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Augmenting PBGD expression in the liver as a Novel Gene therapy for Acute Intermittent Porphyria

Final Report Summary - AIPGENE (Augmenting PBGD expression in the liver as a Novel Gene therapy for Acute Intermittent Porphyria)

Executive Summary:

Acute intermittent porphyria (AIP) is a rare genetic disease in which mutations in the porphobilinogen deaminase (PBGD) gene produce insufficient activity of a protein necessary for heme synthesis. This leads to an accumulation of toxic intermediates resulting in a wide variety of problems including acute, severe abdominal pains, psychiatric and neurological disorders, and muscular weakness. Acute porphyric attacks can be life-threatening and the long-term consequences include irreversible nerve damage, liver cancer and kidney failure. AIP affects 1/10,000 people in the EU and the therapies currently available do not prevent the symptoms or consequences of acute porphyric attacks. The only curative therapy is liver transplantation and thus, new curative options are clearly needed. In 2009, the European Medicines Agency granted Orphan Drug Designation to AAV5-AAT-PBGD for the treatment of AIP. The patent for AAV5-AAT-PBGD was granted AAV is a replication-incompetent virus that has been modified to deliver genes or genetic material into human tissues or cells. AAV5-AAT-PBGD acts by delivering the PBGD expression cassette directly into hepatocytes.
In the initial phases of the project we established a production process as wells as assays to release the AAV5-AAT-PBGD material for clinical, in parallel the safety of the vector was determined in preclinical animal models and AIP patient recruitment started. The observational study in patients with AIP served to establish the clinical and biochemical parameters for each patient.
After that, AIPGENE consortium performed a Phase I clinical trial to test primarily the safety and secondarily the efficacy of AAV5-AAT-PBGD in patients with severe AIP. The Aipgene clinical trial designed with the participation of an External Advisory Board, is a phase I, multicentre, open label, single dose and dose escalation clinical trial (Eudra-CT number 2011-005590-23 and protocol code AAVPBGD-AIP-001. The AEMPS Authorization was received on 25th September 2012.). Eight patients were included in the clinical trial, each received a single dose of the AAV5-AAT-PBGD , the eidht patients were divided in 4 cohorts each cohort received a different dose the first cohort received a dose of 5 x 1011 genome copies/Kg (gc/Kg), second cohort 2 x 1012 gc/Kg, third cohort 6 x 1012 gc/Kg, forth cohort 1.8 x 1013 gc/Kg.

The main conclusions extracted from this study were the following:
1. The treatment with AAV5-AAT-PBGD is well tolerated and safe in patients with Acute Intermittent Porphyria. No signs of toxicity were seen during the administration of the drug or during patient follow up.
2. Humoral immune response was induced in all patients. All the patients developed total and neutralizing antibodies against the vector but no against the recombinant protein.
3. No cellular immune response against the vector or the transgene was detected. Contrary to what was observed in previous clinical trials targeting the liver, for the treatment of hemophilia B, no cellular immune response associated with hepatocyte elimination (liver damage) was observed in the Aipgene trial.
4. AAV5-AAT-PBGD transduced human liver. The analysis of liver biopsies obtained from 6 out 8 patients revealed that the AAV-AAT-PBGD vector transduced the liver.
5. There is some effect on clinical outcomes in certain patients. Some patients have clearly reduced the uptake of medications specific for the treatment of the disease and reduced the number and days of hospitalizations.
In summary, we have gathered critical clinical experience that will contribute greatly to the further understanding of this complex disease. The AIPGENE collaboration has achieved its primary goal in completing a successful Phase I study, providing first and foremost the safety data required for additional clinical study. Finally, we are currently g the final analysis of the data for publication and to defining what the next steps for the further development of AIP gene therapy.

Project Context and Objectives:
Summary description of the project context and the main objectives.
Porphyrias are a group of rare inherited metabolic disorders affecting one of the eight enzymes involved in the heme biosynthesis pathway1-3. Acute Intermittent Porphyria (AIP) is the most common of the acute porphyrias and is caused by mutations in the hydroxymethylbilane (HMBS) gene, mostly known as porphobilinogen deaminase (PBGD) gene, affecting the third enzymatic step in the pathway. Deficient PBGD activity (about 50% of normal) leads, under certain metabolic conditions associated with stressed hepatic heme biosynthesis, to an accumulation of neurotoxic intermediates, aminolevulinic acid (ALA) and porphobilinogen (PBG). This in turn results in clinical manifestations such as acute abdominal pain, psychiatric and neurological disorders, and muscular weakness1-4. AIP, if not recognized or treated properly may be life-threatening5. Long-term consequences may include irreversible nerve damage, hypertension, kidney dysfunction and primary liver cancer6. Severely affected AIP patients have poor quality of life. Treatment is usually palliative, including symptomatic treatment and carbohydrate loading7. Specific treatment involves infusion of human hemin and is used in severe AIP attacks. Hemin restores deficient hepatic heme pool and down regulates stressed hepatic heme synthesis, inhibiting the accumulation of toxic metabolites7. The only proven curative treatment is liver transplantation and thus, new therapeutic options are clearly needed8. We have developed a replication-incompetent, adeno-associated vector-based gene therapy product, named AAV2/5-PBGD for the treatment of this devastating rare disease9; AAV2/5-PBGD was designated as orphan drug by the EMEA in 2009, (EU/3/09/632).
The objective of the EU-funded “Augmenting PBGD expression in the liver as a novel gene therapy for acute intermittent porphyria” (AIPgene) project is to provide an alternative gene therapy option for AIP. Assessment of the safety of this product is the primary goal of the AIPgene project and entails a dose escalation clinical study.
AAV2/5-PBGD is a recombinant adeno-associated virus-based vector containing AAV serotype 2 genome elements and AAV serotype 5 capsid proteins and expressing under the control of a liver specific promoter a codon optimized PBGD gene. The recombinant vector, designed by a group of investigators at CIMA, University of Navarra, was produced under GMP conditions using the baculovirus production system developed and optimized by uniQure biopharma BV.
Pre-clinical “proof of concept” studies were performed in a murine AIP model. Pivotal toxicology of the therapeutic vector was assessed in mice and non-human primates, which included the analysis of the integration pattern of the vector, conducted at the National Center for Tumor Diseases (NCT) in Heidelberg.
An Expert Advisory board composed by European clinician’s expert on Acute Intermittent porphyria and members of the European Porphyria Network (EPNET) was convened and participated in the design of the study including the definition of the target population.
The rarity of the disease and the heterogeneity in the way its clinical manifestations develop mean that interpreting changes before and after treatment could be complex and susceptible to many biases. Thus, an observational study was first conducted in order to record the natural history and clinical management of the disease prior to entering these same patients into an interventional trial. The frequency of their attacks and treatments received, the level of urinary toxic metabolites (ALA and PBG), the variation in other neurological or psychological symptoms and quality of life were recorded. A mathematical model was developed to understand the pattern of metabolite excretion and to predict and/or evaluate objectively the response to gene therapy.
Following these preclinical studies the gene therapy approach to correct PBGD deficiency has been translated to the clinic. The Investigational Medical product Dossier was prepared by DIGNA Biotech (which also sponsors the trial) and uniQure and presented for approval to the Ethical Committees and the Spanish Agency of Medicines and Medical Devices. The trial was designed as a Phase I open label, single dose, dose-escalating study with 4 different vector doses in 4 cohorts consisting of 2 patients each. Eight patients were recruited and received the treatment by intravenous infusion at the Clínica Universidad de Navarra (CUN), and patients were followed up at CUN and at Hospital 12 de Octubre. Besides safety, innate immune response, humoral and T-cell response against capsid proteins and recombinant protein as well as vector shedding were analyzed. Although the study’s primary objective was the assessment of safety and tolerability of treatment, signs of efficacy was also be investigated, including changes in the levels of surrogate markers of activity (PBG and ALA, analyzed at the Porphyria Centre Sweden, Karolinska University Hospital), assessment of AIP symptomatology, psychological changes and quality of life, register of AIP and other concomitant medication, neurophysiological study and cerebral MRI.

