Protocol Number: CT-AMT-010-01
A Study to Determine the Safety and Efficacy in Lipoprotein
Lipase-Deficient Subjects after Intramuscular Administration of AMT-010, an
Adeno-Associated Viral Vector Expressing Human Lipoprotein
Lipase
S447X.
Protocol version: 1.2
Protocol version date: 29 April 2005
Incorporating Amendment number: n/a
Confidential
Protocol: CT-AMT-010-01
SUMMARY INFORMATION
Title:
A Study to Determine the Safety and
Efficacy in Lipoprotein Lipase-Deficient
Subjects after Intramuscular
Administration of AMT-010, an
Adeno-Associated Viral Vector Expressing
Human Lipoprotein Lipase
S447X.
Protocol Number: CT-AMT-010-01
Sponsor: Amsterdam Molecular Therapeutics, Meibergdreef 61, P.O. Box 22506, 1100 DA Amsterdam, The Netherlands Phone: 020-5667580 Fax: 020-5669272
E-mail: [email protected]
Contact person: Janneke Meulenberg PhD, Project Leader Principal
Investigator:
Erik Stroes, MD PhD, Academic Medical Center,
Department of Vascular Medicine, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Phone: 020-5665978 Fax: 020-5669343 E-mail: [email protected]
Clinical Research Monitor:
Rene van Boxtel, PhD, IATEC, Pietersbergweg 9, 1105 BM Amsterdam, The Netherlands
Phone: 020-3149359 Fax: 020-3149399 E-mail: [email protected]
Medical Monitor: Joost Vermeulen, MD, IATEC, Pietersbergweg 9, 1105 BM Amsterdam, The Netherlands
Phone: 020-3149359 Fax: 020-3149399 E-mail: [email protected]
Data Safety
Monitoring Board:
Prof. Joost Hoekstra, MD PhD, Academic Medical Center, Department of Internal Medicine, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
Phone: 020-5666130 Fax: 020-6919658 E-mail: [email protected]
Prof. Anton Hagenbeek, MD PhD, University Medical Center Utrecht, Department of Hematology (G03.647) Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. Phone: 030-2507769 Fax: 030-2511893.
E-mail: [email protected]
Victor Gerdes, MD PhD, Academic Medical Center, Department of Vascular Medicine, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
Phone: 020-5666008 Fax 020-6968833 E-mail: V.E. [email protected] Drug Name: AMT-010
Protocol Agreement
A Study to Determine the Safety and Efficacy in Lipoprotein
Lipase-Deficient Subjects after Intramuscular Administration of
AMT-010, an Adeno-Associated Viral Vector Expressing Human
Lipoprotein Lipase
S447X.
CT-AMT-010-01
Date: 29 April 2005
The signatures of the investigator and representative of the sponsor below constitute their approval of this protocol and provide the necessary assurances that this study will be conducted according to Good Clinical Practice and to all stipulations, clinically and administratively, as stated in the protocol, including all statements as to confidentiality. It is agreed that the conduct and results of this study will be kept confidential and that the case report forms and other pertinent data will become the property of Amsterdam Molecular Therapeutics.
It is agreed that the protocol contains all necessary information required to conduct the study as outlined in the protocol, and that the study will not be initiated without the approval of the national Centrale Commissie Mensgebonden Onderzoek (CCMO) in the Netherlands and the Institutional Review Board (IRB) of the Academic Medical Center (AMC), Amsterdam, the Netherlands.
It is agreed that all participants in this study will provide written informed consent in accordance with the requirements specified in the Declaration of Helsinki/Venice/Tokyo/Hong Kong/South Africa/Edinburgh/Washington. All participants will also be informed that their medical records will be kept confidential except for review by representatives of Amsterdam Molecular Therapeutics or designee and/or appropriate regulatory authorities. Dr Erik Stroes Principal Investigator Signature Date Dr Janneke Meulenberg Sponsor Signature Date
TABLE OF CONTENTS
SUMMARY INFORMATION...2
ABBREVIATIONS ...9
PROTOCOL SYNOPSIS...10
1
BACKGROUND INFORMATION AND RATIONALE...14
1.1
LPL Deficiency ...14
1.2
Rationale for Study...15
1.3
LPL Protein ...15
1.4
Adeno-associated Viral (AAV) Vectors ...16
1.5
Clinical Studies with AAV Vectors ...16
1.6
Study Medication; AMT-010 ...18
1.7
Pre-clinical Proof of Concept Studies ...18
1.8
Pre-clinical Safety Studies...20
1.8.1
Toxicity Study in Mice...20
1.8.2
Biodistribution Studies in Mice and Cats...20
2
OBJECTIVES...21
2.1
Primary Objective...21
2.2
Secondary Objectives ...21
3
STUDY PLAN ...22
3.1
Study Design ...22
3.2
Rationale for Starting Dose and Dose Escalation ...22
3.3
Dose Escalation Plan and Maximum Tolerated Dose (MTD) ...23
4
STUDY POPULATION...24
4.1
Inclusion Criteria ...24
4.1.1
Eligible Population ...24
4.1.2
General Health...24
4.1.3
Age...24
4.1.4
Sex ...24
4.1.5
Compliance...24
4.1.6
Consent ...24
4.2
Exclusion Criteria...24
4.2.1
Disease...24
4.2.2
Laboratory Parameters ...25
4.2.3
Viral Screening...25
4.2.4
Body Mass Index...25
4.2.5
Substance Abuse...25
4.2.6
Medications ...25
4.2.7
Clinical Trials ...25
4.4
Subject Replacement ...26
4.5
Subject Restrictions and Concomitant Medications ...26
5
CLINICAL SUPPLIES ...26
5.1
Packaging and Labelling ...26
5.2
Directions for Use of Study Supplies...27
5.3
Accountability of Study Supplies...27
6
STUDY SCHEDULE...27
6.1
Patient Selection and Pre-administration Evaluation: Pre-monitoring and
Baseline Visit (day -14)...27
6.2
Study Intervention: Day 1 ...29
6.3
Post-administration Evaluation ...29
6.4
Long Term Follow-up ...30
7
ENDPOINTS AND EVALUATION CRITERIA ...30
7.1
Endpoints...30
7.1.1
Safety ...30
7.1.2
Efficacy...30
7.2
Additional Evaluation Criteria ...30
7.2.1
Expression and Biological Activity of the LPL
S447XTransgene Product.30
7.2.2
Immune Responses...31
7.2.3
Assessment of Shedding...31
8
STUDY PROCEDURES AND ASSESSMENTS ...31
8.1
Weight ...31
8.2
Height ...31
8.3
Chest X-ray...31
8.4
ECG ...31
8.5
Heart Rate...31
8.6
MRI...31
8.7
Blood Pressure...32
8.8
Blood Collection...32
8.9
Spinal analgesia ...32
8.10
Laboratory investigations ...33
8.11
Serum, Saliva, Urine and Semen Sampling ...33
9
POTENTIAL RISKS AND BENEFITS ...33
9.1
Potential Benefits Associated with AMT-010 ...33
9.2
Potential risks associated with AMT-010 ...34
9.2.1
General Toxicity and Inflammation ...34
9.2.2
Vector Dissemination to other Tissues and Germline Transmission...35
9.2.3
Integration...36
9.2.4
Overexpression of LPL ...37
9.2.5
Immune Responses...37
10
ADVERSE EVENTS ...38
10.1
Handling of Adverse Events...38
10.2
Serious Adverse Events...39
10.3
Serious Adverse Event Reporting ...39
10.4
Follow-up of Adverse Events...40
11
STATISTICAL CONSIDERATIONS...40
11.1
Design and Endpoints...40
11.2
Sample Size and Analysis Plan ...41
12
ETHICAL AND REGULATORY ASPECTS...41
12.1
Local Regulations/Declaration of Helsinki ...41
12.2
Informed Consent ...41
12.3
Regulatory Approval ...41
12.4
Clinical Trial Insurance for Patients...41
13
STUDY DOCUMENTATION, CRFS, AND RECORD KEEPING ...41
13.1
Investigator’s Files/Retention of Documents...41
13.2
Source Documents/Data ...43
13.3
Case Report Forms (CRFs) ...43
13.4
Data Handling...43
14
MONITORING OF THE STUDY...43
14.1
Data and Safety Monitoring Board ...44
15
CONDITIONS FOR AMENDING THE PROTOCOL...44
16
CONDITIONS FOR TERMINATING THE STUDY...44
17
CONFIDENTIALITY OF STUDY DOCUMENTS AND SUBJECT
