Assessment of lentiviral development as a cure for cystic fibrosis and its potential for the future.

Elizabeth Dodd

Contents

Page

Abstract 2

1. Introduction 31.1 What is cystic fibrosis? 31.2 Causes of CF. 41.3 Aim. 5

2. Gene therapy 62.1 What is gene therapy? 62.2 CF as a candidate for gene therapy 62.3 Monitoring gene therapy effects 7

3. Lentivirus 83.1 Components of lentivirus vector 83.2 Longevity in past studies 11

4. Administration 134.1 Types of administration 134.2 Re-administration 13

5. Integration in gene therapy 145.1 Areas of integration 145.2 Aiding integration 145.3 Translocation and transduction 15

6. Expression of CFTR 156.1 Areas and sufficiency of expression 156.2 Expression of CFTR 16

7. Alternative therapies 178. Future production prospects 18

8.1 Viability of large-scale production of vectors 188.2 Large-scale drug production 208.3 Comparison of gene therapy and drug therapy potential 20

9. Discussion and conclusion 12

Bibliography 23

Appendix 32

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Abstract

Cystic fibrosis (CF) is a genetically inherited recessive disease which effects the cystic

fibrosis transmembrane regulator (CFTR) protein. This thesis is an assessment of lentiviral

development and how successful current gene therapy models are and whether it is possible

to extend the potential of treatment, plus the feasibility of clinical use and large-scale

production. Successful lentiviral vectors would need to successfully operate transcription,

packaging, reverse transcription and integration in order to effectively transduce cells and

express sufficient CFTR protein. Comparison of data collected from past significant studies

suggests that there is high potential for use of HIV-1 vectors pseudotyped with either F/HN

or VSV-G with the addition of LPC pre-treatment as these have demonstrated efficient long-

term expression (up to 2-year murine life-limit) when compared to other vector models. With

insight from an interview with Dr. Bhatt, comparison with drug therapy was made and

revealed that at $294,000 per patient each year drug treatment may prove too expensive and

gene therapy may hold more promise as a sustainable and feasible cure.

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1. Introduction

1.1. What is cystic fibrosis?

Cystic fibrosis (CF) is a genetically inherited recessive disease effecting the formation and

functioning of the cystic fibrosis transmembrane regulator (CFTR) protein. This protein is

expressed on the epithelial apical surface of cells and is responsible for the transport of

chloride and other ions. Dysfunctional or lack of proteins leads to chloride secretion and

sodium absorption causing reduction in airway surface liquid (ASL) (Conese et al, 2011); in

turn this causes the clearance of epithelial mucus to become impaired (Matsui, 1998). The

CFTR also plays a role in host defence, so reduced expression can lead to mucus and anti-

microbial factor secretion being reduced or prevented from sub mucosal glands (Wine, 2004).

Together, these factors produce a dehydrated and thick mucosal environment in which

bacteria can thrive, eventually triggering infection, inflammation and unfortunately organ

failure. Though CF effects the epithelial functioning of many cells and tissues such as the

liver, reproductive organs and pancreas, it is its effects in the lungs which cause the most

damage and mortality.

Since the CFTR gene was first identified and cloned in 1989 (Riordan et al, 1989; Kerem et

al, 1989) there have been many milestones which have enabled research into curing this

debilitating disease, the main highlights of which can been seen in Table 1. As seen in Table

1, the first study to confirm proof-of-principle for gene therapy was conducted in 1990 and

demonstrated that retrovirus-mediated gene transfer of CFTR was able to correct cAMP-

mediated chloride conductance in vivo (Drumm et al, 1990). After trials and discovery of

different vector types the first CF gene therapy trial was carried out on three patients in 1993,

using a first generation adenoviral vector and CFTR cDNA, partial restoration of cAMP-

mediated chloride transport was achieved (Zabner et al, 1993). In more recent years there has

also been a lot of investigation into not only gene therapy but the use of personalised

medicine and drug development which is able to restore CFTR functioning.

Though many strides have been taken towards curing this disease it has proven to be a much

more challenging than first anticipated. Due to the complex lung defence system and the

many ways in which the protein can be effected, CF is one of the most prevalent inherited

diseases amongst Caucasian populations. It has an approximate prevalence of 1 in 2000,

though the frequency ranges significantly between countries, with Ireland having a

prevalence of 1 in 1,800 to Finland having an occurrence of 1 in 25,000. While the UK and

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Northern Ireland have an incidence rate of 1 in 5,350 each European country differs, please

refer to table 2 for more specifics. Unfortunately no matter which country, CF has a severely

reduced life expectancy of approximately 45 years with many sufferers dying in late teens or

early adulthood due to infections and lung complications (FitzSimmons, 1993).

1.2. Causes of CF

Different types of mutations and severity of the disease can be separated into the classes

identified in Table 3; people born with mutations grouped into classes I, II, and III are more

likely to have a severe manifestation of CF. While those in classes IV or V often have

residual function of CFTR and therefore often have less severe manifestations (Rowntree et

al, 2003; Wilschanski and Durie, 2007). A strong correlation has been found between the CF

genotype and phenotype, as seen in figure 1, the severity of the clinical phenotype can be

directly associated with CFTR expression. The presence of two severe CFTR gene mutations

(e.g. F508del/F508del, R553X/G542X) severely limits Cl- transport and can lead to

pancreatic insufficiency, severe lung function deterioration and higher frequency of

meconium ileus. Along with premature mortality, higher incidence of malnutrition and severe

liver disease.