The main objective AIPgene consortium is treating AIP patients using AAV2/5-PBGD to alleviate the negative impact of the disease on the quality of life of patients and their families. This progress would eventually reduce the burden of the disease and the socio-economic cost of this condition. Although our main objective was to implement a scientifically and medically well founded clinical trial to assess the tolerance and effectiveness of gene therapy for patients with AIP, any positive result may benefit immediately other liver metabolic disorders also. The technologies to be developed will be of interest to both the scientific community and the pharmaceutical industry, as they will confirm the advances in a new therapeutic approach for such disorders. We believe our efforts will eventually improve the treatment options available in a number of diseases and in this manner contribute to the reduction of the personal, societal and economic burden to the European Community. The discoveries arising from this project will provide the groundwork to develop improved methods to treat and delay the onset of the liver disorders.
1. Anderson KE. THE PORPHYRIAS. Goldman: Cecil Medicine, 23rd ed. Chapter 229.
2. Kadish K, et al. The Porphyrin Handbook, Volumes 11-20. ACADEMIC PRESS.
3. Thunell S. GeneReviews, Published on NCBI Bookshelf 2010. www.geneclinics.org
4. Elder G, et al. J Inherit Metab Dis. 2013; 36(5): 849-57.
5. Jeans JB, et al. American Journal of Medical Genetics 1996; 65:269-73.
6. Sardh E, et al. J Inherit Metab Dis. 2013; 36(6): 1063-71.
7. Lithner F and Wetterberg L. Acta Med Scand 1984; 215:271-274.
8. Innala E and Andersson C. J Intern Med 2011;269: 538-545.
9. Stein P, et al. Ann Clin Biochem 2013; 50: 217-23.
10. Seth AK, et al. Liver Transpl 2007; 13: 1219-27.
11. Unzu C, et al. Mol Ther. 2011;19(2): 243-50.
12. Pañeda MA, Hum Gene Ther. 2013; 24(12):1007-17.

Project Results:
Description of the main S & T results/foregrounds. The length of this part cannot exceed 25 pages.
AIPGENE is the acronym of the project entitled “Augmenting PBGD expression in the liver as a novel Gene therapy for Acute Intermittent Porphyria (AIP)”. This European Project has the mission to develop the first in human clinical use of the orphan drug AAV5-AAT-PBGD for the treatment of the rare disease AIP. The completion of this project will represent an important advance in the clinical management, physiopathology and therapy not only for this specific disease but potentially for other metabolic liver disorders.
Since the beginning of the project in January of 2011, the scientists and the personnel staff implicated have performed their research on the following six work packages:

• WP1. The production of the clinical AAV5-AAT-PBGD batch
• WP2. Presentation of AAV5-AAT-PBGD IMPD, design of clinical trial and evaluation of the patient
• WP3. Safety of the orphan drug AAV5-AAT-PBGD: Phase I Clinical Trial
• WP4. Efficacy of the orphan drug AIPGENE: Phase I Clinical Trial
• WP5. Project coordination
• WP6. Dissemination

1. Production and release of the AAV5-AAT-PBGD clinical batch
The clinical batch was produced by using uniQure’s proprietary insect cell platform. The company first developed a production process as well as assays for release testing of the clinical material. This vector was tested in Acute Intermittent Porphyria mice to test its efficacy. The results of these assays are used to release the clinical material. uniQure produced the clinical material under Good Manufacturing Practices (GMP); sufficient clinical material to support the clinical trial was available for shipment to the clinical test site. The company also developed and qualified and/or validated assays necessary for release of the batch for clinical use. uniQure is also performed the accompanying and mandatory stability studies of the clinical batch. All these tasks were performed following the specific regulations such as the relevant ICH guidelines and the specific EMA guidelines related to recombinant adeno-associated viral vectors (EMEA/CHMP/GTWP/587488/22007).
Finally, the underlying production process details and the assessment of quality of the final product were reported in the form of an IMPD which was submitted for review to the relevant regulatory bodies, to support the clinical trial application.

The active ingredient in the IMP is an Adeno-Associated Virus-Based Vector Containing Porphobilinogen Deaminase (PBGD) Gene. The selected recombinant vector, rAAV2/5.2-Ealb-hAAT-cohPBGD-polyA PBGD-VD183 - short name AAV5-AAT-PBGD and coded as AMT-021 for the manufacturer - is a recombinant adeno-associated vector containing a single stranded genome from serotype 2 consists of:
• Inverted terminal regions or ITRs of the adeno-associated serotype 2.
• A human codon optimized porphobilinogen deaminase gene or cohPBGD (1085 bp) as the therapeutic gene.
• A liver specific promoter constituted by the albumin enhancer (Ealb) and the alfa-1-antitrypsin promoter (hAAT) (total 689 bp).
• The polyadenilation sequence of the human PBGD gene (44 bp).
• The 3´untraslated terminal region of the human PBGD gene (25 bp).
• A synthetic polyadenilation sequence (67bp) to isolate the expression cassette from the run-through transcription activity of the 5’ ITR.
The characterization results of the AAV5-AAT-PBGD vector are the following.
Appearance: Clourless, liquid
Sterility: No growth
Endotoxin: ≥ 0.12 and < 0.24 EU/mL
Genome copies: 9.1 x 1013 gc/mL
Osmolality: 452 mOSm/kg
Particle size distribution (DLS): 100 %
Ph: 7.0
NuPAGE/Silver stain VP1, VP2, VP3 observed
Residual baculovirus DNA 0.01 µg/mL
Residual baculovirus infectivity < -0.12 log TCID50/mL
Residual Triton X-100 < 1 ppm
Residual VHH 0.4 µg/mL
Sucrose content 5.1 %
Total particles 8.44 x 1013 tp/mL
Total to Full particle ratio 0.9

The manufacturing process was carried out in the GMP facility of uniQure biopharma B.V. After determining the product concentration, the product was prepared to the final concentration, after which it was sterile filtered into 1.2 ml vial aliquots. The final product was stored at -20°C (stability is only ensured in the temperature range -15ºC to -25ºC) in temperature monitored equipment.
AMT-021 was supplied by the manufacturer as a frozen, aseptically prepared product in 1.2 ml glass vials, with a primary label describing the short name and code of the product, lot number, volume per vial, product concentration, route of administration, study code storage conditions and indications for “for clinical trial use only” and “contains GMO”. This label also contains space for patient identifier that pharmacist must complete with Patient Study ID immediately after vial defrost for a complete drug traceability. The final viral drug product was free from biological contamination and meets all the criteria for parenteral drugs with regards to presence of sub-visible particles. The investigational GT product was formulated at a concentration of 1x1014 GC/mL in PBS-/- + 5% sucrose in 1.2 ml vials that allowed a guaranteed extraction of 1 ml of investigational GT product.

2. Proof of concept studies in AIP mice

The efficacy of the therapeutic vector produced under GMP-like condition was tested at FIMA in Acute intermittent porphyria mice.
This study analysed the efficacy in the metabolic correction of PBGD enzyme 12 or 42 week after AAV5-AAT-PBGD administration. The transgene expression was measured as PBGD activity in liver samples. The efficacy of the vector was analysed by the normalization of urinary precursor’s accumulation in animals challenged with phenobarbital in order to induce an acute attack of porphyria.
Three groups of female AIP mice aged between 7 and 15 weeks were used. One group served as the negative control (n=5) and two groups of mice (n = 5 ‐ 6) were injected intravenously (I.V.) with a dose of 5 x 1013 genome copies (gc)/kg body weight of AAV5-AAT-PBGD. An additional group of ten wild type (wt) mice was also included.
PBGD enzymatic activity was determined at 12 or 42 weeks after the AAV5-AAT-PBGD administration. At 3 or 10 months after AAV5-AAT-PBGD administration, an acute attack of porphyria was induced in these animals by challenging them with phenobarbital (Phen). Porphyrin precursor levels are expressed in terms of μg ALA /mg creatinine or μg PBG /mg creatinine. As expected, AAV5-AAT-PBGD -mediated liver gene therapy provided a high expression of functional hPBGD protein in the liver of AIP mice. The significant increase in PBGD activity warranted the production of active PBGD protein. Of interest, comparable PBGD expression were observed in mice sacrificed 3 or 10 months after the vector administration (Figure 2), indicating that long-term and stable expression of hepatic PBGD was achieved with a dose of 5 x 1013 gc/kg body weight of AAV5-AAT-PBGD. Figure 8 showed the results in urine precursor’s accumulation (PBG and ALA) obtained in all the groups of female AIP mice. As expected, the phenobarbital induction produced a high increase of both heme precursors excretion in the non-injected AIP mice. In contrast, phenobarbital challenge in the AIP mice injected with the therapeutic vector did not increase their urinary PBG and ALA concentration, which remained at levels similar to those in the wild type animals (Figure 8). The long-term therapeutic effect of the vector coding for the cohPBGD made that there is no significant differences between the urinary ALA and PBG excretion between animals where biochemical attack was induced 3 or 10 months after the rAAV5 administration.

3. Safety of the AAV5-AAT-PBGD therapeutic vector
The studies to determine the safety and tolerability of AAV5-AAT-PBGD were performed in mice (at Covance) and in Non-human primates (at CIMA).
The toxicological information of the AAV5-AAT-PBGD gene therapy product is summarized in below. The Non Observed Adverse Effects Level for this drug was 5x1014 gc/kg after intravenous injection.