RECORDS...45
18
PUBLICATION OF DATA AND PROTECTION OF TRADE
SECRETS ...45
19
QUALITY CONTROL AND QUALITY ASSURANCE...46
REFERENCES...47
APPENDIX I: STRUCTURE OF AMT-010...52
APPENDIX II: FLOW CHART OF INVESTIGATIONS/STUDY SCHEDULE
...53
APPENDIX III: PRE-MONITORING PROTOCOL LIPOPROTEIN LIPASE
GENE THERAPY ...54
APPENDIX IV: DIET INSTRUCTIONS FOR LPL-DEFICIENT SUBJECTS
PARTICIPATING IN THE STUDY. ...56
APPENDIX V: LPL GENE MUTATIONS OF LPL DEFICIENT PATIENTS57
ABBREVIATIONS
AAV = adeno-associated virus
APTT = activated partial thromboplastin time AMC = academic medical center
BMI = body mass index
Cap = capsid
CFTR = cystic fibrosis transmembrane conductance regulator protein
CMV = cytomegalovirus
CRF = case report form
CPK = creatine phosphokinase
CTL = cytotoxic T-lymphocytes
DLT = dose-limiting toxicity
DSMB = data safety monitoring board ECG = electro cardio gram
FIX = factor IX
gc = genome copies
GLP = good laboratory practice GMO = genetically modified organism HDL = high density lipoprotein HEK = human embryonic kidney IRB = institutional review board ITR = inverted terminal repeat LDL = low density lipoprotein LPL = lipoprotein lipase
MRI = magnetic resonance imaging MTD = maximum tolerated dose
PTT = prothrombin time
Rep = replicase
TG = triglyceride
ULN = upper limit of normal VLDL = very low density lipoprotein
VROM = volkshuisvesting, ruimtelijke ordening en milieubeheer
PROTOCOL SYNOPSIS
Title A Study to Determine the Safety and Efficacy in Lipoprotein Lipase-Deficient Subjects after Intramuscular Administration of AMT-010, an
Adeno-Associated Viral Vector Expressing Human Lipoprotein LipaseS447X
Primary Objective 1) To assess the safety profile of AMT-010.
2) Following administration of AMT-010, to achieve a reduction in individual median fasting plasma triglycerides (TG) to a level equal to or less than 10 mmol/L on top of diet, OR to achieve a reduction in
fasting plasma TG such that the difference in individual median plasma TG observed before and after administration represents a 40% reduction, on top of diet.
Secondary Objectives 1) To determine the biological activity and expression of the transgene product (LPLS447X).
2) To evaluate potential immune responses against the lipoprotein lipase (LPLS447X) transgene product and the adeno-associated viral (AAV) vector.
3) To assess shedding of AMT-010.
Study Design Open label, dose-escalating study of AMT-010. Subjects will be evaluated at days 1 and 2 and weeks 1, 2, 3, 4, 6, 8, 10 and 12. The first two patients will receive a dose of 1 x 1011 genome copies (gc)/kg, according to Schedule A.
There will be at least 2 weeks between dosing of subjects within one dose cohort, and 4 weeks between the dosing of the last subject in a dose cohort and the dosing of the first subject in the subsequent dose cohort, to allow review of the safety and efficacy endpoints, indicated below.
Sample Size At minimum 4 and at maximum 8 subjects.
Summary of Subject Eligibility Criteria
The study population is composed of LPL-deficient subjects on diet, diagnosed by:
1) LPL activity ≤20 % of normal
2) Mutations (homozygous or compound heterozygous) in the gene coding for LPL
3) LPL mass >5% of normal 4) TG levels >10mmol/L.
Dosage See Schedule A Route of
Administration
AMT-010 will be administered at a single dose by intramuscular injection at multiple sites.
Procedures Prior to the study, patients are enrolled in a separate monitoring program. Patients are on diet. The pre-monitoring program is designed to monitor fluctuations in plasma TG levels of each patient, such that effect of add-on administratiadd-on with AMT-010 can be accurately assessed.
Following the baseline visit and administration of AMT-010 at day 1, subjects will remain in the hospital for evaluation until the end of the second day. After administration, they will be monitored by physical examination and assessment of vital signs and body weight, laboratory evaluation of (bio)-chemistry, haematology, and urinalysis at day 2 and weeks 1, 2, 4, 8, and 12. A chest X-ray and ECG will be performed at day 2. Serum, saliva and urine will be screened for the presence of AMT-010 vector DNA at day 2, and this will continue, until three consecutive samples are negative. Semen samples (if possible) will be screened until 3 consecutive samples taken >75 days post-administration are negative. TG will be measured at weeks 1, 2, 3, 4, 6, 8, 10, and 12. Lipid and lipoprotein profile will be measured at weeks 2, 4, 8, and 12. LPL mass and activity will be measured at weeks 4 and 12. Anti-AAV and anti-LPL antibodies at weeks 1, 2, 3, 4, 6, 8, 10, and 12. Cytotoxic T-Lymphocytes (CTL) responses to AAV and LPL are measured at weeks 2, 4, 6, 8, and 12. A physiotherapy program and MRI on injected muscle are performed at week 12. Cytokines are measured at day 2. Adverse events will be recorded at each scheduled assessment.
Dose escalation will proceed according to the following criteria:
• If the two patients in one cohort do not demonstrate dose-limiting toxicity (DLT) and the median fasting TG level of both patients is within the target range (5<TG<10 mmol/L) between week 2 and 4, 2 additional patients will receive the same
dose.
• If the two patients in one cohort do not demonstrate DLT and the median fasting TG level of both patients is lower than the target range (TG<5 mmol/L) between week 2 and 4, 2 additional patients will receive a half log10 lower
dose.
• If the two patients in one cohort do not demonstrate DLT, but the median fasting TG level of only one of these patients is within the target range (5<TG<10 mmol/L) between week 2 and 4, two additional subjects will be accrued at that dose.
• If the two patients in one cohort do not demonstrate DLT and the median TG level of both patients is higher than the target range (TG>10 mmol/L) between week 2 and 4, another 2 patients will be enrolled in the subsequent dose cohort and will receive a half log10 higher dose.
• If one of the two patients in one cohort demonstrates DLT related to vector administration and DLT occurs in one of a possible two additional subjects accrued at that dose, enrollment will be suspended and the available data will be reviewed pending a decision to terminate the study or modify the study design.
Dependent on the number of dose escalations required, a minimum of 4 and a maximum of 8 patients will be treated in this study.
A long-term follow up is scheduled for each patient following administration up to 5 years after this study.
Endpoints 1) Safety will be assessed by recording the possible development of unacceptable DLT defined as any grade III or higher administration-related toxicities and any grade II neurotoxicity occurring at any time during the course of administration and follow-up. This will be assessed by measurement of changes in serum chemistries, haematology, and urinalysis, as well as reported symptoms.
efficacious if the individual median fasting plasma TG is equal to or less than 10 mmol/L after administration, on top of diet, OR if the difference
in individual fasting plasma TG levels observed before and after administration represents a 40% reduction in median fasting plasma TG after administration, on top of diet.