Though there have been thousands of possible mutations identified, many of which can incur

these severe outcomes, the most common found is a deletion of a phenylanine residue at

amino acid position 508 (Kerem et al, 1989). F508del is the most common mutation amongst

CF sufferers and is caused by incorrect protein folding causing retention in the endoplasmic

reticulum (ER) resulting in proteasomal degradation (Rowe et al, 2005), causing a reduced

amount of CFTR present on the cell surface (Welsh et al, 1993). In Britain alone more than

90% of CF sufferers have the F508del, with 70% being homozygous (Dr. Bhatt, 2015).

Though ultimately it is the genotype which defines the presence of CF, environmental factors

can also impact on the manifestation of the disease. Factors such as inhaled pollutants,

exposure to opportunistic micro-organisms, stress, adherence to treatment and level of care

can all influence the disease severity. Genetic modifiers could be equally responsible for

influencing CF lung related disease, at least for homozygous F508del genotypes, as well as

determining prevalence of CF-related diabetes (CFRD) and meconium ileus (Cutting, 2010).

There have been many modifier genes identified, though very few have been substantiated in

groups of over 1,000 CF patients and therefore lack proper evidence of function that can be

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generalised (Cutting, 2010). Some of these modifier genes include: CFM1, cystic fibrosis

modifier; HLA-II, MHC class II antigen; MBL2, mannose-binding lectin; NOS1, nitric oxide

synthase 1; TGFB1, transforming growth factor-a1; TNFA, tumour necrosis factor-a

encoding gene (Badano and Katsanis, 2002).

Figure 1. CFTR function and clinical phenotype correlation showing how the severity of

clinical phenotype is directly correlated with CFTR expression within tissue. Normal

phenotype is associated with fifty percent of CFTR expression (Prickett and Jain, 2013).

1.3. Aim

The aim of this thesis is to assess the current methods being developed to cure CF with focus

on lentiviral development and how successful current vector models are; whether it is

possible to extend the potential of treatment, as well as the feasibility of clinical use and

large-scale production. After conducting an interview with cystic fibrosis specialist Dr. Bhatt,

the transcript of which can be seen in appendix 2, a comparison of gene therapy and drug

therapy and their potentials for large-scale clinical use shall also be debated. The interview

was conducted January 2015 and the main areas of discussion were his experience with gene

and drug therapy and his opinions on their use. The interview brought to attention concerns

with the cost feasibility of these treatments and the fact that gene therapy is still far from use

in clinical settings. The interview personally was insightful and brought to attention the

opinions of those who work in the medical profession and have contact with and treat CF

sufferers on a daily basis. Due to this interview raising the issue of cost feasibility, the cost of

treatments and possibilities of large-scale production and comparison of gene and drug

therapy potentials shall also be discussed.

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2. Gene Therapy

2.1. What is Gene Therapy?

Gene therapy can be defined as ‘the introduction or alteration of genetic material within a cell

or organism with the intention of curing or treating disease’ (Asgt.org). Gene therapy as

treatment for CF has been of great interest since the gene encoding for the CFTR protein was

cloned in 1989 as it has much potential for curing the debilitating disease. Over the years

knowledge has been gained into the functioning of the different components of the genome

and vectors have been developed which aim to deliver corrected CFTR genes into cells to

enable production of functioning CFTR proteins. When CF first became a target for gene

therapy viral vectors were the first developed due to their natural invasive characteristics,

though issues were soon encountered with triggering the immune response. Non-viral vectors

and lipid-based vectors have also been developed though they in themselves have their own

obstacles to overcome.

Though CF has a lot of treatments and the life expectancy can be expanded through many

medications and procedures, the use of gene therapy would eliminate all of the secondary

issues associated with the disease. If perfected then gene therapy would eliminate the need

for sufferers to carry out physiotherapy or to take the amount of medication currently

necessary to address their lung and digestive function. However after many years of research,

due to the evolution of the lung to fight invasion, many obstacles have proven to cause

problems with what originally seemed an easy target. These obstacles include extra- and

intracellular barriers such as sputum obstruction, pathogen defence and immune responses.

For the basis of this thesis viral vectors will be the focus for the potential of gene therapy,

though viral vectors come in a highly diverse range including Adeno virus, Adeno-associated

virus, Sendai virus and lentiviruses. This study will focus mainly on the uses of lentiviruses

as they have been proven to have much potential for future uses.

2.2. CF as a candidate for gene therapy

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CF is a favoured candidate for gene therapy, it is caused by mutations in a single known gene

and therefore can be easily targeted. After years of research the exact nature of the disease

and which biological malfunctions are being caused have been discovered and therefore the

correct pathways and systems can be targeted. Not only have past studies proven proof-of-

principle the fact that some mutations still allow formation of the CFTR protein signifies that

replacing the defective gene will allow correct functioning. Additionally, compared to other

gene therapy candidates, CF treatment can be easily administered, observed and controlled as

the lungs are easily accessible. Though as accessible as it is, the evolution of the lung and its

defence mechanisms have proven to be very difficult barriers.