Toxicological Information
Acute toxicity: NOAEL (intravenous): 5x1014 gc/kg
Oral / Percutaneous: no data available
Irritation and corrosion: Not applicable
Sensitisation Not applicable
Chronic exposure: NOAEL (intravenous): 5x1014 gc/kg
Oral / Percutaneous: no data available
IARC: No component of this product presents at levels greater than or equal to 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.
Signs and Symptoms of exposure: No clinical signs have been observed after intravenous injection in animals.
Inhalation: This product has not been tested for inhalation toxicity. However airway exposure to the wild-type AAV virus is not known to generate any disease.
Skin irritation: This product has not been tested for skin irritation; however, it is not expected to cause any cutaneous effects.
Eye irritation: This product has not been tested for eye irritation; however, it is not expected to cause any ophthalmic effects.
Reproduction toxicity: Based on prenatal development and breeding studies in animals, there is no evidence for any reproductive toxicity of this product.
Carcinogenic effects: Based on non-clinical safety testing, there is no evidence of carcinogenic potential of this product.
Mutagenic effects: This product has been shown to cause low levels of integration of product DNA into chromosomal DNA. The toxicological relevance of these finding, if any, has not been established.
Teratogenic effects: No teratogenic effects were observed in prenatal development studies. NOAEL: 5x1014 gc/kg
Target organs: No target organ toxicity was observed in non clinical studies. A transient and reversible spleen enlargement was observed 7 days after intravenous injection of 5x1014 gc/kg. Some liver vacuolation was observed up to 3-6 months after intravenous.

Integration studies
Six adult NHP of both genders received either 1x1013 gc/kg or 5x1013 gc/kg of AAV5-AAT-PBGD intravenously via the saphenous vein and DNA from four liver sections of each animal were collected 30 days post-injection. Integration site analyses by linear amplification-mediated (LAM-) PCR was performed, resulting in >100000 rAAV LAM-amplicons after 454 sequencing and revealed 752 NHP-specific integration sites (IS). On average, more IS were detected in the animals which received a higher amount of vector (36.9 versus 25.8 IS, respectively). By comparing the location of AAV5-AAT-PBGD IS and their chromosomal distribution to a random data set of 10000 AAV IS we demonstrated a close to random integration pattern. Despite a high number of IS being detected, the presence of a high frequency of concatemers (up to 99%) was demonstrated in each sample. Thus, our IS analyses revealed an almost random integration pattern supporting the good safety profile of AAV5-AAT-PBGD, indicating that AAV5-AAT-PBGD represents a safe therapy to correct the metabolic defect present in AIP patients.
Furthermore, a comparative large scale integration site (IS) analysis has been performed on DNA from tissue (adrenal gland, liver and spleen) obtained from cynomolgus monkeys (Macaca fascicularis) 30 days after injection with the AAV5-AAT-PBGD adeno-associated virus (AAV) vector. In total 18 samples were analyzed by standard and multiplex LAM-PCR, which uses 5 sets of primers, and subsequent high throughput sequencing. IS analyses of these samples revealed a total of 1345 unique IS, whereof 1198 IS were exactly mappable to the monkey genome. There was no preferred integration in the genes MECOM (MDS1-EVI1), or LMO2 previously shown to mediate malignant transformation in clinical retroviral gene therapy. Among the 30 strongest clones (10 from each tissue) with annotated genes there were five genes associated with cancer (adrenal gland: GLIPR1; liver: ING1, PACRG, SAP18; spleen: CCNY) according to cBioPortal. However, because of the high number of cancer genes within annotated Monkey genes (19%) this number is within the expected range compared to random gene selection. None of the eight detected common integrations sites (CIS) with local clustering of 3 IS was close to reported cancer genes.
The following points summarize the results obtained:
• LAM-PCR integration site analysis revealed 752 unique integration sites (IS) in NHP after injection of AAV5-AAT-PBGD and, on average, both NHP and mice showed a tendency to present a higher amount of IS when a higher vector dose was administered.
• No signs of integration hotspots or worrisome integration sites were identified in AAV5-AAT-PBGD injected mice and NHP and, in addition, data showed a close to random integration pattern of AAV5-AAT-PBGD in the two different preclinical settings.
• Despite the high number of IS detected, AAV5-AAT-PBGD persisted predominantly as concatemeric structures (up to 99%) in NHP and mice samples.
• Evaluation of ITR stability and occurrence of preferentially ITR breakpoints in AAV5-AAT-PBGD injected NHP and mice showed the feasibility of full ITR sequencing and the preferential ITR deletion with preferred breakpoints in the C´ region of the ITR.

To conclude, there were no signs for in vivo clone selection or genotoxicity in the analyzed samples.

4. Observational study and clinical trial design for the gene therapy product for the treatment of Acute Intermittent porphyria.
The first step taken by AIPGENE consortium in order to design the observational study and the gene therapy clinical trial for patients with acute intermittent porphyria was the creation of an external advisory board (EAB) with expertise in AIP that actively participate in the design of the observational study (selection of the severe AIP patients and follow-up) and the clinical trial protocol. Five clinician’s experts in AIP from different European countries were contacted and after an informal meeting celebrated in Cardiff, they accepted to participate in the AIPGENE EAB and signed confidential agreements. The first official meeting of the EAB with the AIPGENE consortium was celebrated in Madrid on June of 2011.
This EAB implicates Dr. Deybach (France), Dr. Andersson (Sweden), Dr. Sardh (Sweden), Dr. Badminton (United Kingdom) and Dr. Wilson (Netherlands).

The EAB has being instrumental for the development for the AIPGENE project, they have been actively involved in the design of the clinical protocol and they support with their experience all the essential steps of the project, including in the analysis of the clinical data.

Observational study
The observational studies are regulated in Spain by the new Order SAS3470-2009 since December 2009, so it is mandatory to obtain a study classification by Spanish Medicine and Sanitary Product Agency. The study was classified by AEMPS as a non post-authorization (non-EPA) observational study on 11th May 2011. The non-EPA study code assigned by AEMPS was: DIG-API-2011-01. Then, the observational study protocol was submitted to the Clinical Research Ethics Committee of Hospital 12 de Octubre, as reference EC for the study on 5th May 2011 with code number 11/176. The submission included the following documentation: observational study protocol; insurance policy; draft Case Report Form, and Patient Inform Sheet/Informed Consent. The approval for the observational study untitled “Observational study of acute intermittent porphyria patients” was obtained on 2nd June 2011.
The observational study was designed to record clinical and biochemical baseline parameters for at least 6 moths for each patient in order to compare them with the data obtained after treatment.
The first patient entered the observational study in August 5th 2012. Since them nine patients have been recruited; six at the Clínica Universidad de Navarra and three at the Hospital 12 de Octubre. All of them are severe cases of acute intermittent porphyria. One of the patients evaluated was not included in the study due to the presence of neutralizing antibodies against AAV5 (one of the study exclusion criteria). All of them attended to all the scheduled medical visits (every two months) and data were collected according to the established protocol. Different biochemical and clinical parameters were recorded. Urine samples from these patients were periodically sent to KS for the analysis of porphyrin precursors in urine. Antibody response against transgene and vector was studied at CIMA.

Summary of the Clinical Trial

This is a multicentre, phase I, open label, single dose, dose-ranging clinical trial study conducted at the Clínica Universidad de Navarra (Pamplona, Spain) and at the Hospital Universitario 12 de Octubre (Madrid, Spain), to evaluate the safety and tolerability of a recombinant adeno-associated vector with a liver-specific promoter for the PBGD expression (AAV5-AAT-PBGD), as a potential treatment of Acute Intermittent Porphyria.

Purpose and objectives of the study
The safety, tolerability, and effect of treatment on porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) urinary level changes will be assessed. The maximum therapeutic safe dose of the investigational Gene Therapy (GT) product AAV5-AAT-PBGD will be determined for the treatment of AIP, by registering and evaluating the occurrence of Adverse Events and/or Serious Adverse Events at the doses identified, and the effect of the treatment on symptoms control, psychological involvement and quality of life in AIP patients.
Up to eight patients fulfilling the eligibility criteria will be followed for one year to assess the safety profile of the investigational GT product, and to establish the maximum tolerated dose.

