biojector® immediately followed by dermal electroporation. Vaccinia-based MVA-CMDR boosts including Env gene E and Gag-RT genes A were delivered intramuscularly by needle, once or twice. RESULTS: Both HIVIS-DNA and HIVISopt-DNA primed humoral and cell mediated responses well. When boosted with heterologous MVA-CMDR (subtypes A and E) virus inhibitory neutralizing antibodies were obtained to HIV-1 subtypes A, B, C and AE. Both plasmid compositions boosted with MVA-CMDR generated HIV-1 specific cellular responses directed against HIV-1 Env, Gag and Pol, as measured by IFNγ ELISpot. It was shown that DNA priming augmented the vector MVA immunological boosting effects, the HIVISopt-DNA with a trend to improved (Env) neutralization, the HIVIS-DNA with a trend to better (Gag) cell mediated immune reponses. CONCLUSIONS: HIVIS-DNA was modified to obtain HIVISopt-DNA that had fewer plasmids, and additional epitopes. Even with one DNA prime followed by two MVA-CMDR boosts, humoral and cell-mediated immune responses were readily induced by priming with either DNA construct composition. Priming by HIV-DNA augmented neutralizing antibody responses revealed by boosting with the vaccinia-based heterologous sequences. Cellular and antibody responses covered selected strains representing HIV-1 subtypes A, B, C and CRF01_AE. We assume this is related to the inclusion of heterologous full genes in the vaccine schedule.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Pre-clinical evaluation of a CEA DNA prime/protein boost vaccination strategy against colorectal cancer.

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.

The rationale behind a vaccine based on multiple HIV antigens.

The viral diversity of HIV-1 is likely to require a vaccine strategy that induces broad cellular and humoral anti-HIV-1 immunity. Our strategy is based on multiple HIV-1 DNA immunogens together with adjuvant recombinant granulocyte-macrophage stimulating factor. This article describes pre-clinical and clinical work preceding the initiation of clinical HIV-1 phase I/II trials.

Angiogenic effects of dual gene transfer of bFGF and PDGF-BB after myocardial infarction.

Therapeutic effects of combination of angiogenic growth factors for the treatment of ischemia after myocardial infarction are largely unknown. Plasmids expressing basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB) or their combination with a 1:1 mass ratio were injected into hearts with 7-day-old myocardial infarction. Hearts were harvested after 1 and 4 weeks after gene transfer. The major findings in this chronic myocardial infarction model were that bFGF, PDGF-BB and their combination all had a more pronounced angiogenic effect on the arteriolar than the capillary level. bFGF stimulated both capillary and arteriolar growth while PDGF-BB preferentially stimulated arterioles. The combination increased the amount of both capillaries and arterioles and in addition gave rise to stable capillaries compared to single factor transfer but did not further enhance angiogenesis. No cardiovascular side effects were observed after gene transfer.

Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium.

We hypothesised that angiopoietin-1 (Ang-1), in conjunction with vascular endothelial growth factor (VEGF) gene therapy, can enhance arteriogenesis and angiogenesis during myocardial ischemia. Mice were given a single intramyocardial injection of saline, phVEGF-A(165) and phAng-1 or a combination thereof into the non-ischemic normal heart or into the ischemic border zone of the infarcted heart. In the normal and the ischemic myocardium, gene transfer of phVEGF-A(165) alone increased the myocardial capillary density by 16% and 36%, respectively, and phAng-1 had a similar effect. In the normal heart, the ratio of arteriolar to capillary densities increased with phVEGF-A(165) and more so in the ischemic myocardium where phAng-1 also had an effect. Furthermore, the combination of plasmids induced an up to 7.5-fold increase. Transient overexpression of VEGF-A(165) boosts endogenous arteriogenesis in addition to capillary angiogenesis. Ang-1 further boosts this effect at the arteriolar level.

Protein and angiogenic dose-response expression of phVEGF-A(165) gene in rat myocardium.

Therapeutic myocardial angiogenesis by means of transient overexpression of angiogenic growth factors is a potential treatment modality for severe ischemic heart disease. This study was undertaken in the rat to examine effects of phVEGF-A(165) myocardial transfection in terms of dose-response as regards the number of hVEGF-A expressing cells on one hand and on the other angiogenesis. Non-surgical echocardiography-guided intramyocardial injection of phVEGF-A(165) was done into normoxic or hypoxic (10% O(2)) rats. Cardiomyocytes expressing VEGF-A protein, capillary morphology and density were determined after 5 days. VEGF protein expression was seen in rat cardiomyocytes located around the tip of the injection scar and increased dose-dependently (p<0.05). Microvessel density also increased dose-dependently with phVEGF(165) (p<0.05) and with hypoxia (p<0.05). No vascular tumours were observed. In conclusion, direct intramyocardial injection of phVEGF-A(165) in the rat results in a dose-dependent increase both in transfected hVEGF-A protein producing cells and in angiogenesis.