SCHEDULE A
Administration scheme illustrating maximum and minimum number of dose escalations in this study. NB: this schedule is an example, and not all possible permutations have been included. N=2 1e11 gc/kg No DLT TG>10 N=2 3e11 gc/kg No DLT TG>10 N=2 1e12 gc/kg No DLT 5<TG<10 N=2 1e11 gc/kg No DLT 5<TG<10 No DLT 5<TG<10 N=2 1e12 gc/kg No DLT 5<TG<10 N=2 1e11 gc/kg No DLT TG>10 N=2 3e11 gc/kg No DLT TG>10 N=2 1e12 gc/kg No DLT 5<TG<10 N=2 1e11 gc/kg No DLT 5<TG<10 No DLT 5<TG<10 N=2 1e12 gc/kg No DLT 5<TG<10
Dose escalation and assignment to cohorts.
Cohort Study intervention Patients Injection Sites Dose/Site (gc) Dose/kg (gc)
Total dose1) Injection volume
(mL)
Muscles injected
1 AMT-010 2-4 40 1.7 x 1011 1 x 1011 7 x 1012 20 upper leg muscles
2 AMT-010 2-4 60 3.5 x 1011 3 x 1011 2.1 x 1013 30 upper + lower leg muscles 3 AMT-010 2-4 80 8.8x 1011 1 x 1012 7 x 1013 40 upper + lower
leg muscles 1) assuming a 70 kg adult
1
BACKGROUND INFORMATION AND RATIONALE
1.1
LPL Deficiency
Lipoprotein lipase (LPL) is the key enzyme in the metabolism of triglyceride-rich lipoproteins. LPL protects the human body against the excessive rise of triglycerides (TG) after every meal by mediating hydrolysis of TG in chylomicrons and very low density lipoproteins (VLDL). Consequently, these TG-rich lipoproteins are rapidly cleared and the TG levels in the circulation are reduced.
LPL deficiency is an autosomal recessive inherited condition caused by homozygosity or compound heterozygosity for mutations in the LPL gene. The LPL gene is located on chromosome 8p22 and comprises ten exons. To date, more than 70 LPL gene mutations have been described, most of them associated with loss of catalytic function. LPL deficiency results in extremely high concentrations of circulating TG-rich lipoproteins. LPL deficient patients usually present with fasting plasma TG greater than 11 mmol/L (1000 mg/dL) and those levels may exceed 113 mmol/l (10,000mg/dL) (Santamarina-Fojo et al, 1998). Fasting
TG levels of normal individuals range between 1-2.3 mmol/L. The disease usually presents in infancy or childhood with typical complaints of severe abdominal pain, repetitive colicky pains, repeated episodes of pancreatitis and often ´failure-to-thrive´ (Black and Sprecher, 1993; Santamarina-Fojo et al, 1998). On physical examination eruptive xanthomas
(accumulation of fat under the skin), lipaemia retinalis and hepatosplenomegaly may be detected. Approximately 30% of LPL deficient cases are detected in the first year of life. Unfortunately, the condition is not always diagnosed early and may only become evident after several episodes of pancreatitis. Laboratory investigation reveals genuine lactescent plasma (lipemia) due to the increased chylomicron concentrations. The severity of the symptoms is proportional to the degree of chylomicronemia, which in turn is dependent on dietary fat intake.
The most severe complication associated with LPL deficiency is pancreatitis (Fortson et al,
1995). High concentrations of circulating chylomicrons are degraded by small amounts of lipases present in the pancreatic microcirculation, thereby eliciting a vicious cycle of free fatty acid production, inflammation and enhanced leakage of pancreatic enzymes. Pancreatitis in an LPL deficient patient often leads to prolonged admissions (sometimes up to weeks) to an intensive care unit. Patients who survive their first episode may develop chronic pancreatitis, ultimately resulting in endocrine and exocrine pancreatic insufficiency (Brunzell and Deeb, 2000; Fortson et al, 1995). Therefore, every attempt should be made to maintain
the fasting plasma TG level below 11-23 mmol/L (1000-2000 mg/dL) to prevent pancreatitis (Brunzell and Deeb, 2000). Treatment of LPL deficient patients currently consists of severe reduction in dietary fat to less than 20% of caloric intake and the use of medium-chain TG. For these patients it is almost impossible to comply with such a dietary regimen and as a consequence they will remain at increased risk for potentially lethal pancreatitis. As there is currently no drug or any specific therapy available to modulate the course of the illness, these patients are at high risk of morbidity and mortality. Enzyme replacement therapy is not
expected to be effective, due to the short half life of the LPL protein (approximately 15-30 minutes). Therefore we propose gene therapy for LPL deficient subjects.
1.2
Rationale for Study
Several features make LPL deficiency a good model for gene therapy. First, we can target the skeletal muscle, a natural site of LPL production, easily through routine intramuscular injection. Intramuscular injection is expected to reduce systemic distribution of the vector as compared to intravascular administration. Second, pre-clinical data indicated that LPL activity levels of 10% of normal already resulted in (partial) resolution of lipemia in LPL-/- mice, suggesting that the therapeutic range is wide (section 1.7). Third, animal studies have shown that biologically active LPL can be produced by other cell types, including hepatocytes (Excoffon et al., 1997; Liu et al., 2000), which underlines that tissue-specific
regulation of the transgene is not required. Finally, determination of biological response is straightforward and unequivocal since plasma TG are easy to measure and correlate well with clinical severity of disease.
1.3
LPL Protein
LPL is a glycoprotein that is primarily expressed in parenchymal cells, including adipocytes, skeletal muscle cells (the target for our therapy), and cardiac muscle cells. After intracellular dimerisation, the enzyme is transported to the luminal side of the blood vessel. Here it is bound to the endothelium through heparan sulfate proteoglycans where it catalyses the hydrolysis of TG in VLDL and chylomicrons. This provides free fatty acids for the delivery of energy to the muscle and for storage in fats in adipose tissue. LPL’s catalytic function is largely dependent on the presence of its activator apolipoprotein CII. Through a ligand and bridging function LPL is also involved in the hepatic removal of atherogenic lipoproteins from the circulation.
LPL levels in terms of mass and activity are measured in post-heparin plasma. LPL is liberated from the heparansulfate proteoglycans by administration of heparin (50U/kg). After 10 minutes, plasma samples are taken and LPL mass is measured by ELISA, whereas LPL activity is measured by conversion of a radiolabeled triolein into free fatty acids. LPL mass can also be measured in non-heparanised plasma, but levels are low compared with those obtained in post-heparin samples.
LPLS447X is a naturally occurring variant, containing a stop codon at position 447 which is
found in 20% of caucasians, and is associated with lower plasma TG levels, higher HDL cholesterol concentrations and lower rates of cardiovascular disease than comparable controls carrying other variants of the LPL gene (Wittrup et al, 1999). Preliminary data have shown
that subjects with the S447X variant have increased turnover of TG-rich particles, as well as enhanced removal of pro-atherogenic apoB100-containing particles, including LDL-cholesterol (Meimar et al., in press). Based on these findings, the LPLS447X gene is assumed
1.4
Adeno-associated Viral (AAV) Vectors
AAV is a pathogenic virus that is ubiquitous in the environment. The virus is a non-enveloped, replication-defective parvovirus and consists of a single stranded genome encapsidated in a protein coat. The AAV genome consists of three elements: the replicase (rep) gene, the capsid (cap) gene and the inverted terminal repeats (ITRs). The rep gene
directs production of the proteins that enable the virus to replicate its genome. The cap gene directs production of the protein coat. AAV vectors are derived from the parent virus by removing all of the viral elements except for the ITRs and inserting the gene of interest and specific regulatory elements. AAV vectors efficiently transduce post-mitotic cells such as muscle in vivo. Because all of the viral genes have been removed, there is no immune response directed against the transduced cell due to viral gene expression. This accounts, at least in part, for the long-term expression (several years) observed after single intramuscular administration (Manno et al., 2003). Up to now the AAV vector most extensively used in
animal and clinical studies is derived from AAV serotype 2 (AAV2). However, other
serotypes have been identified, which transduce certain cell types more efficiently. For instance, AAV1 has been shown to transduce muscle cells of mice and dogs more efficiently than AAV2 (Rabinowitz et al, 2002; Arruda et al., 2004; Ross et al., 2003 and 2004). These
studies show that AAV1 is an excellent vector for gene therapy strategies directed at transduction of muscle tissue.