2.3. Monitoring gene therapy effects

The main techniques used to monitor progression of CF are; pulmonary exacerbation, quality

of life, spirometry, infant lung function, and chest computed tomography (CT). Each comes

with their own advantages and pitfalls. In the cases where children <6 years are involved,

then the most accurate and preferred methods may include respiratory cultures and possible

chest CT as these are available to all ages. Though when looking at the literature, the

preferred method overall appears to be either number of pulmonary exacerbation events in

which the lung function worsens and FEV1 drops; or spirometry in which breathing apparatus

measures the amount of air inhaled, exhaled and speed of exhalation allowing calculation of

FEV1. Spirometry reveals changes within a matter of minutes, however is not suitable for

those <6 years and pulmonary exacerbation has yet to have a standardized definition (Stenbit

and Flume, 2012). Quality of life is often recommended to assess patients >6, and is

measured with a point system using the Cystic Fibrosis Questionnaire revised (CFQ-R).

Perfecting chest CT scans and imaging techniques could improve potential for being an

accurate and reliable method for function monitoring in future trials.

Though sweat chloride is used as a reliable diagnostic tool it was revealed to not be reflective

of patient lung function (Durmowicz et al, 2013) and therefore is not suitable for use as an

end point of clinical trials. Sweat chloride can be improved but is only reflective of sweat

epithelial cell function and cannot be reflective of those CFTR functions within other organs

such as the lung.

One of the most efficient and reliable ways to assess the lung function is spirometry,

measuring the forced expiratory volume in 1 second, FEV1 as seen in figure 2. This is the

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volume exhaled during the first second of a forced expiratory manoeuvre started from the

level of the total lung capacity (Morris et al, 1973); FEV1 expressed as a percentage of the

vital capacity (VC) is standard for assessing and quantifying airflow limitation. In healthy

individuals VC is measured according to inspiratory vital capacity (IVC) and expiratory vital

capacity (EVC) and are similar levels. In patients with obstructive lung disease, the VC

obtained during expiratory manoeuvers is smaller the when obtained upon inspiration:

IVC>EVC>FVC. (Yernault, 1997).

Figure 2. Graph demonstrating the proportion of exhaled air volume which allows calculation

of the FEV1.

3. Lentivirus.

Lentiviruses are a favoured option for use in gene therapy for CF as they are able to infect

non-dividing cells such as those found in the lungs (Cooray et al, 2012). Due to their natural

ability to infect host cells they are the most likely to incur large amounts of transfection,

particularly those vectors derived from the human immunodeficiency virus (HIV). Contrary

to initial beliefs, although they are derived from highly infectious diseases they appear to

possess a lower immunogenicity than other viral vectors (Griesenbach et al, 2002;

Griesenbach et al, 2004).

3.1. Components of lentivirus vector.

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The basis on which lentiviruses are built is to create a replicative-deficient recombinant

particle using three main components: genomic RNA, internal structural and enzymatic

proteins and an envelope glycoprotein (Cooray et al, 2012). The components of a lentivirus

need to successfully operate transcription, packaging, reverse transcription and integration in

order to effectively transduce cells and express sufficient CFTR protein.

‘First generation’ lentiviruses using HIV were built with all but the envelope genes (Naldini

et al, 1996). Later came development of ‘second generation’ lenitiviral vectors which have all

but four HIV-1 genes removed and have a much simpler construct. In this system the gag

reading frame encodes for the structural components, while the pol reading frame encodes for

the enzymatic components of the virion. Also left are the tat and rev genes which ensure

transcriptional and post-transcriptional roles are fulfilled (Zufferey et al, 1997). More

recently the ‘third-generation’ system has been developed for clinical purposes which encode

only the gag, pol and rev genes along with a chimeric 5’ long terminal repeat (LTR) which

ensures the ability of transcription in the absence of tat.

Though third generation systems have been developed for safer and more efficient clinical

therapeutic uses it has not completely written off the use of second generation systems. The

second generation packaging system has not only proven to be safe but also works in both

second and third generation vectors. For example, the pCMVR8.74 packaging plasmid

encodes for the HIV-1 Gag, gag/pol, tat and rev proteins (Rohrlich et al, 2005); in many

cases it is easier to use the second generation packaging system as an all-purpose packaging

plasmid.

Evidence has been collected which so far shows no sign that second and third generation

lentivirus packaging systems create replication-competent-recombinants (RCRs) which

means they present low safety risk (Escarpe et al, 2003). Past studies into the effects of RCRs

cause concern that they would have detrimental effects in humans, particularly when using

HIV-based vectors. In particular a study involving retroviral vector insertions found

overexpression of oncogene EV11 and led to genomic instability, causing progression of

myelodysplasia (Stein et al, 2010). The region responsible for replication within wild type

viruses is the 3’ LTR U3 region. Within the more recent vectors produced this region is

deleted which renders the vectors replication-defective and unable to reconstitute their

promoter. This type of design is referred to as a Self-inactivating (SIN) vector. These vectors

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also increase safety by inactivating viral promoter/enhancer elements following reverse

transcription, halting any effect it has on surrounding cells (Thornhill et al, 2008).