Patient population
Patient’s may participate if they are aged between 18 and 64 years, they have provided written Informed Consent and who have a confirmed diagnosis of severe AIP (as confirmed by clinical, biochemical data and genetic confirmation of porphobilinogen deaminase (PBGD) gene mutation), and who have participated in observational study of acute intermittent porphyria patients for at least six months.
Pregnant women, or females with an intention of becoming pregnant, females of childbearing potential who are not using barrier contraception, or male patients with partners of child bearing potential who are not using barrier contraceptive methods will not participate.
Patients cannot participate if they have evidence of positive titre of neutralizing antibodies against adeno-associated serotype 5 (AAV5).
Patients can also not participate if they suffer from acute or chronic liver disease of viral, autoimmune or metabolic cause, if they have a history of acute or chronic severe gastrointestinal dysfunction, patients with kidney disorder, severe respiratory disease, severe autoimmune disease or severe acute active infection including HBV, HCV, HIV, or if they have evidence of drug use or alcohol abuse or addiction during the three months preceding the selection visit.

Duration of the study, number of visits
The study duration per patient is approximately 52 weeks. In this period, patients will visit the hospital approximately 22 times. Patients will be hospitalized for 3 days for the IMP administration and early close follow up. Patients are expected to visit the follow up site weekly for 8 weeks. From week 8 onwards, patients should attend the hospital every two weeks. From week 12 onwards, patients should return to the sites every 4 weeks. The follow up will be performed in one of the two hospital sites, the one selected by the patient at the beginning of the study based on the geographical preference.

The phase I clinical trial protocol was submitted to Spanish Agency of Medicines and Medical Devices (AEMPS) on June 11th 2012. The phase I clinical trial protocol was submitted to the Ethical Committees (ECs) of the Clínica Universidad de Navarra and Hospital 12 de Octubre for the ethical evaluation as first step for the global clinical trial authorization. In this case, considering that all patients will receive the treatment at Clínica Universidad de Navarra, the reference ethical committee was assigned to Navarra Ethical Committee with code number 5/2012. The AEMPS Authorization was received on 25th September 2012. AMT-021 drug was qualified as product on clinical research phase, with PEI number 12-074.

The initial visit to Clinica Universidad de Navarra was performed on 15th November 2012. The initial visit to University Hospital 12 de Octubre was performed on 23rd January 2013.
Patients performed one inclusion visit followed by three hospitalization days for treatment administration and preliminary follow up, a total of eighteen follow up visit and a final visit. In order to centralize patient’s recruitment, all the inclusion and final visits take place at the Clínica Universidad de Navarra in Pamplona. A total of sixteen of the follow-up visits were performed at the most appropriate centre for each patient, which was established at the beginning of the study. Follow up visit number 13, including optional intermediate MRI and electrophysiological study; and follow up Visit 18, including optional liver biopsy procedure, were optionally performed at CUN if patients consent with voluntary procedures.

The complete study evaluation included the following assessments performed by the clinical teams (partner 3 and 7) and partner 1 divided in three main sections:

Safety and tolerability assessment
• Medical evaluation.
• Routine laboratory tests.
• Immunogenicity analysis. Cellular and Humoral Immunogenicity analysis.
Peripheral blood lymphocytes are obtained and T-cell response against AAV5 capsid protein, AAV5 capsid-derived peptides and PBGD protein was determined.

Total antibodies against AAV capsid and PBGD protein and neutralysing antibodies against AAV5 virus was assessed.
• Liver ultrasonography. As mentioned for observational study.
• Cytokines. Blood samples obtained from the patients before and after treatment are collected to determine the levels of a pool of cytokines by multiplex assay.
• Vector shedding. Blood, urine, oral swabs/sputum, nasal swabs, faeces and semen are taken from the patients for vector shedding analysis. Determination performed at the CIMA in the University of Navarra (partner 1).

Efficacy assessment
• Measurement of ALA and PBG urinary levels.
• Questionnaire to determine AIP symptomatology
• Psychological and quality of life evaluation.
• Medical evaluation.

Pharmacokinetic assessment
• Vector shedding pharmacokinetic analysis. All data corresponding to vector shedding in serum, urine, oral swabs, nasal swabs, faeces and semen (if applicable), measured over the complete trial period are going to be analysed by means of compartmental modelling and pharmacokinetics parameter evaluation by partner 3.

Exploratory assessment
• Biological markers.
• Neurological evaluation. Cerebral MRI and electrophysiological assessment of extremities are being performed at specific visits to better assess neurological compromise.
• Pharmacokinetic modelling response analysis. Two types of responses will be modelled: ALA/PGB measurements in urine, and acute-attacks (expressed as number of events during a specific period of time for an attack to occur and the severity of the attack).
• Vector integration. Characterization of vector integration in the human genome will be optionally studied in leftover PBLs samples and in liver biopsy samples available.
• Vector genome and transgene quantification in liver biopsy. Quantification of vector DNA genomes and RNA copies in liver tissue biopsies were performed to characterize the vector transduction and transgene expression.

5. SAFETY

Side effects observed after AAV5-AAT-PBGD administration in Phase I trial
A total of thirty adverse events (AEs) have been reported on this phase I clinical trial for the eight patients included in the study. Patients’s treatment: CUN-GT-01 and CUN-GT-02 were treated with the first lowest vector dose, CUN-GT-03 and 12H-GT-01 were treated with the second lowest vector dose, 12H-GT-02 and CUN-GT-04 were treated with the intermediate vector dose, 12H-GT-03 and CUNGT- 06 were treated with the highest vector dose.
• Nine AEs were reported for the first vector dose cohort: five occurred to CUNGT- 02 and four to CUN-GT-01.
• Twelve AEs were reported for the second vector dose cohort: four occurred to CUN-GT-03 and eight to 12H-GT-01.
• Three AEs were reported were reported for the third vector dose cohort: two occurred to 12H-GT-02 and one to CUN-GT-04.
• Six AEs were reported were reported for the fourth vector dose cohort: six occurred to 12H-GT-03 and none to CUN-GT-06.

Four of the adverse events have been considered as serious, and a more detailed explanation about these four SAEs (highlighted in red colour) is described in table 16.
Three of the adverse events have been considered as unlikely related to study treatment: diarrhoea, hypersomnia, rash cutaneous. The rest of adverse events have been considered as unrelated to study treatment by investigators.

Viral shedding after AAV5-AAT-PBGD administration

Vector shedding analysis in serum showed maximum vector concentrations 8 hours after vector administration, decreasing thereafter and being below detectable levels 30 days post-injection. Presence of the AAV5-AAT-PBGD in serum was more prolonged in patients receiving the highest doses. Very low levels of AAV5-AAT-PBGD can be transiently detected in saliva, urine, nasal secretion and faeces, but was undetectable in all patients by day 30. No vector was detected in semen samples.

Immune response
All the patients included in the trial developed a humoral immune response against the vector capsid but not against the recombinant PBGD protein. All the patients have total and neutralizing antibodies against AAV5 virus and in general there was a dose response except for patient CUN01 that showed the highest immune response.
However no cellular immune response was detected against the viral capsid or against the recombinant PBGD protein the patients. This result is in clear contrast with previous clinical trials targeting the liver using AAV serotypes 2 and 8 from the treatment of factor IX deficiency were it has been hypothesized that the development of a T cell immune response against viral capsid’s peptides was associated with the elimination of transduced hepatocytes and reduction/disappearance of transgene expression. Elimination of transduced hepatocytes was associated with transaminase elevation.
In our study we did not observed T cell immune response against viral capsid neither transaminase elevation.

Integration of AAV5-AAT-PBGD genome
A comparative large-scale integration site (IS) analysis has been performed on DNA from peripheral blood lymphocytes (PBLs) and liver biopsies obtained from patients treated with the AAV5-AAT-PBGD. In total 22 samples have been analyzed by standard and multiplex LAM-PCR, which simultaneously uses 5 different sets of primers, and subsequent high throughput sequencing.
IS analyses of these samples revealed a total of 3221 unique IS, whereof 2054 IS were exactly mappable to the human genome. There was no preferred integration in the genes MECOM (MDS1-EVI1) or LMO2 previously shown to mediate malignant transformation in clinical retroviral gene therapy. Two patients (12H-GT-03, CUN-GT-03) displayed a strong clone (>99.5% of the identified IS in the respective samples) in the PBLs taken 10 weeks after vector injection. There were no preferred integration loci found in the analyzed samples.
Among all annotated genes in NCBI around 20% are associated with cancer in one of the three cancer data bases used here. Among the top 10 clones from each patient and time point with a total of 62 associated genes there were 11 clones (17%) who’s genes are associated with cancer (EPHA4, RBPJ, MSI2, 2 x RORA, DCC, DLL1, RAB6C, ETS1, PTPRU, MAPK4) according to cBioPortal. This number is below the expected number of expected cancer genes.
Among the eight common integrations sites (CIS) with local clustering of more than three IS seven had at least one IS close to a gene associated with cancer.
To conclude, there were no direct signs for in vivo clone selection and no indications found for genotoxicity in the analyzed samples.