1.5
Clinical Studies with AAV Vectors
To date, two different AAV vectors have been tested in humans: AAV-cystic fibrosis transmembrane conductance regulator protein (CFTR) in patients with cystic fibrosis, and AAV-factor IX in patients with hemophilia B. The design of three additional studies using AAV-sarcoglycan, AAV-alpha 1-antitrypsin (AAT), and AAV-aspartoacyclase have been reported (Stedman et al., 2000; Flotte et al., 2004; Janson et al., 2002) but data from these
studies are not yet available
AAV-FIX was administered intramuscularly to 8 patients, at three dose levels, i.e. 2 x 1011, 6 x 1011, 1.8 x 1012gc/kg (Kay et al., 2000; Manno et al., 2003).The number of injection sites
varied between 10 (lowest dose) and 90 (highest dose). There was no evidence of local or systemic toxicity up to 40 months after injection. Laboratory studies revealed no abnormalities in serum chemistries, save for in one subject a 5-fold elevation in creatine phosphokinase (CPK) levels, which returned to baseline 1 week after injection. Complete blood counts also demonstrated no abnormalities except for one patient with a history of thrombocytopenia. This patient had a transient reduction of platelet count. Four out of 8 subjects developed transient minor abnormalities at the site of muscle biopsy. Five out of 8 subjects developed small hematomas or pain at one or more vector injection sites, which resolved uneventfully. Routine histology on muscle biopsies showed no evidence of inflammation or muscle injury. Serum, saliva, and urine were positive for vector DNA, early after administration, but were negative after day 7, except for the serum of one patient, which was positive up to 12 weeks, and the urine of another patient which was positive up to 2 weeks after vector administration. Most importantly, there was no evidence of vector DNA in
semen. Strong evidence for gene transfer and expression at the injection site was obtained in all 8 subjects, irrespective of the level of anti-AAV2 antibodies before injection. No inhibitory antibodies were formed against FIX, probably because all patients had missense mutations. At the vector doses administered in this trial, efficacy was quite limited, with 2 of 8 subjects demonstrating a small elevation of FIX levels, reducing the use of FIX concentrate by at least half for periods of more than 1 year. These patients had received the lowest vector dose (2 x 1011 gc/kg). Levels of circulating FIX and efficacy did not improve at higher doses. Since pre-clinical data indicated that gene transfer to the liver, the natural site of FIX production, was more efficient than to the muscle, a new clinical study of intra hepatic artery delivery was initiated. To date, seven subjects have been treated with doses up to 2 x 1012 gc/kg. Concerns were raised due to the transient detection of vector sequence in the seminal fluid of the first subjects. However, vector detection in the semen was transient for all subjects, and no dose dependence was observed. Furthermore, the motile sperm fraction of one of these individuals was negative and therefore the study was continued (Department of Health and Human services, National Institutes of Health Recombinant DNA Advisory committee, Minutes of meeting. National Institutes of Health, Bethesda, MD, December 6, 2001 and March 7-8 2002; www4.od.nih.gov/oba). Of two subjects receiving the highest dose (2 x 1012 gc/kg), one displayed transient therapeutic levels of FIX. Both subjects had transient increases in serum transaminase levels, most likely as a result of cell mediated immune responses against AAV2 capsid proteins (High et al., 2004). No inhibitory antibodies against
FIX were detected (High et al., 2004). Immune responses are currently being investigated in
more detail.
Three phase I studies and two phase II studies have been performed with AAV-CFTR (Flotte
et al., 2004; Flotte et al., 2003; Aitken et al., 2001, Wagner et al., 1998, 1999, 2000). In total,
90 subjects have received this vector at doses between 6 x 104 and1 x 1013 genome copies, without evidence of significant toxicity. AAV-CFTR has been instilled into the nose, maxillary sinus, and single lung lobe, as well as delivered as an aerosol by oral inhalation to the entire lung. In one study, multiple doses were administered. Administration of AAV-CFTR did not result in significant changes in the complete blood count, white blood count differential, liver function tests, renal function tests, or other clinical chemistries. Adverse events and serious adverse events were noted prior to and/or after vector delivery, but most of them appeared to be related to the endogenous CF lung disease or a result of bronchoscopic procedures and could not be attributed to vector administration. Vector shedding was limited. Stool and urine were negative, whereas sputum was transiently positive for vector DNA between day 1 and 7 post vector administration. Neutralizing antibodies against AAV were detected after endobronchial, intranasal and aerozolised administration of AAV-CFTR but not after (repeated) administration to the maxillary sinus. A clear dose-response relationship was observed in vector gene transfer, but gene transfer was too low to confirm transgene expression or to demonstrate efficacy. Although there is a trend to indicate that the vector is biologically active for the correction of chloride secretion, further studies potentially incorporating transient anti-inflammatory and anti-protease therapies are planned to improve airway surface delivery of AAV-CFTR.
1.6
Study Medication; AMT-010
AMT-010 [scientific name; AAV1-LPLS447X] is an investigational new drug, expressing the human LPLS447X protein from an AAV vector. A diagram of the vector is included in Appendix I. The LPLS447X transgene is expressed from the cytomegalovirus (CMV) immediate early promoter and a bovine growth hormone polyadenylation sequence is inserted downstream of this gene. In addition a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been incorporated to enhance transgene expression (Loeb et al., 1999). The expression cassette is flanked by two ITRs derived from AAV2. Small
intervening non-functional DNA sequences are derived in the process of assembling the genetic elements through recombinant DNA techniques. This vector is pseudotyped with AAV1 capsids, because it has been shown that AAV vectors pseudotyped with capsids of serotype 1 transduce skeletal muscle cells of different animal species more efficiently (Rabinowitz et al., 2002; Ross et al., 2003 and submitted), than AAV vectors coated with
AAV2 capsids.
AMT-010 is produced by transient expression of two co-transfected plasmids in human embryonic kidney (HEK293) cells. One plasmid carrying the vector genome consisting of the LPLS447X expression cassette flanked by the AAV2 ITRs (Appendix I) and a second helper plasmid carrying the required AAV2 rep genes (encoding replicase proteins Rep78, Rep68, Rep52, and Rep40), AAV1 cap gene (encoding VP1, VP2, and VP3) and adenovirus helper genes (E2A, E4, and VA) are introduced (transfected) by calcium phosphate precipitation into HEK293 cells (Grimm et al., 2003). Following an incubation period, AMT-010 is
liberated from the cells by lysis. AMT-010 is purified by a process including anion and hydrophobic interaction chromatography and filtrations steps. Finally, AMT-010 is ultrafiltered/diafiltered against PBS/5% sucrose.
1.7
Pre-clinical Proof of Concept Studies
LPL deficient (LPL-/-) mice are an established model to study LPL deficiency, and display plasma hypertriglyceridemia and low HDL-C similar to human LPL deficiency (>200-fold increase in fasting plasma TG, up to 99 mmol/L; HDL-C <10% of normal, 0.2 mmol/L; LPL activity <5% of normal; Strauss et al., 2001; Ross et al., 2002 and 2004).