The ability for lentiviruses to transduce non-dividing cells is due to the presence of the pre-

integration complex (PIC) and its mechanism of entering the nucleus without disrupting the

nuclear membrane. The main structural proteins which aid this are encoded in the gag gene

and are matrix (MA) and nuclearcapsid (NC). MA and NC proteins remain associated with

PIC and as they contain nuclear localization signals they are able to facilitate nuclear

translocation. Some studies have found that MA binds directly to importinα, allowing entry

through the nuclear pores (Bukrinsky et al, 1993; Gallay et al, 1997). This entry process is

also facilitated by the accessory protein vpr which has been shown to cause transient

herniations in the nuclear membrane by binding directly to the nuclear pore (Noronha et al,

2001).

Additional proteins are also encoded for in secondary generations, accessory proteins

include: vif, vpu, tat, rev and nef; though tat and rev are the only ones proven necessary for

replication. Studies have confirmed the role of tat in activation of the promoter of the HIV

LTR, allowing viral RNA to be efficiently produced (Karn et al, 1997). Rev then interacts

with the rev response element (RRE) region of the RNA to promote its transport from

nucleus into the cytoplasm (Brenner and Malech, 2003). However, in third generation

systems the presence of a chimeric 5’LTR construct has enabled the lentivirus promoter to

become tat-independent. This is achieved by replacing the U3 region of the 5’LTR with

alternatives such as the CMV enhancer (CCL LTR) (Dull et al, 1998). These highly efficient

promoters also allow much higher titers to be produced without the tat HIV transactivator.

In addition to accessory proteins, the much reduced third generation lentivirus has proved to

be much more effective when pseudotyped through transient transfection. In the past the most

commonly used is the vesicular stomatitis virus (VSV-G) envelope protein. Pseudotyping the

vector in this way has allowed the host cell tropism to be increased and the viral envelope

gains stability. As well as being able to be concentrated to titers of 108-109 IU/ml (Yamada

et al, 2003; Kafri et al, 1999). Though the VSV-G envelop can be toxic to cells this can been

overcome by transient transfection of VSV-G onto separate packaging expression constructs

(Schambach et al, 2000). Though the use of VSV-G proteins for pseudotyping has been

favoured, some studies have shown that this envelope protein is unable to transduce intact

polarised airway epithelia efficiently through in situ administration to the apical surface

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(Johnson et al, 2000; Johnson, 2001). Entry into the cell when tested in vivo was only

achieved if agents were used to open tight junctions and allow access to the basolateral side,

however this is not practical for clinical applications (Wang et al, 1999; Johnson et al, 2000).

Other apical membrane-binding envelope proteins are available which are able to overcome

this issue, the most promising of which appears to be the filovirus-pseudotyped feline

lentiviral vector which has a tropism for respiratory epithelium (Kobinger, 2001).

3.2 Longevity in past studies

There have been a large number of studies carried out into the uses of lentiviruses, their

safety, efficacy and their possible use within clinical trials. Though clinical trials using

lentiviruses have not yet been conducted, they have been extensively trialled on animal

models and human epithelial cell cultures producing promising results which could indicate

their future successfulness.

Tarantal et al (2005) conducted a study using rhesus monkeys which successfully transfected

cells with a SIN HIV-1-derived vector pseudotyped with VSV-G protein. A titre of 107

infectious particle/foetus was used and expression was shown for 2-3 months with the

animals having normal growth and development (P>0.05) when compared to the control.

Another early study, also conducted in 2005 by Sinn et al, used GP64 pseudotyped FIV

vectors to transfect mice through direct installation demonstrating very promising results.

Using a titer of 1.25x107 TU of FIV in a 50µl volume, transduction of both human airway

epithelial cells (P<0.01) and murine respiratory epithelia in vivo occurred (P<0.0001). This

study also shows that expression remained constant over the 50 week test period which was

found to be significant (P<0.0001) compared to the naïve control (Sinn et al, 2005).

Sinn et al (2008) carried out later studies into re-administration effects, again using a GP64

pseudotyped FIV vector at 1.25x107 TU/50µl vol. Though in this study the administration

was conducted on seven consecutive days, and demonstrated that repeated administration was

both effective and feasible. Though this study only tested expression up until 13 weeks, there

were no adverse effects or any sign of discontinued expression. In order for gene therapy to

be successful a vector would need to transduce the cells effectively and show sustained

transgene expression.

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A study into long-term transgene expression was conducted by Buckley et al (2008), using a

GP64/HIV-luciferase vector. A single dose of 3x107 IU was delivered via intra-amniotic

administration to 1 day neonatal mice. Results from this study reveal expression being

detected throughout the 390 days the trial was conducted. However this study was overtaken

by Stocker et al (2009), who also tested whether long-term expression was achievable. Using

a VSV-G/HIV-1 vector at a titer of 2.45x108 TU/ml on mice, expression was achieved for the

24 month lifetime limit available. Though expression had been reduced by this point a single

dose was able to achieve partial correction at the tested 1 and 12 month points (P<0.05).

Though these results showed a lot of promise, the use of VSV-G proteins as discussed

previously were proved to be complicated for use in clinical trials so studies were conducted

which investigated the use of lysophatidylcholine (LPC) (Cmielewski et al, 2010; Kremer et

al, 2007). LPC was used as pre-treatment before HIV-1 vector treatment on mice with the

intention of opening the tight junctions which restrict transduction for VSV-G vectors.

Expression improved when pre-treatment was used when compared to control groups. Re-

emergence of expression was found at 12 months which may indicate that the LPC allows

access and transduction of progenitor cells which could allow more persistent expression

(Rock et al, 2009). Studies then moved on to alternative envelope proteins.