6. EFFICACY
Porphyrin precursor levels in urine

Laboratory studies can reliably establish the diagnosis and follow up of Acute Intermittent Porphyria evolution, as the disease is characterized by increased urinary Pprphobilinogen (PBG) and to a lesser extent Aminolevulinc acid (ALA). PBG urine excretion ten times > 1.6 mmol per mol of creatinine and ALA urine excretion two times > 3.9 mmol per mol of creatinine are the reference levels used for AIP laboratory diagnose. Quantitative determination based on anion exchange separation is the preferred method to determine PBG levels in urine. ALA can be quantified simultaneously by sequential elution on a second column. The Porphyria Centre Sweden (PCS) at the Karolinska University Hospital is the partner of the AIPGENE consortium responsible of the ALA and PBG determinations of the urine samples collected along the observational study and the phase I clinical trial. PCS is the institutions with the highest expertise in the world for the management and characterization of samples for porphyric patients.
First, the analysis of porphyrin precursor PBG and ALA in the patients included in the clinical trial showed that all the patients showed a sustained elevation of both precursors that further increase during porphyric attacks.
The comparison of precursors levels in urine before (observational study) and after (clinical trial) revealed no significant differences between the two periods, indicating that the administration of AAV5-AAT-PBGD has no effect over precursor excretion.

A model was established to describe the pharmacokinetic/pharmacodynamic relationship of the effect of haem arginate on ALA and PBG in acute porphyric patients in steady-state disease conditions. The model described the time course of urine metabolite concentrations in absence and under treatment with Normosang. This model was use to explore a synergistic effect during gene therapy treatment. However, the PD model developed using historical data and the data from the observational study could not be used to characterize time profiles of ALA and PBG and no effect was observed after treatment.

Frequency of porphyria acute attacks

Porphyria acute attack is a clinical condition characterized by an episode of clinical symptoms requiring hospitalization and specific treatment. The attacks are provoked by precipitating factors that induce cytochrome P450 and/or ALA-S1 activity and by increasing haem-demand in the liver.
Therefore AIP evolves as acute crises and in between each crisis the patient may either feel normal or they may suffer persistent symptoms. The frequency and intensity of acute crises varies between patients, but usually an episode of clinical symptom requires hospitalization and specific treatment.
The most common symptoms are nausea, vomiting and abdominal pain without peritoneal signs. Hypertension and tachycardia are generally present. Motor neuropathy is common and engagement of respiratory functions may lead to respiratory arrest. Hyponatremia may take place. Seizures can be a consequence of hyponatremia or hypomagnesemia or be secondary to central nervous system involvement. Psychiatric manifestations such as depression, insomnia, agitation, confusion and hallucinosis may be present. The attack of acute porphyria is by definition accompanied by increased concentrations of porphyrin precursors in plasma and urine.

Considering this is a phase I trial to measure safety of the gene therapy product but also preliminary efficacy, the frequency of porphyria attacks has been recorded in the observational study and during the clinical trial by registration of AIP related hospitalization and uptake of medications associated with an acute attack such as hemin or glucose as well as pain killers.

We observed a reduction higher than 50% in the number of hospitalizations in two of the patients, CUNGT02, CUNGT03 and 12H01, when we compared the number of hospitalization before and after the administration of the vector. Furthermore, we observed a reduction higher than 50% in the number of days hospitalized in two of the patients, CUN02 and CUN03, and higher that 30% in three more patients CUN01, 12H01 and 12H03 when we compared the number of hospitalization before and after the administration of the vector.

Secondly, a reduction of hemin uptake was observed in patients as well as a reduction of glucose uptake in the same patients indicating a less frequent incidence of AIP symptoms on these patients.

Taken together all the data we observed that patients showing a clinical improvement (CUN01, CUN03 and 12H01) see below, presented a more stable ALA and PBG pattern, basal PBG values were lower than 40 mmol/mol creatinine. In comparison with patients that did not show any improvement after treatment that showed basal values higher that 40 units, and reaching maximun values of 70-100. In these particular patients, PBG values fluctuate about 20 units, whilst in patients that did not show any improvement after treatment, fluctuation can reach 50 units and PBG/ALA ratio is <2.

Liver biopsies

From the eight patients included in the trial 6 agreed to donate a liver biopsy to analyse vector transduction efficiency. We analysed the presence of vector genome by quantitative PCR. Vector genomes were detected in all the samples analysed. Furthermore, the analysis of the presence of transgene messenger RNA revealed the presence of the recombinant transcript.

Psychological evaluation of the patients.

Psychological evaluation of the patient’s was assessed through the BAI and BDI-II questionnaires. Patients completed the questionnaires in every study visit, during the Observational study, and at the beginning monthly and then every two months, in the clinical trial.
Beck Anxiety Inventory (BAI) assesses anxiety severity in adults and adolescents. It is a 21-question multiple-choice self-report inventory that is used for measuring the severity of an individual's anxiety. The different categories evaluated subjective somatic or panic-related symptoms of anxiety. BAI discriminated anxious diagnostic groups (panic disorder, generalized anxiety disorder, etc.) from non-anxious diagnostic groups major depression, dysthymic disorder.
The data indicate a clear trend to improvement in both parameters in all the patients. However to reach to conclusions Statistical Analysis shouls be performed.

The main conclusions extracted from this study are the following:
1. The treatment with AAV5-AAT-PBGD is well tolerated and safe in patients with Acute Intermittent Porphyria. No signs of toxicity were seen during the administration of the drug or during patient follow up.
2. Humoral immune response was induced in all patients. All the patients developed total and neutralizing antibodies against the vector but no against the recombinant protein.
3. No cellular immune response against the vector or the transgene was detected. Contrary to what was observed in previous clinical trials targeting the liver, for the treatment of hemophilia B, no cellular immune response associated with hepatocyte elimination (liver damage) was observed in the Aipgene trial.
4. AAV5-AAT-PBGD transduced human liver. The analysis of liver biopsies obtained from 6 out 8 patients revealed that the AAV5-AAT-PBGD vector transduced the liver.
5. There is some effect on clinical outcomes in certain patients. Some patients have clearly reduced the uptake of medications specific for the treatment of the disease and reduced the number and days of hospitalizations.
In summary, we have gathered critical clinical experience that will contribute greatly to the further understanding of this complex disease. The AIPGENE collaboration has achieved its primary goal in completing a successful Phase I study, providing first and foremost the safety data required for additional clinical study. Finally, we are currently performing the final analysis of the data for publication and to defining what the next steps for the further development of AIP gene therapy.
Furthermore, the consortium believes that increasing the level of liver transduction i.e. making the patients liver produce even more PBGD would likely have a much bigger benefit. This in turn could be achieved by increasing the dose and/or generating an improved version of the gene therapy product. These options and potential implications are currently being carefully considered in order to generate realistic and solid plans for further exploitation.

Even though a detailed technology implementation plan cannot be drafted at present, all partners remain determined to support further development of the gene therapy product for AIP. Based on the encouraging data from the Phase I/II, uniQure as the manufacturer and partner who can bring the product further forward into development and even commercialization, is currently considering how to design the next clinical trial. Its clinical team has engaged with the consortium partners and experts and is seeking advice on the future clinical program.

Potential Impact:

Description of the potential impact
Acute Intermittent Porphyria is a severe rare condition lacking effective treatment. AIP is a highly disabling disease and it represents a life limiting condition for the patients and their families. Furthermore, Acute porphyric attacks can be life-threatening and the long-term consequences may include irreversible nerve damage, liver cancer and kidney failure. Curative therapy based on liver transplantation has only been tried on a few occasions. This limited experience showed that organ replacement is associated with a high risk of mortality in AIP patients.
The AIPGene consortium aims to treat AIP patients using the orphan drug AAV5-AAT-PBGD to alleviate the negative impact of the disease on the quality of life of the patients and their families. This progress would in turn lead to an improvement in both the duration and the quality of life of the patients, and eventually reduce the burden of the disease and the socio-economic cost of this condition.
Our data has unequivocally demonstrated that gene therapy in patients with Acute intermittent porphyria using a recombinant adenoassociated vector is SAFE. High doses of the vector have been intravenously administered and no side effects related to the treatment have been observed. Furthermore, some of the patients have benefit from the treatment, reducing medication uptake. Although a rigorous statistical analysis is pending a trend to an improvement in the quality of life of the patients can be observed.
Although our objective is to implement a scientifically and medically well founded clinical trial to assess tolerance and effectiveness of gene therapy for patients with AIP, our result may benefit immediately to other liver metabolic disorders. The technologies developed are of interest to both the scientific community and pharmaceutical industry, as they will confirm the advances in a new therapeutic approach for such disorders. The importance of this element is reflected by the participation of the SMEs. We believe our efforts will eventually improve the treatment options available in a number of diseases and in this manner contribute to the reduction of the personal, societal and economic burden to the European Community. This is particularly relevant when considering treating childhood diseases and the ageing European population, since the discoveries arising from this project will provide the groundwork to develop improved methods to treat and delay the onset of the liver disorders.
Furthermore, the design of the clinical trial using AAV5-AAT-PBGD required the organization of a group of European AIP experts capable of providing useful advice on the management and follow-up of the patients included in the trial and on the best way to proceed after completion of the study. Because AIP is rare disease, the study of the natural course of the disease in a patient cohort is limited when the follow-up is restricted to a single country. It is only by merging patient cohorts at the European level that clinical studies will yield significant data. The participation of patient associations to this goal will be paramount. The repercussions of these studies in the daily management of patients will only be possible if groups of clinical experts are created at the European level. Some of the most important experts on liver disorders and Porphyrias can be found in Europe and it is important for European research to remain competitive in this area, it is necessary to build on the existing knowledge base to further our understanding of these disorders. The proposed Consortium will increase European competitiveness by integrating research capacities and uniting the critical mass necessary to fully address the problems at hand. The state-of-the-art technologies that will be combined would not otherwise be available in each of the individual laboratories, or in the laboratories that already maintain ongoing collaborations. Thus, the constitution of this consortium will serve to further integrate and advance the work carried out in this area in a coordinated manner. At present, our consortium includes investigators from 4 countries but we are confident that building-up a coordinated European expertise on AIP gene therapy treatment will create a dynamic process and become highly attractive for clinicians and scientists based in other European countries. In this way the competitive nature of European research in this field will be maintained
Since the research that has been carried out in this project is multidisciplinary, the proposal builds on this strength to translate basic research to the clinic in partnership with SMEs. The partners involved offer a diverse array of skills, the combination of which serves to reinforce the competitiveness of EU and contribute to define it as a potential leader in this field. The results and technology derived from this combined effort have a realistic potential for future commercial applications. We believe that a joint task force of European groups provides a clear competitive advantage, and will enhance the returns from recent innovative findings obtained in the laboratories of the consortium. This collaboration will foster innovation, sustainable development, and the transfer of information and know-how to European industry, thereby enhancing its competitiveness in an increasingly globalised market. In particular, the project will notably contribute to the development and growth of European SMEs by increasing their RTD activities. In turn, the new knowledge acquired will strengthen the scientific and technological basis of community industry encouraging it to become more competitive at an international level. This is particularly important because modern biotech based industries should continue to be a major factor in the economic growth of the European Union, increasing the value of products and promoting job creation. The results from our combined efforts will have a potential for commercial applications. It is therefore essential that we develop this area of research as rapidly as possible in order to compete with US and Japanese companies who are also involved in related activities.

Main dissemination activities
The consortium has been and is highly sensitive of the need to communicate the results obtained from research to the scientific community and to the general public, as the research undertaken in the project is highly relevant and applicable to current research interests in the field. Dissemination of the results of the research have been will be made swiftly through the normal channels of original papers in peer reviewed, international scientific journals, oral presentations and posters at conferences, workshops and seminars, as well as being described in reports to the European Commission. In all cases, the financial support of the VII Programme has been and will be recognised in the appropriate section of the communication. Members of the Consortium have been and will be encouraged to participate in conferences and scientific meetings in the area in order to disseminate the progress of the project but also to receive the opinions of other experts in the field.
Prior to the publication or presentation of any results from the project, they were and will be evaluated by the SC in order to decide whether they merit protection, patenting and further exploitation or not. This approval for publication of manuscripts/results have been and will be initially be made on the basis of abstracts sent by the Partners to the CO (prior to submission for publication or presentation). No manuscripts have been will be submitted until this evaluation has been performed.
The CO has participated and will participate in forums to discuss and foster the transfer of results and technology, for example of compounds considered to be of further interest for development. Each institution also has a technology transfer office experienced in securing patent rights and in contacting additional partners. Decisions on the exploitation and dissemination strategy has been taken mostly during the meetings of the SC.
The AIPGENE Coordinator has ensured an optimal communication within and outside the consortium, by the periodical consortium meetings, local clinicians-sponsor meetings, teleconferences and emails. Has informed and assessed all members of AIPGENE project accomplish well through attendance at scientific meetings, inviting also an external advisory board group composed by some Porphyria experts.
The AIPGENE consortium has informed the general public about the activities and results emerging from the project. The consortium have done the necessary steps to inform third parties of its activities and have participated in European groups or initiatives where the particular expertise in the consortium may contribute to improving public awareness or making more informed policy decisions. Also, the Consortium has also included information about its activities on the webpage informing about the consortium and its activities.
An important component of dissemination have been channeled through the public part of the consortium website, which will serve as an informative forum, both for scientific and healthcare professionals and for the public at large, for issues surrounding the scientific and technological aspects in the programme, as well as the novel therapeutic advances.

The AIPGENE project has participated in the following events:
The 47th Meeting of the European Association for the Study of the Liver (EASL). Oral presentation entitled “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria” exposed by Dr. Astrid Pañeda (DIGNA). This meeting was held in Barcelona during the 18-22th of April of 2012.
The XXXVII Annual Congress of the Asociación Española para el Estudio del Higado (AEEH). Oral presentation exposed by Dr. Astrid Pañeda (DIGNA) entitled “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria”. This meeting was held in Madrid during the 26th of February of 2012.
The 15th Annual Meeting and Training Course of the ASGCT (American Society of Gene & Cell Therapy. Oral presentation exposed by Dr. Christine Kaeppel (DKFZ-NCT) entitled “Absence of Integration Hotspots in Non-Human Primates after Intravenous Injection of AAV2/5-AAT-coPBGD”. Poster presentation entitled “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria” exposed by Dr. Delia D’Avola (UNAV). This meeting was held in Philadelphia during the 16-19th of May of 2012.
The European Conference on Rare Disease & Orphan Drugs. Poster presentation entitled “Augmenting PBGD expression in the liver as a Novel Gene therapy for Acute Intermittent Porphyria” was exposed by Dr. Carlos Camozzi (uniQure). The meeting was celebrated in Brussels during 24th of May of 2012.
XXI European Society of Gene and Cell Therapy (ESGCT). AIPGENE attended to the European Society of Gene and Cell Therapy (ESGCT) conference that was held on October 2012 in Versailles, France. uniQure presented the several posters
The International Rare Diseases Research Consortium International Rare Diseases (IRDiRC). AIPGENE attended to the first International Rare Disease Research Consortium (IRDiRC) conference that was held on April 16-17 2013 in Dublin, Ireland. A Poster untitled, "AIPGENE: Augmenting PBGD expression in the liver as a Novel Gene Therapy for Acute Intermittent Porphyria was presented at the first IRDiRC conference. Furthermore the information about the AIPGENE consortium in the book published by the European Commission: "Rare diseases, How Europe is meeting the challenges".
International Congress of Phorphyrins and Porphyrias & International Meeting of Porphyria Patients. AIPGENE members were pleased to participate in the International Congress of Porphyrins and Porphyrias & the International Meeting of Porphyria patients convened in a meeting with expert clinicians, researchers and patients suffering from these rare diseases. The meeting was held at the KKL Lucerne convention centre in May of 2013, with the purpose to exchange the latest information about these diseases knowledge and future strategies. Dr. Prieto (UNAV), as AIPGENE Study Coordinator, was invited for a lecture about Gene Therapy as a potential treatment for Acute Intermittent Porphyrias. Dr Fontanellas and Dr Pañeda presented the consortium results as oral communications.
XXII European Society of Gene and Cell Therapy (ESGCT). AIPGENE project participated as sponsorship in the European Society of Gene & Cell Therapy Collaborative Congress, where several researchers, clinicians, and patients associations working or interested in the gene and cell therapy field, joined together. The event was held at the Palacio Municipal de Congresos in Madrid.
17th Annual Meeting of the American Society of Gene & Cell Therapy. Dr. González attended as invited speaker and presented an oral presentation entitled “Liver Directed Delivery of AAV5 for Acute Intermittent Porphyria“. Also Dr. D’Avola presented an oral presentation entitled “Phase 1 Clinical Trial of Liver Directed Gene Therapy With rAAV5-PBGD in Acute Intermittent Porphyria: Preliminary Safety Data”; Dr. Schmidt presented two oral presentations entitled “Topology of Wildtype AAV Integration” and “Double Barcoded LAM-PCR as Quality Control for Accurate Deep-Sequencing of Vector Genome Junctions” respectively. Finally, two posters were exposed by Dr. Schmidt entitled “Bioinformatical Analysis in the Era of High Throughput Sequencing Integromics” and “Targeted Sequencing for Mapping Vector Integration Sites”. Meeting was celebrated in Washington, United States. The event was celebrated in Washington, United States.