LPL-/- mice were used to test AMT-010, demonstrating dose- dependent AAV1-mediated expression of LPLS447X, and biological effect for more than a year following a single intramuscular administration at multiple sites (Ross et al., 2003 and 2004). At a dose of
8x1012 gc/kg, visible lipemia (i.e. milky plasma) was resolved completely within 1 week, corroborated by a 97-98% reduction of fasting TG to near-normal levels (1.8-2.0 ± 0.2 mmol/L). In addition, total cholesterol (Tchol) levels were normalised, and HDL-C levels were improved by 2.4 to 7.5 fold to 35-50% of normal (to 0.7 ± 0.2 and 1.1 ± 0.02 mmol/L in a long term and short term study, respectively). Administration of a 10-fold lower dose (8x1011 gc/kg) resulted in 68% reduction of TG levels to 26.7 ± 2.3 mmol/L. Full correction of dyslipidemia in LPL-/- mice (using 8x1012 gc/kg AMT-010) was achieved with LPL
activity levels reaching ~30% of normal. As mentioned, partial correction (using 8x1011 gc/kg AMT-010) was achieved with LPL activity levels <10% of normal. Following intravenous administration of a fat challenge, LPL-/- mice treated previously with AMT-010 showed a near-normal TG clearance, in contrast with untreated LPL-/- mice that displayed a slow clearance of this TG load. These results indicate enhanced post-prandial TG clearance following administration with AMT-010.
Further testing was performed in LPL-/- cats, which also display hypertriglyceridemia (fasting TG up to 200 mmol/l) and lipemic plasma, but near-normal levels of HDL-C. In contrast to LPL deficient mice and humans, however, these animals do not have any LPL mass (i.e. a null-mutation) and therefore their immune system is essentially naïve towards LPL (Ginzinger et al., 1996). Using doses between 5x1011and 1.7x1012 gc/kg of AMT-010,
fasting TG were normalised within 1 week (> 99% reduction from up to 200 mmol/L down to 0.14 ± 0.5 mmol/L). Efficacy was abrogated by an immune response, as demonstrated by the induction of inhibitory anti-LPL antibodies. Oral co-administration of cyclophosphamide (between 90-200 mg/m2) attenuated this response. It is important to emphasize that LPL deficient patients display a limited number of similar mutations, and a majority of these show very low LPL activity but detectable levels of LPL mass (Brunzell and Deeb, 2000; Merkel et al., 2002). This will minimise the likelihood that immune responses against LPLS447X arise in
these patients. Still, the conformation of the LPLS447X might be different from that of the inactive protein of the patients and therefore immune responses cannot be totally excluded. Further reduction of dose to 1x1011 gc/kg resulted in approximately 90.8 ± 6.2% reduction of fasting plasma TG (down to 1.3 ± 0.7 mmol/L) in LPL-/- cats co-treated with cyclophosphamide. In these cats reduction of TG appeared to last longer than the reduction observed at doses between 5x1011and 1.7x1012 gc/kg. In two cats that had received this low dose and no heparin treatment, the anti-LPL response was weaker. Possibly, reduction of dose and absence of heparin together may reduce the immune response against LPL. Since full correction of hypertriglyceridemia was not achieved at 1x1011 gc/kg (a plasma TG level of 1.3 mmol/L remains 11-fold above normal feline levels), a limiting dose level may have been reached.
By varying the number of intramuscular injection sites at a given dose, between 6x108 and 1x1012 gc/site were administered to LPL-/- cats, showing a linear relationship between dose/injection site and expression of LPLS447X protein/injection site. This demonstrated that the capacity of muscle tissue to express the LPLS447X protein is not a limiting factor at doses up to 1x1012 gc/site. Efficacy following the administration of 2 injections of AMT-010 in two separate muscles (2 injections of 1ml each in the vastus lateralis and gastrocnemius of a single hind limb of LPL-/- cats) indicated that transduction of a very limited muscle mass was sufficient to fully correct systemic hypertriglyceridemia. Similar results were obtained in LPL-/- mice, when reducing the number of injection sites (and muscles) from 40 to 4.
As mentioned, administration of AMT-010 in LPL-/- mice (at a dose of 8x1011 gc/kg) indicated that ~10% of normal LPL activity (~40 mU/ml) and a corresponding level of ~200 ng/ml LPLS447X protein already has substantial effects on plasma TG (68% reduction). A
lower level of transgene expression (~100 ng/ml protein, <5% of normal activity or 20 mU/ml), as found in untreated LPL-/- mice, was unable to correct hypertriglyceridemia. A dose of 1x1011 gc/kg in LPL-/- cats resulted in plasma levels of LPLS447X protein and activity amounting to 189.5 ± 43.4 ng/ml and 104 ± 125 mU/ml, respectively, resulting in fasting plasma TG levels of 1.3 ± 0.7 mmol/L (a ~90% reduction in plasma TG). The results thus indicate a higher transduction efficiency and/or transgene expression in the larger animal model. If similar transduction efficiencies can be obtained in humans, injection of 1x1011 gc/kg (total dose of 7x1012 gc based on 70 kg total body weight) in 40 sites would result in LPL activity levels sufficient to reduce fasting TG below the target level of 10 mmol/L.
1.8
Pre-clinical Safety Studies
1.8.1 Toxicity Study in Mice
The toxicity profile of AMT-010 was investigated in mice in compliance with Good Laboratory Practices (GLP) regulations. AMT-010 was administered intramuscularly according to the following dosing scheme: buffer (control), 1x1011 gc/kg (low dose), 1x1012 gc/kg (mid dose), and 1x1013 gc/kg (high dose). Of all groups, male (n=6) and female (n=6) mice were analysed at days 8, 29 or 91. Clinical observations showed a slight reduction in body weight gain in the high dose female group. This was not associated with a reduction in food consumption. No consistent effect of administration could be observed in the haematological and clinical chemistry profile for each dosing group, except for a transient increase in serum amyloid A levels. This acute inflammatory response dissipated within 24 hours. There were no macroscopic findings. Microscopic findings included a transient minimal lymphoid hyperplasia in the spleen in the highest dose group at days 8 and 29, which had disappeared by day 91. Minimal (grade 1) myositis was observed in both control and high dose animals at day 8, consistent with needle track lesions expected after intramuscular injection. Histology of the injection area of all groups was normal at day 29. Analysis of the animals at day 91 indicated a slight (grade 2) myositis at the injection area of all high dose animals, minimal myositis in some animals at the injection area with the medium dose of AMT-010, whereas histology of the injection area was normal following administration of the low dose and buffer control. In summary, both short- and long-term inflammatory events were observed in rodents at a dose that exceeds the starting dose in humans 10 to100-fold. These observations have prompted us to focus on vector–related muscle toxicity as part of the safety assessment within this clinical trial (see also section 9.2).
1.8.2 Biodistribution Studies in Mice and Cats
The biodistribution of AMT-010 was investigated in mice in compliance with GLP regulations. AMT-010 was administered intramuscularly according to the following dosing scheme: buffer (control), 1x1011 gc/kg (low dose), 1x1013 gc/kg (high dose). Tissues were harvested at day 8, 29 and 91 from males (n=5) and females (n=5) per group. The presence of AMT-010 vector DNA was detected using a quantitative PCR method with a detection limit of 10 copies vector DNA per µg genomic DNA. Vector leakage from the injected muscle was observed shortly after administration. Vector was transiently detected in the blood circulation. At day 8 after administration, high levels of vector DNA sequence were detected
in injected muscle, and filtering organs/tissues (particularly liver, spleen, and marrow). High levels of vector DNA were also found in the draining lymph nodes close to the injection sites (i.e. inguinal lymph nodes). At later time points (29 and 91 days after administration),
residual vector DNA levels in filtering organs declined rapidly (indicating degradation), remaining high in injected muscle and inguinal lymph nodes.