Mitomo et al (2010) used SIV pseudotyped with SeV (F/HN) proteins on around 100 mice. A

titer of 4x109 TU/ml per animal was administered to the nasal epithelial cells and expression

was achieved for up to 15 months, though many only showed expression to 12 months. This

expression was significantly higher than the control virus (P<0.001) and the study also

confirmed production of CFTR protein. Griesnbach et al (2012) also conducted a study using

F/HN pseudotyped SIN vectors investigating re-administration on not only mice but also used

human AL interface cultures, primary human nasal epithelia and human and sheep lung

slices. Daily administration of a 106TU/day in 100µl was delivered for 10 consecutive days

via nasal inhalation. Though expression decreased by 60% between 15-22 months, expression

was seen in some mice until the 22 month trial termination. Though not all mice survived

until the end of the study, the mortalities were not specified to be caused by the treatment and

previous studies have indicated no adverse effects from using these vectors.

The details and statistical information from the studies discussed in this section can be seen in

table 4, in which it is clear to see that it is possible to have successful expression of

transgenes within airway epithelium and many of the barriers posed by the lung defence

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system have been overcome. However, we have yet to see any clinical trials successfully

conducted in order to assess the safety of these vectors and their possible uses for large-scale

production to be used as a curative therapy. Effective comparison of results is difficult as the

studies conducted were terminated at varying times so expression length may depend on

when the trial ended. However comparison of the significant P values of each treatment

compared to their control group can be made. When looking at these values it is clear to see

that there was much more significant change in those trials using the FIV/GP64 vectors, and

these therefore show much promise for further research.

4. Administration

4.1. Types of administration

For future uses in clinical trials an advantageous administration technique would enable

access to target areas and enable the optimal transfection. However, when considering future

uses for CF sufferers the ease of administration and how often a treatment needs to be

administered are important factors that need to be considered.

Nasal installation has been the favoured technique of vector delivery in the studies using

lentiviruses carried out so far. This is the simplest and least invasive delivery method, in most

situations the mice are anesthetised and the vector is delivered through inhalation; the vector

coming into contact with nasal airway epithelial cells. In gene therapy clinical trials using

different vectors, nasal installation has been used and has been found to allow successful

administration (Alton et al, 1999). There have been studies into administering vectors into

progenitor cells and administration into foetal and neonatal subjects; Buckley et al (2008) in

particular carried out trials with mice to see the effect of infection into both foetal, neonatal

and adults. Their study found that expression could be achieved and sustained through

administration to 1-day old neonatal mice.

4.2. Re-administration

As expression decreases over time there is need for re-administration and the need for safe

repeat treatments. In the past studies have demonstrated humoral immune responses to

vectors, particularly viral vectors. This has caused many issues for repeated administration as

the additional dosages have either not been tolerated well causing adverse effects, or the

immune system prevents effective transfection on secondary administrations. More recent

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studies have shown that modified lentiviruses have been able to be successfully re-

administered without causing any adverse effects and also have shown correlation between

administration and expression (Sinn et al, 2008). Gene expression has been found to last a

number of months, so it can be hypothesised that if used in the future administration of

vectors would only need to be carried out every few months.

5. Integration in gene therapy

5.1 Areas of integration

Once the vector has been administered and traversed the obstacles presented by the host lung

defence, it then has the task of correctly integrating into the host genome. When introducing a

new gene into a genome there is the chance that it may be inserted into unpredictable areas

and possibly activate oncogenes which would have extremely detrimental effects. There has

been a lot of evidence that suggests lentiviruses, particularly the HIV-1, has preferred areas

for integration. Past studies have compiled evidence that these favoured areas are a subset of

transcriptionally active genes within the host genome (Schroder et al, 2002; Sherril-mix et al,

2013). A more recent study conducted by Marini et al (2015) has narrowed done the areas in

which HIV-1 integrates into the host cell genome. This study found that that integration of

HIV-1 occurred mainly in the outer regions of the nucleus, close to the nuclear pores. In these

areas there is the presence of active transcription chromatin which is thought to identify the

area for viral infection. Lentiviruses have a much lower propensity for integrating into

oncogenic regions than other possible vectors such as gamma-retroviral vectors (Cattoglio et

al, 2007). This allows them to integrate more predictably and increases their safety potential

within clinical therapeutic use.

5.2 Aiding integration

Essential for the transcription process is viral integrase which is coded for by the pol region

and is translated as a section of the gag-pol polyprotein (Van Maele et al, 2005). Viral

protease processes the pol region into its mature form and controls activities including

reverse transcription, nuclear import and viral particle assembly (Zhu et al, 2004; Gallay et

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al, 1997). This enzyme binds to viral cDNA within the U3 region of the 5’LTR and the U5

region of the 3’LTR (Zhou et al, 2001) and processes the 3’ end to create an overhang which

is in turn attached to the 5’ phosphorylated double stranded cut in the target genome. The

enzyme then repairs the cuts and results in an inserted vector genome flanked by a 5 base-pair

repeat (Mizuuchi, 1992).