Patients Associations Events
Working closely with experts of the mentioned fields and with the Patients Associations. During June of 2011, an official letter presenting the AIPGENE project was sent to all the main community interested. Also the AIPGENE project was officially presented in the following meetings:
The International Porphyrins and Porphyrias Congress. The project was exposed by Dr. Gloria Gonzalez (FIMA) to clinicians, researchers and other experts to inform about the principal objectives of the project. Oral presentation entitled “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria” exposed by Dr. Astrid Pañeda (DIGNA). Last day, Dr. Gloria González also presented the project to the 1st International Porphyria Patient Meeting. This event was celebrated during 12th of April of 2011 in Cardiff, Wales. Also, Dr. Fontanellas (FIMA) exposed a poster entitled “Decreased glucose uptake in the primary somatosensorial cortex in the brain of female mice with acute intermittent porphyria” in the mentioned Congress.
Porphyria Spanish Association (AEP) Meeting. The AEP convened a meeting with patients suffering from these rare diseases together with relatives, expert clinicians and researchers on a ‘Porphyrias Today: Challenges and Prospects’ meeting. The meeting was held at the State Reference Centre for Care of Persons with Rare Diseases and Their Families (CREER) in Burgos (Spain) last 28th of April of 2012. Furthermore, Dr. Enríquez de Salamanca (SERMAS), Head of Porphyria Unit of the Hospital Universitario Doce de Octubre in Madrid, underscored the relevance of incorporating an early screening to make possible an efficient diagnosis. The first day of the meeting concluded with the intervention of Dr. Delia D'Avola (UNAV) and Dra. González-Aseguinolaza (FIMA). They make two presentations to describe how gene therapy could be a promising treatment that is likely to provide a definitive cure for this disease, and how the AIPGENE consortium is moving to the clinical development.
Porphyria Spanish Association (AEP) Meeting. Last 23rd November 2013 the North Delegation of Spanish Porphyria Patient Association organized a first meeting in Bilbao. The main objective was to establish contacts between clinicians and researchers working in the porphyria field, with affected patients and their families. Four invited speakers presented different aspects of acute and cutaneous porphyria's. Presentations addressed last advances in basic research (Dr. Millet), clinical diagnose (Dr. Enríquez), dermatological implications (Dr. Ratón) and neurological involvement (Dra. Aranzabal). Dr. Enríquez, as AIPGENE member, presented also an update on the AIPGENE therapy clinical trial and answered questions from attendees.