AMT-010 vector DNA could be detected just above threshold (0-10 copies/µg) in the male gonads at day 90 in the low dose group in 3 out of 5 animals. No vector DNA was detectable in the female gonads in the low dose group at day 91. In the high dose group at day 91, all male and female gonads were positive for AMT-010 vector DNA. Vector sequence levels in testis and epididymis, in terms of copies per μg of total genomic DNA, were at least 1,000-fold lower compared to the injected muscles. These results indicate that dissemination of AMT-010 to gonadal tissue is limited. To obtain additional information of vector dissemination to reproductive organs, epididymis, testis and motile sperm of LPL deficient cats used in pre-clinical proof of concept studies (section 1.7) were tested for the presence of AMT-010 vector DNA. Other organs that were tested included liver, injected muscle (triceps and gastrocnemius) and uninjected muscle (deltoid). The highest levels of AMT-010 vector DNA were found in the injected muscles and liver at all time points and dosages. Levels of AMT-010 DNA in testis and epididymis were very low to undetectable (<10-170 copies/µg). Importantly, no significant levels of AMT-010 sequences were detected in the motile sperm fraction. Sperm samples of half the injected animals were negative, and in the other animals less than 10 copies per reaction were detected. In summary, the results indicate that despite detection of vector DNA in gonadal tissue, very low levels of vector DNA are found in the actual germ cells. Nevertheless, as it is of importance to determine the risk for germline transmission of the vector in human subjects, the biodistribution and shedding of AMT-010 will be closely monitored in this clinical study.
2
OBJECTIVES
2.1
Primary Objective
1) To assess the safety profile of AMT-010.
2) Following administration of AMT-010, to achieve a reduction in individual median fasting plasma TG to a level equal to or less than 10 mmol/L on top of diet, OR to
achieve a reduction in fasting plasma TG such that the difference in individual median plasma TG observed before and after administration represents a 40% reduction, on top of diet.
2.2
Secondary Objectives
1) To determine the biological activity and expression of the lipoprotein lipase (LPLS447X) transgene product.
2) To evaluate potential immune responses against the lipoprotein lipase (LPLS447X) transgene product and the adeno-associated viral (AAV) vector.
3) To assess shedding of AMT-010.
3
STUDY PLAN
3.1
Study Design
This study is an open-label, dose-escalating study evaluating the safety and efficacy of a single intramuscular administration of AMT-010 (at multiple sites). The study will be performed in the Academic Medical Center (AMC) Amsterdam, the Netherlands, under the supervision of their local clinical biosafety and medical ethical committees. The study participants will be treated under the responsibility of a Principal Investigator specialised in the treatment of lipid disorders. Dependent on the number of escalations required, a minimum of 4 and a maximum of 8 patients will be treated (Schedule A in protocol synopsis). Participants will be screened 2 weeks prior to administration of AMT-010 and will be evaluated for 12 weeks post administration in this study. The procedures to be performed and timelines are summarised in the Flow Chart/Study Schedule (Appendix II).
3.2
Rationale for Starting Dose and Dose Escalation
Based on pre-clinical studies in LPL deficient mice and cats described in detail in section 1.7, it is anticipated that the effective dose in humans will be ≥ 1 x 1011 gc/kg. The aim is to minimise the number of patients receiving low, non-therapeutic doses, as there is currently no drug or other therapy available for these subjects and it is still unknown whether re-administration of AAV is efficacious. Therefore, it is proposed to start at a dose of 1 x 1011 gc/kg in a cohort of two subjects (schedule A in protocol synopsis). Toxicity is not expected at this dose, given the results from the toxicity study performed with a similar, 10- and 100-fold higher dose (see section 1.8). However, the slight myositis (grade 2, see section 9.2) observed at this 100-fold higher dose in mice has prompted special focus on AMT-010 vector-related muscle toxicity within this clinical study and during follow up. The dose and number of injection sites have been chosen as such that the local vector dose remains low thereby decreasing the chance of a local (toxic) response to administration.
The first dose (1x1011 gc/kg) cohort will receive 40 injections distributed over the upper leg muscles of both legs. Dependent on the primary safety and efficacy outcome, two additional patients will be enrolled in the same dose cohort or in subsequent dose cohorts, as described in detail in section 3.3. In subsequent dose cohorts, injections will be in additional muscle groups of the lower leg (mid-dose: 3x1011 gc/kg and high dose: 1x1012 gc/kg). The number of sites is increased at each dose cohort to keep the dose per site to ≤ 1 x 1012 gc, as pre-clinical studies in LPL-/- cats indicated that up to this dose/site, production of LPLS447X protein was still linear. Patients will be evaluated for 12 weeks in this clinical study, and are subsequently followed up for 5 years.
3.3
Dose Escalation Plan and Maximum Tolerated Dose (MTD)
Endpoints for dose-escalation are dose-limiting toxicity (DLT) and efficacy, as defined in section 7.1
There will be at least 2 weeks between dosing of subjects within one dose cohort and 4 weeks between the dosing of the last subject in a dose cohort and the dosing of the first subject in the subsequent dose cohort, to allow review of the safety and efficacy endpoints. Dose escalation will proceed according to the following criteria:
• If the two patients in one cohort do not demonstrate DLT and the median fasting plasma TG level of both patients is within the target range (5<TG<10 mmol/L) between week 2 and 4 post-administration, 2 additional patients will receive the same dose.
• If the two patients in one cohort do not demonstrate DLT and the median TG level of both patients is lower than the target range (TG<5 mmol/L) between week 2 and 4 post-administration, 2 additional patients will receive a half log10 lower dose.
• If the two patients in one cohort do not demonstrate DLT, but the median fasting TG level, of only one of these patients is within the target range (5<TG<10 mmol/L) between week 2 and 4 post-administration, two additional subjects will be accrued at that dose.
• If the two patients in one cohort do not demonstrate DLT and the median TG level of both patients is higher than the target range (TG>10 mmol/L) between week 2 and 4 post-administration, 2 additional patients will be enrolled in the subsequent dose cohort and will receive a half log10 higher dose.
• If the first of the two patients in one cohort demonstrate DLT related to vector administration, and DLT occurs in one of a possible two additional subjects accrued at that dose, enrollment will be suspended and the available data will be reviewed pending a decision to terminate the study or modify the study design.
• If 2 patients within a dose cohort experience unacceptable DLT related to vector administration, the MTD has been exceeded. The MTD is defined as the next lower dose level.
4
STUDY POPULATION
4.1
Inclusion Criteria
4.1.1 Eligible Population
Study participants must be diagnosed with lipoprotein lipase deficiency, meeting the following criteria: (I) Their lipoprotein lipase activity levels in post-heparin plasma should be ≤20 % of normal; (II) Confirmed homozygocity or compound heterozygocity for mutations in the LPL gene; (III) Post-heparin plasma LPL mass should be >5% of normal; (IV) Median fasting plasma TG concentrations >10mmol/L, as determined during the pre-monitoring period.
4.1.2 General Health
The participant must be in good general physical health with, in the opinion of the investigator, no other clinically significant and relevant abnormalities of medical history, and no abnormalities at the physical examination and routine laboratory evaluation performed prior to the trial.
4.1.3 Age
Age ≥18 years old.
4.1.4 Sex
Male or female. Females must be of non-child bearing potential, or with a negative pregnancy test and not breast feeding. Female subjects must use appropriate contraception (if relevant) and their spouse must use barrier contraception for the duration of the study (12 weeks). Males must practice barrier birth control and their spouse should use appropriate contraception until three consecutive semen samples, taken at least 75 days after administration, are negative for AMT-010 vector DNA.
4.1.5 Compliance
The participant is willing to fully comply with all study procedures and requirements of the trial, such as restrictions to a diet (see also Appendix IV).