Co-factor is the term used to describe those cellular proteins which aid integration of the

provirus into the host cell. There are a few co-factors which have been identified over the

years, these include: integrase interactor 1 (Kalpana et al, 1994) and more recently lens-

epithelium-derived growth factor (LEDGF, also known as p75) (Turlure et al, 2004; Emiliani

et al, 2005). Addition of recombinant LEDGF/p75 has been shown to enhance strand-transfer

activity of HIV-1 integrase (Cherepanov et al, 2003) and investigation suggests that it plays

an important role in tethering integrase to chromosomal DNA (Van Maele et al, 2005). This

is thought to be done by increasing integrase affinity for DNA through targeting the lentiviral

PIC to actively transcribed regions. This theory is supported by the evidence of HIV-1

preferred integration sites being transcriptionally active regions. Once integrated transcription

needs to be regulated and is aided by the HIV-1 DNA associating with various nucleoporins

(Marini, 2015).

5.3. Translocation and transduction.

As previously mentioned there are a number of limiting factors and variables which can

dramatically affect the ability of lentiviruses to successfully infect cells and incur sufficient

CFTR expression. One of these is nuclear translocation of the genome which has been found

to slow infection (Follenzi, 2000). One of the effects found from deleting the majority of the

viral sequences within lentiviruses was that nuclear translocation and transduction was

limited. Restoration of 118bp from HIV-1 pol cis region greatly improved the efficiency of

gene transfer of the vector genome. Follenzi (2000) suggested that the sequence when

inserted into a SIN lentiviral vector upstream of an expression cassette in the sense direction

(cPPT), was able to promote nuclear translocation of the viral genome.

6. Expression of CFTR

6.1. Areas and sufficiency of expression

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In early studies it was thought that the predominant areas of CFTR expression were in the

bronchial sub-mucosal glands. Over the years there have been many studies into trying to

find the optimum target area for therapies and to this day there are still debates between

epithelium and submucosal glands or both (Zhou, 1994; Joo, 2010). A particular study carried

out by Englehardt (1992) detected CFTR expression in primary nasal epithelial cells through

use of western blotting and in situ hybridisation. This was contradicted in more recent

studies, in particular a study carried out by Kreda (2005). The evidence compiled suggests

that it is ciliated cells that are involved in transepithelial ion transport and Kerda suggests that

any therapeutic approaches to CF should have a focus on re-establishing CFTR function to

the superficial epithelium. Evidence also suggests that CFTR encoded chloride channels are

also expressed on the apical plasma membrane (Kartner et al, 1992). Improvements have also

been proposed to be achieved by enhancing the maturation and apical plasma membrane

localization for F508del (Kreda, 2005).

There has been much debate over the amount of expression required to enable correct

functioning, it has been speculated that less than 50 % gene transfer would be sufficient to

engage normal lung function (Caplen, 1999). Though the level of gene transfer achieved

during clinical trials has not been confirmed to be sufficient for generating improvement

there is a large amount of literature supporting the theory that even small amounts of transfer

would be suffice (Griesenbach and Alton, 2013). Some studies have found that patients with

mild mutations which acquire only 10% of normal CFTR function in each cell have achieved

reduced lung disease (Chu et al, 1993). Others have found that using ad-mediated CFTR gene

transfer into 20-30% of sinus epithelial cells is able to correct chloride transport to 50% of

non-CF cells within CF knockout pigs. Even levels as low as 7% were seen to produce some

correction (Potash et al, 2013; Bombieri et al, 2011). On average, from the literature

composed, it appears that the target amount of expression to achieve correct CFTR

functioning should be around 20%.

6.2. Expression of CFTR

When planning research and clinical trials marker genes are recommended for inclusion as

they allow any expression to be easily monitored. It is also advisable to test the vector with

the marker before inclusion of the transgene to allow for repairing any problems caused by

components other than the transgene. One of the more popular markers used is enhanced

16 | P a g e

green fluorescent protein (eGFP) as it can be easily monitored by flow cytometry or confocal

microscopy (Bierhuizen et al, 1997). A luciferase reporter gene would be more useful in an

in vivo clinical trial situation as it tracks gene expression in real time and therefore the

progression of expression can be measured over the length of a trial.

It has been observed that on numerous occasions lentiviral vectors are subjected to epigenetic

silencing associated with DNA methylation of CpG sequences (Bestor, 2000; Ellis, 2005),

which can drastically reduce expression of the transgenes (Zhang et al, 2010). This issue can

be partially protected by flanking transgenes with DNA insulators, though this may cause

reduced titers (Gaszner and Felgenfeld, 2006; Urbinati, 2009). Other modifications may also

improve levels of expression, Zhang et al (2007; 2010) suggest a novel enhancer-less

ubiquitous chromatin opening element may resist DNA methylation and silencing and could

be viable for future applications.

7. Alternative therapies

Drug therapy, with respect to cystic fibrosis, would be a great example of ‘personalised

medicine’ as the type of drug developed would have to address the different ways in which

the different mutations effect the CFTR pathway. As discussed, mutations vary in their

effects on the CFTR protein; they may prevent trafficking of the protein to the apical

membrane or hinder the gating of the channel preventing correct chloride transport. There

have been a number of studies investigating how the use of drugs can improve the

functioning of the CFTR protein. One use of drug therapy which has proven to be very

successful is the use of VX-700 in correcting CFTR function in those with the G551D

mutation. More commonly known as kalydeco or Ivacaftor, the drug was first approved by

the FDA on January 31st 2012 in USA and Canada for use on patients above the age of 6.