Summary of dissemination activities
31-01-2011, uniQure, Press release, Published in different internet webpage links
03-02-2011, DIGNA and FIMA, Press release, Published in different internet webpage links
10-02-2011, FIMA, Press release, Published in CIMA’s official webpage
25-05-2011, FIMA, Webpage, Webpage launch. http://cima.es/aipgene/ 27-06-2011, Consortium as a whole, A Project Letter was sent to most of the official Associations and Societies of patients. Also was sent to clinicians and researcher experts on the field.
12-04-2011, FIMA, Poster presentation entitled: “Decreased glucose uptake in the primary somatosensorial cortex in the brain of female mice with acute intermittent porphyria”, Cardiff, UK.
12-04-2011, FIMA, DIGNA and UNAV, Conference at the International Porphyrins and Porphyrias Meeting. Worldwide scientists and medical doctors working on porphyrias. Cardiff, UK.
12-04-2011, FIMA, DIGNA and UNAV, Conference presented to the Porphyria patient Association. Cardiff, UK.
16-02-2012, DIGNA, FIMA, uniQure, UNAV Conference at the XXXVII congreso anual de la Asociación Española para el Estudio del Higado (AEEH). Madrid. “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria”
06-03-2012, FIMA, Scientific article, entitled “Renal failure affects the enzymatic activities of the three first steps in hepatic heme biosynthesis in the acute intermittent porphyria mouse” has been published in Plos One Journal.
18-03-2012, FIMA Conference at the The 47th Meeting of the European Association for the Study of the Liver (EASL) entitled “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria”. This meeting was held in Barcelona, Spain.
28-04-2012, FIMA, UNAV and SERMAS Conference at thePorphyria Spanish Association Meeting. Dr. Enríquez de Salamanca (SERMAS), Head of Porphyria Unit of the Hospital Doce de Octubre in Madrid, underscored the relevance of incorporating an early screening to make possible an efficient diagnosis and Dr. Delia D'Avola (UNAV) and Dr. González-Aseguinolaza (FIMA) presented the project. This meeting was celebrated in Burgos, Spain.
02-05-2012, FIMA, Press release entitled “La progresión de la IR empeora la PAI” was published in Diario Médico.
06-05-2012, UNAV and DIGNA, Press release entitled “La terapia Génica sale del túnel” was published in the newspaper El País.
17-05-2012, NCT and UNAV, Conference and poster presentation at The 15th Annual Meeting and Training Course of the ASGCT. Oral presentation exposed by Dr. C. Kaeppel entitled “Absence of Integration Hotspots in Non-Human Primates after Intravenous Injection of AAV2/5-AAT-coPBGD”. Poster presentation exposed by Dr. Delia D’Avola (UNAV) entitled “Toxicology and Liver Transduction Efficacy Evaluation of a Recombinant Adeno-Associated Viral Vector in Non-Human Primates as a Potential Treatment for Acute Intermittent Porphyria”. This meeting was held in Philadelphia, USA.
24-05-2012, uniQure and DIGNA, Poster presentation at The European Conference on Rare Disease & Orphan Drugs. Dr. Camozzi (AMT) presented a poster entitled “Augmenting PBGD expression in the liver as a Novel Gene therapy for Acute Intermittent Porphyria”. The meeting was celebrated in Brussels, Belgium.
15-06-2012, FIMA, UNAV, uniQure and SERMAS,Scientific article entitled “Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates” has been published in the Journal of Translational Medicine.
17-09-2012, UNAV, EPNET Meeting. Dr. D’Avola. Project Presentation.
20-09-2012UniQure and DIGNA Conference Meeting at BioSpain roundtable. BioSpain press release. Advanced Therapy Medicinal Products: “Introduction BioSpain 2012 supera sus expectativas y mejora las estadísticas de la edición anterior”. Asebio 93-99.
27-10-2012, uniQure, The European Society of Gene and Cell Therapy. Dr. H. Petry presented a poster entitled “No longitudinal transmission of AAV5-PBGD vector DNA in mice“ and “Non-clinical safety evaluation of AAV5-PBGD in mice and Cynomolgus Macaques”, and Dr. C. Kaeppel presented a poster entitled “Safe AAV profile in LPLD gene therapy”. The meeting was celebrated in Versailles, France.
Oct 2012, AIPGENE Consortium Press release (AIPGENE web), Spanish Medicines and healthcare products agency provides authorization for first gene therapy clinical trial in acute intermittent porphyria.
Nov 2012, NCT and uniQure, Scientific Article entitled “A largely random AAV integration profile after LPLD gene therapy” has been published in Nature Medicine.
11-12-2012, uniQure, Press release, uniQure initiates Phase I in acute intermittent porphyria - AIPGENE consortium sets first step towards cure for rare disease. (http://www.uniqure.com/news/171/182/uniQure-initiates-Phase-I-in-acute-intermittent-porphyria.html).
Jan 2013, FIMA, UNAV, uniQure and DIGNA, press release entitled “Primer tratamiento con terapia génica en España para una enfermedad metabólica rara” was published in the Spanish Newspaper El Diario de Navarra and in CIMA’s institutional journal.
01-03-2013, NCT, The Annual Meeting of DG-GT. Oral presentation exposed by Ms. Christine Kaeppel entitled “AAV1-LPLS447X gene therapy results in a random distribution of integration throughout chromosomal and mitochondrial DNA”. This meeting was held in Hamburg. Germany.
Mar 2013, SERMAS, DIGNA and FIMA, press release entitled “El Hospital participa en el primer ensayo clínico mundial con terapia génica probado en pacientes para la porfiria aguda intermitente” was published in the Hospital 12 de Octubre’s institutional journal.
Mar 2013, FIMA, press release entitled “Un virus directo contra la porfiria” was published in the Spanish Newspaper El País.
Apr 2013, FIMA , Scientific Article entitled “Helper-dependent adenoviral liver gene therapy protects against induced attacks and corrects protein folding stress in acute intermittent porphyria mice” has been published in Human Molecular Genetics.
16-04-2013, FIMA, International Conference,The International Rare Diseases Research Consortium conference. Dr. González presented a poster entitled “Augmenting PBGD expression in the liver as a Novel Gene therapy for Acute Intermittent Porphyria”. The meeting was celebrated in Dublin, Ireland.
May 2013, SERMAS and DIGNA Press release entitled “Primer ensayo mundial con terapia génica para porfiria aguda” was published in the local Journals Diario Médico and Acta Sanitaria and link websites consalud.es; El distrito.es; Madrid informativo.
16-05-2013, NCT, International Congress The 16th Annual Meeting and Training Course of the ASGCT. Oral presentation exposed by Dr. C. Kaeppel entitled “AAV integrates randomly into the nuclear and the mitochondrial genome after LPLD gene therapy”. This meeting was held in Salt Lake City, USA.
17-05-2013, UNAV, FIMA, KS and DIGNA, International Congress The International Congress of Phorphyrins and Porphyrias & International Meeting of Porphyria Patients. Dr. Prieto, Dr. Fontanellas presented oral presentations entitled “Gene therapy of Acute Intermittent Porphyria“ and “OC16 Helper-Dependent Adenoviral Liver Gene Therapy Protects Against Induced Attacks and Corrects Protein Folding Stress in Acute Intermittent Porphyria Mice“, respectively. Dr. Pañeda presented two posters entitled “Non-clinical safety evaluation of AAV5-PBGD in mice and cynomologus macaques” and “No longitudinal transmission of AAV5-PBGD vector DNA in mice” and Dr. Harper presented a poster entitled “Quantitative Description of Heme Arginate Effects on ALA and PBG Urine Profiles in Acute Porphyric Patients During a Screening-Type Observational Period”. The meeting was celebrated in Lucerne, Switzerland.
Jun 2013, uniQure and DIGNA, An editorial article entitled “A Gene therapy for AIP” was published in Pan European Networks.
01-06-2013, SERMAS and DIGNA, Spanish Patient Association. An informative session for questions and answers of the AIPGENE project was included as part of the session that the Spanish association of porphyric patients in Spain organized in Madrid in the ONCE building.
02-07-2013, FIMA and UNAV, Scientific Article entitled “IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over pre-existing AAV transgene expression” has been published in Plos One.
26-10-2013, AIPGENE Consortium, International congress, The European Society of Gene and Cell Therapy. Dr. González presented an oral presentation entitled “AIPGENE: Augmenting PBGD expression in the liver as a novel gene therapy for acute intermittent porphyria“. Also Dr. López-Franco presented a poster entitled “Preliminary safety data from phase I clinical trial in acute intermittent porphyria“. Meeting was celebrated in Madrid, Spain.
26-10-2013, AIPGENE Consortium, International Congress, AIPGENE Project participated as a sponsor in The European Society of Gene and Cell Therapy Congress.
26-10-2013, AIPGENE Consortium, Dissemination tools During the European Society of Gene and Cell Therapy Congress, the coordinator distributed leaflets informing about the project evolution.
23-11-2013, SERMAS, Patient Association Meeting, Spanish Patient Association. Dr. Enriquez de Salamanca presented an update on the AIPGENE therapy clinical trial and answered questions from attendees, Bilbao, Spain.
24-12-2013, AIPGENE Consortium, Scientific Article entitled “Safety and Liver Transduction Efficacy of rAAV5-cohPBGD in Nonhuman Primates: A Potential Therapy for Acute Intermittent Porphyria” has been published in Human Gene Therapy.
20-02-2014, FIMA and UNAV, Press releaseentitled “Pasos de gigante en la terapia génica” was published in the national newspaper “El Mundo”.
05-05-2014, NCT, Scientific Articleentitled “Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats” has been published in Biotechniques.
23-05-2014, FIMA, UNAV and NCT, International Congress, 17th Annual Meeting of the American Society of Gene & Cell Therapy. Dr. González attended as a invited speaker and presented an oral presentation entitled “Liver Directed Delivery of AAV5 for Acute Intermittent Porphyria“. Also Dr. D’Avola presented an oral presentation entitled “Phase 1 Clinical Trial of Liver Directed Gene Therapy With rAAV5-PBGD in Acute Intermittent Porphyria: Preliminary Safety Data”; Dr. Schmidt presented two oral presentations entitled “Topology of Wildtype AAV Integration” and “Double Barcoded LAM-PCR as Quality Control for Accurate Deep-Sequencing of Vector Genome Junctions” respectively. Finally, two posters were exposed by Dr. Schmidt entitled “Bioinformatical Analysis in the Era of High Throughput Sequencing Integromics” and “Targeted Sequencing for Mapping Vector Integration Sites”. Meeting was celebrated in Washington, United States.
27-05-2014, uniQure, Press Release, UniQure published in their official webpage the following press release “uniQure Announces Successful Transfection of Liver Cells with AAV5 and Strong Safety Data from Acute Intermittent Porphyria Clinical Trial”.
06-06-2014, FIMA, International CongressV International congress on glucogenosis. Dr. González presented an oral presentation entitled “Gene therapy for hepatic monogenic hereditary diseases”. The event was celebrated in Barcelona, Spain.
20-06-2014, NCT, Scientific Comment, A scientific reply entitled “NGS library preparation may generate artifactual integration sites of AAV vectors” has been published in Nature Medicines.
01-07-2014, FIMA, Press release entitled “Positive signs for curative treatment against Porphyria” was published in the www.youris.com (European Research Media Center)
01-07-2014, FIMA, A video entitled “Gene Correction for a rare disease” was published in the www.youris.com (European Research Media Center).
15-10-2014, uniQure, A poster entitled “Gene therapy for Acute Intermittent Porphyria” was presented by Dr. Grosios in the 2nd Rare Diseases Summer School radiz - Rare Disease Initiative Zürich. Switzerland.
08-10-2014, UNAV, Scientific event, Jornada Terapias Avanzadas. Fundación Sermes. Dr. D’Avola presente dan oral presentation entitled “Ensayo clínico de Terapia Génica para una enfermedad Metabólica huérfana”. The event was celebrated in Madrid, Spain.
08-10-2014, FIMA, Social event, The coordinator of the project presented a general view of the objectives of the project to society. The event “Aula de Salud” was organized by the local newspaper “Diario de Navarra”.
09-10-2014, AIPGENE Consortium, International Symposium AIPGENE partners organized an International Symposium entitled “Liver directed gene therapy for rare diseases” in Pamplona, Spain.
14-10-2014, FIMA, Press release entitled “Un experto ve en la terapia génica el futuro de la medicina” was published in the regional journal “Diario de Navarra”.
16-10-2014, FIMA, Press reléase entitled “Los avances en terapia génica sugieren una 'nueva era' de la medicina molecular” was published in the national newspaper“ABC”.
23-10-2014, FIMA, uniQure and UNAV International Congress. The European Society of Gene and Cell Therapy. Dr Gonzalez Aseguinolaza and Dr D’Avola presented the latest data of the AIPGNE clinical trial. Dr. Majowicz from uniQure presented an oral presentation entitled “Successful repeated hepatic gene delivery in mice and non-human primates achieved by sequential administration of AAV2/5 and AAV1 vector serotypes”. Meeting was celebrated in The Hague, The Netherlands.
16-11-2014, FIMA, National Congress, UPV BioForo. Coroideremia: Conocimiento y terapia. Dr. González atteended as a speaker with the following speech “Liver Directed Delivery of AAV5 for Acute Intermittent Porphyria”. The event was celebrated in Bilbao, Spain.

During 2011 and 2012, The consortium has distributed USB memory sticks in International Congresses related to the AIPGENE scientific field, in order to disseminate information regarding AIPGENE project.

Exploitation of results.
The consortium has made significant efforts in raising awareness about the disease via its various dissemination activities. Furthermore, it has established strong contacts with patients, patient’s organizations and professionals in the field. In terms of wider societal implications, it is still too early to say that the specific gene therapy tested in this Phase I clinical trial will be commercialized as such. It is clear that evidence of clinical benefit have been observed. However this needs to be verified in a Phase II/III setting. At the same time and as previously mentioned the consortium believes that increasing the level of liver transduction i.e. making the patients liver produce even more PBGD would likely have a much bigger benefit. Nevertheless, the phase I/II data provides some indication that an AAV-based PBGD gene therapy could have major positive impact on the life of patients with AIP and their families.

List of Websites:

Project Web site:
http://cima.es/aipgene/

Partners websites:
www.cima.es
www.uniqure.com
www.unav.edu
www.cun.es
www.karolinska.se/val-av-webbplats
www.dkfz.de/en/index.html
www.dignabiotech.com
www.imas12.es