4.1.6 Consent
The participant has the mental ability to give voluntary written informed consent to participate in the study.
4.2
Exclusion Criteria
4.2.1 Disease
a) Apolipoprotein CII deficiency.
c) Any current or relevant previous history of serious, severe or unstable physical or psychiatric illness, any medical disorder that may make the participant unlikely to fully complete the study, or any condition that presents undue risk from the study medication or procedures (e.g. malignant neoplasia).
d) Active infectious disease of any nature, including clinically active viral infections.
4.2.2 Laboratory Parameters
The following blood screenings tests will result in exclusion from participation:
a) Platelet count < 100 x 109 /L.
b) Hemoglobin < 7.0 mmol/l.
c) Liver function disturbances (bilirubin >2.50 x normal, transaminases >3 x ULN).
d) CPK > 3 x ULN.
e) Creatinine > 3 x ULN.
f) Abnormal bleeding time, PTT and APTT outside normal range.
4.2.3 Viral Screening
Seropositive for HIV, Hepatitis C, Hepatitis B.
4.2.4 Body Mass Index
Severe obesity defined as Body Mass Index (BMI) > 30 kg/m2.
4.2.5 Substance Abuse
The participant has a recent history of alcohol abuse or other substances such as barbiturates, cannaboids and amphetamines. The participant is positive in a urine screen for drugs of abuse.
4.2.6 Medications
a) Use of medication known to have immunosuppressive effects. b) Use of anti-platelet agents or other anti-coagulants.
4.2.7 Clinical Trials
The participant has participated in another clinical trial or received an investigational drug within 30 days of screening.
4.3
Subject Withdrawal Criteria
Subjects have the right to withdraw from the study at any time for any reason. Should a subject decide to withdraw, all efforts will be made to complete and report the observations
as thoroughly as possible. The investigator also has the right to withdraw subjects from the study in the event of intercurrent illness, intolerable adverse events, investigator’s decision that withdrawal from further participation would be in the subject’s interest, unacceptable protocol violations, or other reasons. A complete final evaluation at the time of the subject’s withdrawal should be made with an explanation of why the subject is withdrawing/withdrawn from the study.
4.4
Subject Replacement
Any subject who withdraws prior to administration of study agent will be replaced.
4.5
Subject Restrictions and Concomitant Medications
Participants are to be maintained throughout the study on the same type and dose of any pharmacological agent that they were taking at study entry. In case of initiation or cessation of medication, or change in dose of medication during the study period, the participant should notify the study investigator directly. All details will be recorded on the CRF.
During both the pre-monitoring (Appendix III) and the entire study period, all participants are placed on a low-fat diet (Appendix IV), in accordance with existing guidelines. During the pre-monitoring and study period, patients will document their dietary habits in a diary of food intake. These diaries will be evaluated, together with the individual patient, by a study dietician.
5
CLINICAL SUPPLIES
5.1
Packaging and Labelling
Amsterdam Molecular Therapeutics will manufacture the study material, in compliance with EU GMP guidelines at their facility in Amsterdam, The Netherlands. AMT-010 is supplied as frozen sterile formulation in vials with a label describing the name of sponsor, product code, lot number, concentration, volume and expiry date. The diluent, a phosphate buffered solution containing 5% sucrose, is supplied as frozen sterile formulation in vials with a label describing the name of the sponsor, product name, lot number, volume, and expiry date. Technical Release of the GMP manufactured AMT-010 and diluent is the responsibility of AMT’s Qualified Person. The technical release is based on the compliance of the production areas, raw materials and documentation used for the manufacture of AMT-010 and diluent to the pre-set requirements (as stated in EU directive 91/356/EEC and the Master Formulation Sheets).The proper amount of AMT-010 is dispensed and supplied on demand for each patient. It is dispensed in syringes, and transported in a double sealed leak proof and unbreakable container to the hospital under the responsibility of the hospital pharmacist. Release of AMT-010 and diluent to be used for this specific clinical trial in the AMC is the responsibility of the Qualified Person of the Pharmacy of the AMC. The syringes contain a label indicating product code, trial subject identification number and trial reference code. The outer container contains a label indicating name of the sponsor, product code, volume,
concentration, number of syringes, trial subject identification number, name of the investigator, trial reference code, “for clinical trial use only”, route of administration, expiry date, storage conditions, “contains GMO” and a biohazard label. The pharmacist will supply the study material to the investigator prior to administration
5.2
Directions for Use of Study Supplies
AMT-010 is supplied on demand for each individual patient. AMT-010 and diluent are thawed approximately 3 hours prior to use. Syringes for AMT-010 administration will be filled in a biosafety hood according to the containment procedures as agreed with the hospital Infection Control Unit and the Ministry of Volkshuisvesting, Ruimtelijke Ordening en Milieubeheer (VROM). Syringes containing the appropriate dose of AMT-010 for an individual patient are transported to the hospital in a double sealed container. The product is administered within 8 hours of thawing to assure maximum potency. Prepared study material will be kept at 2-8 °C prior to administration, but will be injected at room temperature. Any product remaining in the vial or syringes will be discarded as biohazardous waste. Detailed instructions will be described in the study operating manual.
5.3
Accountability of Study Supplies
All material supplied is for use only in this clinical study and should not be used for any other purpose.
The investigator or designee will maintain a full record of drug accountability. A Drug Dispensing Log must be kept current and will contain the following information:
• the date and quantity of drug received by the pharmacist
• the identification of the subject to whom the drug was dispensed;
• the date(s) and quantity of the drug dispensed to the subject;
• the date and quantity of drug sent for final destruction
The inventory must be available for inspection by the study monitor during the study. If the study is terminated, suspended, discontinued or completed, the monitor will verify drug supplies. Drug supplies will then be returned by the investigator or designee to Amsterdam Molecular Therapeutics. Empty vials shall be incinerated.
6
STUDY SCHEDULE
6.1
Patient Selection and Pre-administration Evaluation: Pre-monitoring
and Baseline Visit (day -14)
Study participants are to be recruited from a database of LPL deficient subjects present at the Department of Vascular Medicine at the AMC. The mutations of potential participants,
whose LPL gene has been sequenced are listed in Appendix V. LPL deficient subjects will have completed a pre-monitoring program, a protocol separate from the current clinical protocol (Appendix III) designed to establish a median fasting TG level for each patient. Eligible patients will be selected at random from this group, and invited to attend the clinical site on day -14. The participant will be informed on the purpose and procedures, including any potential hazards of the study by the Principal Investigator or designee. If the patient agrees to participate, written informed consent will be obtained. A participant is considered to be enrolled into the study after the informed consent has been signed and witnessed. A CRF will be completed for each enrolled participant.
Suitability of the participant will be assessed based on the study inclusion/exclusion criteria. Potential participants will undergo clinical and laboratory evaluation as indicated in the flow chart of investigations/study schedule (Appendix II). Patient’s medical history is recorded, and participants undergo physical examination. Participants will be assessed by an anesthesiologist. Participant’s weight and height are measured to calculate BMI. Vital signs will be recorded, and a chest X-ray and ECG will be performed. Urine is collected for urinalysis, testing for drugs of abuse, alcohol, and pregnancy, if appropriate. Blood is collected for haematology, hemostasis, biochemistry and screen for HIV and Hepatitis B/C. Concomitant medication use will be documented. Participants will also undergo lipoprotein lipase deficiency assessment: LPL mass and activity levels will be determined in post-heparin plasma and the LPL gene mutations will be confirmed. In view of the natural variability of the TG values, administration TG levels are measured several times during the pre-monitoring period, at baseline (day –14), and on day 1 (prior to administration). For these measurements, participants should be fasted for 10 hours. Lipid and lipoprotein profile, and pre-existing immunity to AAV1 and LPL will also be assessed. Serum, saliva, urine and semen (if possible) will be collected to screen for AMT-010 vector DNA by PCR. Male patients who are considering reproduction will be counselled to consider banking sperm prior to administration of AMT-010. Patients are strongly advised to practice appropriate barrier birth control, as described in section 4.1.4. Dietary instructions are given, both during the pre-monitoring and clinical trial period, and potential participants are requested to document their food intake in a diary, under close supervision of a dedicated dietician.