After further testing it has now been approved for ages 2-5 and for a range of different class

three gating mutations (Davis et al, 2012) and has now been in use as a CF treatment for the

last 2 years (Dr. Bhatt, 2015). Ivacaftor is a CFTR potentiator which targets gating mutations

by improving the probability that the CFTR channel will open on the apical surface. This is

why it is specific to those class III mutations in which the CFTR protein is present on the

apical surface but complications are caused by gating malfunctions, particularly G551D.

Correction of the channel gating allows the probability of the channel opening to be increased

and therefore there is higher chance of ion transportation. There have been two significant

17 | P a g e

studies into its uses and are known as the ‘STRIVE’ and ‘ENVISION’ projects, statistical

data from these and other studies can be seen in table 5.

As seen in table 5 the two most recent Phase III studies were both conducted as randomized,

double-blind, placebo-controlled trials but were carried out on different age groups. The

‘STRIVE’ study was conducted by Ramsey et al (2011) and was carried out using

participants over the age of 12, with a mean age of 25.5. The participants were given 150mg

of Ivacaftor every 12 hours for 48 weeks and their change from baseline in FEV1 was

observed. Significant change in the treatment group when compared to the placebo (P<0.001)

and change was observed from 2 weeks continuing until 48 weeks. Sweat chloride levels

were also measured and significant improvement was also seen here from 15 days through to

the end of the 48 weeks (P<0.001). Though some adverse events occurred none were

relatable to the treatment and Ivacaftor was therefore deemed safe and effective for those

over 12 (FDA).

The second study was conducted using participants between six and eleven years old and was

named the ‘ENVISION’ project. Davies et al (2013), conducted the study using the same

method as the STRIVE study, administering 150mg every 12 hours for 48 weeks. This study

also produced very positive results with change in FEV1 increasing significantly when

compared to placebo (P<0.001) and was maintained until 48 weeks. The sweat chloride levels

in this study also showed improvement (P<0.001) and no clinically relevant adverse effects

were observed. Leading to the approval of Ivacaftor as a treatment for CF sufferers over the

age of six (Davies et al, 2013).

Other drugs aimed at more common mutations have also been developed, Goor et al (2011)

found the use of agent VX-809 (lumacaftor) was able to restore some channel misfolding

instigated by the F508del. This has further been developed and Phase II trials have been

completed which did not show any clinical significance (Clancy et al, 2012) though

improvement was observed when used in tandem with Ivacaftor (Boyle et al, 2012) and

phase III trials are under way.

8. Future production prospects

8.1. Viability of large-scale production of vectors

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Vectors have been in use in laboratory and experimental situations for many years, but

production scale of vectors has not yet been expanded to the size required for clinical

purposes. As the most expensive part of gene therapy development, the initial investment into

research and the large-scale manufacturing of pseudotyped vectors for use in clinical trials

requires a lot of consideration. Particularly when assessing costs of production and materials

required, including patient number, amount of vector needed per patient and approximated

vector titre. Therefore calculations are needed before starting a trial in order to assess whether

it is technically possible and how much it would cost, which have been described by Leath

and Cornetta (2012). To start, vector dose needed per patient needs to be determined, also

referred to as infectious unit per patient (IU/Pt):

IU/pt = MOI(IU/cell) x no. cells treated/kg x mean pt weight /9kg) x no. transductions

Where MOI is multiplicity of infection. This calculation requires determination of the

number of cells to be exposed to the vector and the ratio of infectious particles per cell. The

age of participants has a significant impact on vector required as the number of cells is based

on weight, for example 5x106 CD34+ cells/kg.

Determining the total number of particles needed (X) requires the number of patients and

dose per patient to be considered, along with additional amounts for testing for replication

competent lentivirus (RCL) and 5% reserve to be held if repetition is required. This leads to:

X = (IU/pt x pt) + (X x 0.1 for RCL) + release testing needs.

The total volume of un-concentrated vector necessary for production can now be calculated,

central to this is the anticipated titer of the unprocessed vector. Another thing to be

considered is loss of product during processing, Merten et al (2010) describe how yield can

be affected severely through purification and concentration. The use of tangential flow filters

for diafiltration and concentration can yield around 70-80% of the un-concentrated particles.

While purification through ion exchange chromatography further decreases this to 30-50%.

Additional decrease from size chromatography creates a yield of approximately 15%.

Anything produced with the intention of being used in clinical trials requires testing, usually

carried out by the FDA, therefor samples will need to be taken for sterility testing. If testing

is conducted by the FDA then this will most likely be the first, last and every tenth vial. As

well as losing product through discarding excess material in partially used vials, how

19 | P a g e

efficiently the vector is vialed needs to be considered to minimize loss of product. Therefore

any losses should be calculated into vector amount needed:

Production volume (ml) = X(IU) / yield (%) x un-concentrated IU/mL x (estimated vialing

loss)

All of these calculations must be taken into account in order to determine the viability of

vector production. Though the most expensive part of the process is the development of the

new vector and getting the vector through clinical trials, once a new treatment has been

established then manufacturing should be at a low enough cost that production and

distribution are achievable. These costs need to be justified by how many people would

benefit and the level of how beneficial the treatment actually is. Things such as the social

value of treatment, the amount of costs saved and gain of quality of life should be considered.