The in vitro activity and specificity of human endothelial cell-specific promoters in porcine cells.

The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Therefore, it is generally agreed that successful pig to human xenotransplantation requires donor pigs to be genetically modified. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications. Luciferase assays were used to evaluate the activity and specificity of human endothelial-cell specific promoters in porcine aortic-, microvascular- and nonendothelial cells. The promoters for human Flk-1 (fetal liver kinase-1), Flt-1 (fms-like tyrosine kinase), ICAM-2 (intercellular adhesion molecule-2), thrombomodulin and vWf (von Willebrand factor) supported similar levels of luciferase expression in human and porcine aortic endothelial cells, with the Flk-1 promoter being the strongest followed by the thrombomodulin promoter. Relative to the activity of the CMV promoter, the human endothelial cell-specific promoters all showed less activity in porcine kidney microvascular endothelial cells than in liver or brain microvascular endothelial cells. The thrombomodulin and Flk-1 promoters exhibited similar activity in liver and kidney microvascular endothelial cells, whereas the Flk-1 promoter was stronger in aortic and brain microvascular endothelial cells. Human endothelial cell-specific promoters also showed some degree of specificity in pig, because they supported less luciferase activity in porcine nonendothelial cell lines. Based on the in vitro data and previously published in vivo data, the human Flk-1 and thrombomodulin promoters are good candidate promoters for strong endothelial cell-specific gene expression in transgenic pigs.

Myocardial Doppler tissue velocity improves following myocardial gene therapy with VEGF-A165 plasmid in patients with inoperable angina pectoris.

BACKGROUND: Myocardial tissue velocity and perfusion were studied in patients with severe angina pectoris following gene therapy by intramyocardial injection of phVEGF-A165 via thoracotomy. Plasma concentrations of VEGF-A increased postoperatively. Two months after treatment anginal status and myocardial tissue velocity improved and perfusion showed a tendency to improve. Tissue velocity imaging appears to be a sensitive, objective method for detecting changes in myocardial function following gene therapy. OBJECTIVE: To study effects on myocardial tissue velocity and perfusion in patients with angina pectoris following intramyocardial injection of phVEGF-A165 via thoracotomy. DESIGN: Open label, phase I/II. METHODS: Six patients with Canadian Cardiovascular Society (CCS) angina pectoris functional class III - IV and with major defects at adenosine stress single-photon emission computerized tomography (SPECT) were studied. In addition to SPECT, coronary angiography and dobutamine stress echocardiography with tissue Doppler velocity imaging were performed before and two months after gene transfer. RESULTS: Plasma concentrations of VEGF-A increased 2 to 3 times (P < 0.04) over baseline from 2 to 14 days after injection with normalization after 4 weeks. The CCS class improved about 40%, from 3.3 +/- 0.2 to 2.0 +/- 0.3 (P < 0.02) and nitroglycerine consumption decreased 30 - 40%, from 44 +/- 17 to 15 +/- 5 tablets per week (P < 0.05). The maximal systolic myocardial tissue velocity increased in all patients about 25% (P < 0.02) but did not reach the reference range. Myocardial perfusion at SPECT improved in four of the six patients. CONCLUSIONS: Anginal status, myocardial tissue velocity and perfusion can be improved by phVEGF-A165 intramyocardial injection. Tissue velocity imaging appears to be a sensitive, objective method for detecting changes in myocardial function following gene therapy.

Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease--12-month follow-up: angiogenic gene therapy.

OBJECTIVE: To test the safety and bioactivity of phVEGF-A165 after intramyocardial injection during 12-month follow-up. DESIGN: Open-labelled study. SUBJECTS: Inclusion criteria were angina pectoris, Canadian Cardiovascular Society (CCS) class III-IV, unamenable to further revascularization, ejection fraction (EF) >30%, perfusion defects extending over >10% of the anterolateral left ventricle wall detectable with adenosine single photon emission computerized tomography (SPECT) and at least one patent vessel visible by coronary angiography. Seven of 39 patients referred for gene therapy were included. INTERVENTION: Via a mini-thoracotomy under general anaesthesia. phVEGF-A165 was injected directly into the myocardium at four sites in the anterolateral region of the left ventricle. RESULTS: Operative procedures were uneventful. Perioperative release of myocardial markers and electrocardiogram (ECG) changes were detected in two patients. There were no perioperative deaths but one patient died 7 months postoperatively because of myocardial infarction. Plasma vascular endothelial growth factor (VEGF)-A levels increased two to threefold peaking 6 days postoperatively (P < 0.004) and returning to baseline by day 30. A significant reduction in angina pectoris was reported. The CCS class improved from 3.3+/-0.2 to 1.9+/-0.3 (P < 0.01) and nitroglycerine intake decreased from 39+/-15 to 12+/-5 tablets week(-1) (P < 0.001) 2 months after gene transfer. Improvements remained after 12 months when nitroglycerine consumption approached zero. Improved myocardial function in the phVEGF-A165 injection region was documented in all patients (P < 0.016) by tissue velocity imaging (TVI). Reduced reversible ischaemia was detected by adenosine SPECT in four patients. Improved collateralization was detected in four patients with coronary angiography. CONCLUSION: Intramyocardial injection of phVEGF-A165 is safe and may lead to improved myocardial perfusion and function with longstanding symptomatic relief in end-stage angina pectoris. Based on these results this therapeutic potential is being tested in a double-blind placebo controlled multicentre trial.

Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies.

The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.

Identification of viable myocardium by perfusion imaging with intravenous contrast echocardiography after acute myocardial infarction.

Myocardial perfusion contrast echocardiography is evolving into an effective method for the evaluation of myocardial blood flow after acute coronary events. The direct injection of ultrasound contrast agents into the aortic and coronary circulation has been shown to accurately identify areas of viable myocardial tissue. Recently, intravenous ultrasound contrast has been found to be useful in detecting microvascular blood flow after the restoration of blood flow in patients with myocardial infarction. We present the case of a patient in whom intravenous ultrasound contrast assisted in the detection of viable myocardial tissue after an acute ischemic syndrome.

Transient myocardial dysfunction associated with angiotensin-converting enzyme inhibitor-induced angioedema: recognition by serial echocardiographic studies.

We report a case of a 58-year-old woman who had angiotensin converting enzyme inhibitor-induced angioedema after she underwent a biopsy of a hypopharyngeal mass. The angioedema was associated with severe transient myocardial dysfunction documented on echocardiography. She did not have anaphylaxis or coronary artery disease. To our knowledge this is the first reported case of transient myocardial dysfunction in the setting of angiotensin converting enzyme inhibitor-induced angioedema without anaphylaxis.

Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila.

We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA. These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (approximately 900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription. It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously. Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required. In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

The intranuclear organization of normal, hemizygous and excision-deficient rRNA genes during developmental amplification in Tetrahymena thermophila.

In the ciliated protozoan, Tetrahymena thermophila, the diploid germinal micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA). During genesis of new somatic macronuclei the germline rDNA gene is excised by developmentally programmed chromosome breakage and preferentially amplified to approximately 9, 000 copies. We have studied this process by fluorescence in situ hybridization. We find that initially rDNA amplification is restricted to two separate and highly confined regions of the nucleus. Analysis of nuclei that are hemizygous for the rDNA locus reveals that each focus of hybridization is derived from a single allele of the rDNA. As rDNA amplification progresses these two foci of hybridization disperse and spread throughout the macronucleus, eventually forming approximately 100-500 new nucleoli. These events are correlated with morphologically distinct developmental stages. We investigated the amplification of the C3 allele of the rDNA that confers a replication advantage over the B allele during vegetative propagation, and find no evidence for preferential amplification of the C3 early in rDNA maturation. We also show that the rmm 11 rDNA mutant allele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and heterozygotes, but fails to amplify further and disperse into multiple nucleoli. These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occur. We propose that rDNA amplification is a two-step process. First, the two rDNA alleles are independently amplified, while allelic copies remain closely associated. Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, generating fully mature rDNA minichromosomes that are able to replicate to high copy number.

An academic centre for gene therapy research with clinical grade manufacturing capability.

Huddinge University Hospital is a major teaching hospital affiliated with the Karolinska Institute in Southern Stockholm. For the past few years several groups have been working there in different areas of gene therapy relating to cancer, genetic and infectious diseases. However, a facility to produce clinical grade material under good manufacturing practice was lacking. To this end, Huddinge University Hospital has taken the initiative to open a Gene Therapy Research Center in 1996. This facility, which is unique of its kind in Scandinavia, is located in the Novum Research Park, Huddinge, and is a part of the existing Clinical Research Center. The newly built centre will allow clinicians and researchers to develop and produce vectors (viral and nonviral) for clinical trials and do basic research to understand the mechanisms of diseases. Although the centre will primarily serve the academic institutions it will also extend its facilities to other investigators in this field. The production unit is run in collaboration with the Faculty of Medicine, University of Lund. On-going projects include production of plasmid vectors for prevention of postangioplasty restenosis, DNA vaccine for HIV-1, cationic liposome DNA complexes for cystic fibrosis and retroviral vectors for HIV-1.