With special focus on the administration area, a physiotherapy program will be set up to monitor relevant muscle function prior to- and 12 weeks after administration. In addition, MRI will be used to monitor the general state of muscle, as well as to quantify the total triglyceride content of the injected muscles compared to non-injected muscles. In the event signs of local muscle toxicity as a consequence of AMT-010 administration are observed, or in the event that efficacy or transgene expression is not observed in any of the dose groups, muscle biopsies may be taken for further analysis.
On confirmation that the study participant remains eligible for inclusion, he or she will be asked to return within 14 days for study administration.
6.2
Study Intervention: Day 1
The evening before the study intervention, patients will enter the hospital. The next morning, i.e. study day 1, the inclusion and exclusion criteria will be checked and confirmed. Participants will undergo physical exam, vital signs are assessed, and urine and blood samples are taken for the laboratory investigations indicated in Appendix II. Spinal analgesia will be given to all study participants prior to the study intervention. AMT-010 will be injected into the muscle groups of both upper legs in the first dose cohort, in subsequent dose
cohorts injections will be in additional muscle groups, as indicated in Schedule A. AMT-010 will be administered in a special isolation unit. Containment procedures will be as agreed with the hospital Infection Control Unit and the Ministry of Volkshuisvesting, Ruimtelijke Ordening en Milieubeheer (VROM) and summarised in the “AMC Richtlijn Gentherapie, January 2005”. Injections will be divided equally between both extremities. Total dose will be calculated using a weight obtained at baseline screen. Documentation of drug allocation will be recorded in a drug dispensing log. Volume per injection site will not exceed 0.5 mL. The muscle will be visualized prior to injection using ultrasound to identify major vessels and will be injected using a 27 gauge needle. Injections will be spread evenly over the various selected muscle groups, and within each muscle. Skin above two injection sites will be marked by a tattoo to facilitate the localisation of injection sites at a later time point.
6.3
Post-administration Evaluation
Following study drug administration, isolation will be discontinued. Throughout the day, subjects will be evaluated for vital signs on a 2-hourly basis. It is expected that most patients will be discharged at the end of the second day. Safety and efficacy will be evaluated during 12 weeks post administration according to the flow chart of investigations/study schedule (Appendix II). Participants will have physical examination and assessment of vital signs and body weight at day 2 and weeks 1, 2, 4, 8, and 12. A chest X-ray and ECG will be performed at day 2. Laboratory evaluation of (bio)-chemistry, haematology, and urinalysis is scheduled at day 2 and weeks 1, 2, 4, 8, and 12. In addition serum, saliva, and urine will be screened at day 2 and semen (if possible) will be screened at week 1 for the presence of AMT-010 vector DNA. Screening of these samples for vector DNA will continue, until three consecutive samples are negative. Fasting plasma TG will be measured at weeks 1, 2, 3, 4, 6, 8, 10 and 12. Lipid and lipoprotein profile will be measured at weeks 2, 4, 8, and 12. LPL mass and activity will be measured in post-heparin plasma at weeks 4 and 12. Humoral responses against AAV1 capsid and the LPLS447X transgene product will be monitored by measuring anti-AAV, anti-LPL antibodies at weeks 1, 2, 3, 4, 6, 8, 10, and 12. Cell-mediated immunity will be monitored by measuring CTL responses at weeks 2, 4, 6, 8, and 12. More acute inflammatory events will be monitored by measuring cytokine levels in plasma at day 2. The food intake of the patients will be evaluated at weeks 6 and 12. Adverse events will be collected and recorded at each scheduled assessment.
As indicated in 6.1, injected muscles will be closely monitored after intramuscular administration of AMT-010 for signs of toxicity during physical examination and by measuring CPK levels in plasma. Physiotherapy screen and MRI is performed at week 12. In
the event that signs of local toxicity are obtained, biopsies (open biopsies) from the injected muscle (sites identified by tattoo) may be taken for further investigations. Biopsies are also considered in the event of a lack of efficacy and transgene expression.
6.4
Long Term Follow-up
After the study, patients will be followed up long term with particular emphasis on the safety and efficacy aspects of LPL gene therapy using AMT-010. Patients will be evaluated at the clinical site every three months up to one year, every 6 months up to two years, and yearly up to 5 years after administration of AMT-010. The evaluation will include physical exam and medical history, vital signs, body weight, serum (bio) chemistry, immune responses, TG and lipids and lipoprotein profile. Semen samples will be taken until three samples are negative for AMT-010 DNA (provided the male subjects are able to generate this sample).
7
ENDPOINTS AND EVALUATION CRITERIA
7.1
Endpoints
7.1.1 Safety
The primary endpoint on which the criteria to stop the study are based, is the development of unacceptable toxicity graded according to the modified common toxicity criteria outlined in Appendix VI. Dose-limiting toxicity (DLT) is defined as the occurrence of any grade III or higher administration-related toxicities or any grade II neurotoxicity during the course of administration. Adverse events from spontaneous reports and information elicited by direct questions will be recorded in the Case Report Form (CRF).
7.1.2 Efficacy
Efficacy of AMT-010 will be assessed by measuring TG levels in plasma from fasted subjects. The administration will be considered efficacious if the individual median fasting plasma TG after administration of AMT-010 is equal to or less than 10 mmol/L on top of diet, OR if the difference in individual fasting plasma TG levels observed before and after
administration of AMT-010 represents a 40% reduction in median fasting plasma TG after administration, on top of diet.
7.2
Additional Evaluation Criteria
7.2.1 Expression and Biological Activity of the LPLS447X Transgene Product
Biological activity of the transgene product LPLS447X will be measured directly by determining the LPL activity in post-heparin plasma samples from the participants. In addition, LPL mass will be measured in these samples by ELISA. However, since patients were selected that do have LPL mass in their circulation, it is presently unknown whether the expression of LPLS447X mass will be detectable over the endogenous LPL mass levels.
7.2.2 Immune Responses
Patient’s serum will be assayed for humoral and cellular immune responses against the AAV1 capsid of AMT-010 and against the LPLS447X transgene product.
7.2.3 Assessment of Shedding
Shedding of AMT-010 will be assessed by detection of vector DNA in body fluids (serum, saliva, urine) using quantitative-PCR (Q-PCR), until 3 consecutive samples are negative for AMT-010 vector DNA. To evaluate the potential for germ line transmission, semen samples will be tested until 3 consecutive samples. During follow up after the study additional semen samples will be taken at least 75 days after administration, unless three samples are negative for AMT-010 DNA (provided the male subjects in the study are able to generate this sample).
8
STUDY PROCEDURES AND ASSESSMENTS
8.1
Weight
Participants will be weighed at least twice (until 2 consecutive measurements are within 0.5 kg of each other), and the average of the two will be recorded in the CRF.
8.2
Height
The participant's standing height will be measured at least twice (until two consecutive measurements are within 0.5 cm) at the screening visit.
8.3
Chest X-ray
An X-ray will be performed during rest at screening, and 24 hrs post-administration. Any new clinically significant abnormalities will be recorded on the CRF.
8.4
ECG
An ECG will be performed during rest at screening, and 24 hrs post-administration. Any new clinically significant abnormalities will be recorded on the CRF.
8.5
Heart Rate
Heart rate will be measured after the participant has been seated for at least five minutes and recorded on the CRF.
8.6
MRI
MRI will be used to monitor the general state of muscle, and muscle fat content according to the methods described by Maas et al. (2002)
