Essay: Effect of lentiviral-NAGLU injections with IV injections of enzyme on its own

Aims
Mucopolysaccharide IIIB (Trudel et al., 2015) is a form of rare autosomal recessive inborn-error of metabolism called mucopolysaccharides (MPS) III, and is caused due to deficiency in N-acetyl-alpha-D-glucosaminidase one of the four enzymes needed to break down heparan sulphate, a type of glycosaminoglycan. The four types of MPSIII are caused due to mutations in different genes but are clinically identical. Heparan sulphate (Schiattarella et al., 2015) is found in the extracellular matrix and on cell surface of glycoproteins in all animal cells. It binds to a number of proteins and aids in regulating variety of biological processes such as angiogenesis and blood coagulation. Overexpression of heparan sulphate causes accumulation in the lysosomes therefore resulting in abnormal signalling pathways, haemostasis, and degradation; eventually leading to neurological disorders (Andrade et al., 2015) such as MPSIIIB. The main symptoms of MPSIIIB are behavioural problems in children with mild physical abnormalities, such as ear infections and toothaches. The children affected by MPSIIIB have high threshold for pain tolerance so are not affected by simple bumps and bruises which would be very painful for normal children. Although, clinically manufactured enzyme can be intravenously injected into the children, it has proven to have no effect on the disease (Gilkes and Heldermon, 2014). This is because the enzyme cannot penetrate the blood-brain barrier preventing treatment of neurological manifestations of the disease.  The average life expectancy of affected children is between late teens to early twenties. Therefore, researchers have designed and modified lentiviral vectors which can carry the missing enzyme through the blood brain-barrier into the neurons.
Research has been carried out on knockout mice treated with lentiviral-NAGLU injections (Di Natale et al., 2005) after going through haematopoietic stem cell transplantation (HSCT) over a period of one year. HSCT is a procedure where multi-potent stem cells from a donor or the patient’s own bone marrow, peripheral blood or umbilical cord blood, are transplanted intravenously. HSCT has been reported to be ineffective on its own in patients with all MPS diseases therefore more research is being carried out in search of a more suitable treatment. Research has shown that enzyme replacement therapy (ERT) (Gilkes and Heldermon, 2014) is ineffective in treating the neurodegenerative symptoms for MPSIIIB disease but enzyme increases the expression of deficient enzyme NAGLU in the body.
The purpose of this research study is to evaluate and compare the effects of lentiviral-NAGLU injections with the intravenous injections of enzyme on its own.
2. Study Design
• The research study will be randomised in order to reduce any chance of bias occurring. These include allocation bias which occurs when the measured treatment effects are different than the true treatment effects because of the way the patients were assigned to either the control group or the treatment group. Assessment bias is also another type of bias that could occur when the researcher takes co-variates other than the effects of lentiviral HSCT injections into account while recording results. Therefore, when a randomised research study is carried out all the possible intervention are controlled by the researcher and both groups are presented with similar conditions except the treatment that they are given.
• This study is a parallel research because it compares the effects of a new therapy of lentiviral vectors transfected with NAGLU administered every month for 2 years with intravenous injections of the deficient enzyme via enzyme replacement therapy. Enzyme replacement therapy has been proven to be ineffective in crossing the blood-brain barrier so is used as a control in this study because it increases deficient levels of the enzyme.
• This research study will use double blind masking as both researcher and the patients would not be aware of patient allocation and which treatment is being administered therefore preventing different care or treatment being given to a patient based on personal favour or beliefs. Double-blinding also affects how the patients responds to the treatment, for example, emotional stress will be reduced as the patients wouldn’t know what to expect because they would have been briefed about any drawbacks before the start of research study.
3. Study Population
 Inclusion Criteria:
To be eligible for this study, patients must meet the following criteria:
• The patients must be clinically diagnosed with MPSIIIB which will be confirmed by the investigator via series of clinical health and psychological tests carried out prior to any procedures being performed.
• The patients must be 5 years old or under at the time of enrolment.
• Both male and female patients are accepted.
• Healthy volunteers for the research study are not accepted as the purpose of this study is to look for treatment for patients suffering from MPSIIIB disease.
• The parents or legal guardians of the patients must provide written consent to show the investigator that they understand the purpose and benefits of the study and also are aware of any potential harm that could come to their children. Informed consent also confirms that the parents understand the protocol of the study and any procedures that would be carried out during the study. The informed consent must be signed before any procedures can be performed on the patients involved in the study. The informed consent must be approved by Ethics committee before any research could be carried out.
• The parents or the legal guardians must provide complete medical record of the patients to ensure that they are aware of the potential risks and benefits of the haematopoietic stem cell transplantation treatment which will be carried out with the lentiviral vector injection transfected with the missing NAGLU enzymes. The medical records are also needed to ensure the investigator that the patients are capable of taking part in any of the procedures involved in the study such as they are not suffering from spinal cord compression which would affect the delivery of the injections.
• Patients with heparan sulphate excretion in the urine. This can be found from the health records but that is not necessary as the investigator can perform a test prior to the initiation of the research study.
• Patients with severe behavioural problems and cognitive development without any probable or identifiable cause.
• Patients suffering from Dysostosis multiplex without any cause. Dysostosis multiplex is a hereditary disease which is caused due to mutations in beta-galactosidase. It is very common in patients with MPS diseases; symptoms include bone and mental retardation, severe skeletal cartilage abnormalities.
• Patients suffering from hepatomegaly or splenomegaly without any identifiable cause. Hepatomegaly is where the patients have enlarged liver and presents itself as an abdominal mass. Splenomegaly is the enlargement of spleen. These two conditions are common in patients with MPSIIIB disease.
• Parents and legal guardians understand that this is a long term study of 2 years and will involve follow- up procedures.
• Patients must not be on any medications that could interfere or react with the injections administered during the study.
 Exclusion Criteria:
Patients who do not meet the following criteria will be excluded from the research study:
• The parents/legal guardians refuse to provide informed consent or comply with the study’s protocol.
• Patients are suffering from other medical conditions that could interfere with the study therefore they may not be able to take part in some of the procedures that are important for this clinical research therefore preventing the patients from completing the research study.
• Patients have received treatment with other conventional therapies for MPSIIIB or different disease in the past 2 months.
• Patients are not involved in any other clinical trial at the time or 60 days prior to the study.
• Patients have a history of poorly controlled seizure disorder which could interfere with the results obtained from this study.
• Patients are on any medication that can react with injections given during the course of 2 years in this study.
• The patient has a severe organ disease such as cardiovascular, pulmonary, hepatic or neurologic etc., these exclude symptoms of MPSIIIB. Presence of any of these diseases could potentially decrease the chances of survival during the study and may also prevent the patients from fully complying to the aims and objectives of the study as these medical problems could stop them from taking part in some of the procedures needed for this research study.
4. Treatment/Intervention
• This is an interventional research study as the purpose is to inject MPSIIIB patients with lentiviral HSCT injection transfected with the expression of NAGLU, an enzyme needed to break down heparan sulphate.
• The patients in one treatment group were injected with 12 modified lentiviral vectors transfected with NAGLU every month for 2 years. Lentiviral vectors deliver NAGLU through the blood brain barrier into the brain and alter the neurodegenerative structural and disorders caused due to MPSIIIB.
• Patients in the control group were intravenously injected with the manufactured NAGLU enzyme, this treatment is known as enzyme replacement therapy. The patients were also given 12 injections every month for 2 years, similarly to the treatment group. Although ERT has been proven to be ineffective in treating neurodegenerative disorders of MPSIIIB because the enzyme cannot cross through the blood-brain barrier, it increases the level of the deficient enzyme in the body. Therefore, it can be used as a control in this study as all the patients would receive NAGLU infusion.
5. Randomisation
Stratified randomisation method was used in this research study because this method allows maximum control and influence over the independent variable being measured, in this instance it would be the effects of 12 lentiviral HSCT injections compared with 12 ERT enzyme injections administered every month over two years. The researcher must identify specific variables which can have effects on the dependent variables therefore influencing the results from this research study. The researcher designs separate randomly sized blocks for each variable and the participants are assigned to the ones that apply to them. After patients are separated according to the variables that apply to them, simple randomisation is carried out within each block to allocate the patients in one of the two groups; treatment group with 12 injections and control group with 12 ERT injections. Simple randomisation is achieved via a random number tables or coin toss so it is very difficult to assign wrong groups to patients. A major disadvantage of using simple randomisation method alone, is chance bias which means that patients may be allocated in groups with unbalanced co-variates by chance, providing unbalanced results. Therefore, the use of stratification randomisation method in combination with simple randomisation method allows for unbiased results with reduced chances of unbalanced results. Blocking is a way to provide restrictions in randomisation and has major advances in clinical trials.
6. Outcome measures
 Primary outcome measures are used to calculate sample size for a specific randomised controlled trial. These measures are used to answer the main questions being asked in the research for example, the effects on the patients receiving 12 injections every month for 2 years and the effects ERT driven injections in the control group. The primary outcome measures used in this research study are:
• The researcher should carry out neurocognitive function test, every 24 weeks from the first treatment injection, for the both treatment groups and control group. This test must be performed using standardised test for quantifiable measures of the patient’s neurological functions.
• The researcher should also assess the changes in size of organs such as liver and spleen every 24 weeks, using MRI. The researcher should make sure that they avoid exposing the patients to radiation as they are very young so it would be easier for them to be affected.
• Patients’ dysmorphic features, physical and mental structures must be assessed at 24 week intervals using the multiple QOL tool, this is carried out to determine if the symptoms of MPSIIIB are decreasing.
• The behavioural functions of the patients must be assessed every 12 weeks using an MPSIIIB specific behaviour rating scale.
• The patients must also undergo clinical laboratory tests every 24 weeks for the duration of 2 years; these include urine, blood and cerebral spinal fluid sample testing. In the tests, the researcher must look for the presence of heparan sulphate and any specific MPSIIIB disease markers.
 Secondary outcome measures are used by the researchers to determine if the research has effects other than the main aims. It measures if there is reduction in other symptoms of disease as well as the chances of death. The secondary outcome measures used in this research study are:
• Measurement of urinary and CSF GAG levels at the beginning of research study (Baseline) and the end (week 104). Compare the differences to determine if there is reduction in the expression heparan sulphate and the MPSIIIB disease biomarkers.
• Measurements must be taken of the change in heart size and function from Baseline to week 104. This is to ensure that the cardiac activity has increased in patients because of this treatment.
• Measurements of neurological structures and function (Baseline to week 52 and then week 104).
• Measure in behaviour of the MPSIIIB affected patients by the MPSIIIB specific measure rate (measurements taken at Baseline, 26, 52, 78 and 104 weeks). More measurements are taken for this variable because behaviour can be learnt and adapted so measurements at different intervals would ensure that the problems which occurred due to MPSIIIB have been corrected.
• The researcher must provide a clinical analysis at the end of the study which explains overall clinical status from baseline to end of the research study at week 104. The researcher should highlight the significant changes that occurred during the 2 years of the study.