For large-scale production the costs of producing and delivering the product will be of

concern to companies manufacturing the product, and the number of patients receiving it will

have a large impact. As CF is one of the most prevalent inherited diseases among Caucasian

populations then the number of people that would benefit from successful treatment would be

vast and would greatly justify many costs.

8.2 Large-scale drug production

As an alternative to gene therapy, drug therapy has shown much promise and has already

been in use in the US, it also gives an idea as to how achievable production of such needed

treatment can be. The large-scale production of Ivacaftor has been under way for the last 3

years as the drug has been administered and in use in the US for over 24 months (Dr. Bhatt,

2015). However there has been a lot of debate of the pricing of the drug and whether it is

financially available to all those who would benefit from its effects. Ivacaftor was produced

by Vertex Pharmaceuticals and during its development the company received $75 million of

funding from the Cystic Fibrosis Foundation (Babaian, 2014). Once the drug had been

approved and released for use then the annual cost per patient was estimated as $294,000

(Condren and Bradshaw, 2013; Babaian, 2014). As the medical care system in the US differs

from that here in the UK, commercially insured patients were estimated to expect personal

payments of up to $88,200 per year (Condren and Bradshaw, 2013). There are schemes in

place to aid those who are unable to afford these costs and Ivacaftor is freely available to

those who are uninsured and have a household income below $15,000 per year. However,

20 | P a g e

these prices are still among the highest for any drug treatment available on the market right

now and is causing a lot of problems with the availability to patients who would drastically

benefit from this treatment. Another issue with these costs for production is that Ivacaftor is

only available for those with gating mutations which constitutes only around 4-5% of CF

sufferers and is not applicable to the F508del mutation that effects the majority of sufferers.

Therefore the target market is rather small in the eyes of the companies that produce the drug.

8.3. Comparison of gene therapy and drug therapy potential

Since the discovery of the CFTR gene, research has managed to achieve evidence of not only

proof-of-principle but has provided definitive evidence that gene transfer is achievable and

sustainable for extended periods of time. If perfected gene therapy has many advantages over

drug therapy that would make it the prime treatment for cystic fibrosis and possibly many

more genetic diseases. As the therapy targets the initial cause of the disease, correction would

eliminate all secondary effects and symptoms. Treatment sufferers currently have to

undertake and all the medication they currently depend on for correct digestion and other

organ functioning, would not be required. This would lower treatment costs and balance the

cost needed to produce the vectors. Also, though the gene therapy treatment is not as easily

administered as drug therapy, it is not considered invasive as it does not harm the patient.

Additionally, as expression can now be achieved for extended periods of time, the gap in-

between treatments could be up to around 3 months. As CF sufferers already attend hospital

check-ups at 3 month intervals, this would not cause any extra inconvenience to the patients.

Also, in between visits no other treatment or medication would be needed so they could carry

out ‘normal’ lives without any complications.

The cost of this drug therapy is proving to be an important issue and additional medication

which patients require to aid digestion still needs to be taken, meaning the cost of the drug

treatment is just additional cost to that already required to treat patients. This means that not

only do patients require the medication already taken they would now need to take two

additional tablets each day. The administration process of drug therapy is simpler than that of

gene therapy as it is just a small tablet taken orally and administration needs to be taken every

twelve hours. Effective taking of the required medication could be effected by the patient’s

life style and would require a lot of maintenance. Additionally the drug therapy currently

available to the public only addresses the minor mutation group which effects gating function

21 | P a g e

of the CFTR. Development of the drug therapy which addresses the more prevalent F508del

has yet to reach the same point and is still under development so is still around the same

stages of gene therapy. This means that there is only a small number of people who benefit

from this treatment compared to the number of people who would benefit from gene therapy

which would correct not only the major mutations but also the extremely rare ones which

may not have been studied effectively yet.

Drug therapy has been proven to be an extremely revolutionary and effective treatment and a

great example of ‘personalised medicine’, it still has many hurdles to overcome. Particularly

the cost of production and the range of mutations targeted. In comparison, gene therapy has a

much higher potential for being effective at treating a larger range of sufferers of not only

cystic fibrosis but once perfected it could also be applied to many other diseases. Once

available for clinical use it also has much higher prospects for being cost effective as it would

eradicate all other treatments currently needed to address secondary symptoms. Therefore,

when looking at evidence it may be thought that there is more potential for gene therapy.

9. Discussion and conclusion.

At this point in time there is no cure for cystic fibrosis and though drug therapy has been

developed to correct functioning of the CFTR of a small population of sufferers it is proving

to be rather unfeasible for a permanent solution. Therefore it is still highly important that

research continues into developing gene therapy as it has proven to have incredible benefits if

perfected. The most effective and prospectively viable vector under development at the

moment is the lentivirus which has proven to be able to sustain expression for extended

periods of time. It has been proven that it is able to be developed to evade an immune

response and through pseudotyping it is able to be produced at effective titers. As well as

effectively transducing the correct target cells and hopefully progenitor cells. This has

resulted in effective expression of the CFTR protein which has been estimated to be required

for reversal of CF mutation effects, as well as sustained expression. Additionally its ability to

be re-administered demonstrates a high level of potential for future use as this is required for

life-long treatment of patients and has so far proven safe to use with no adverse effects being

associated with its use.

From the evidence that past studies have demonstrated it seems that the most success has

come from HIV-1 vectors pseudotyped with either F/HN or VSV-G with the addition of LPC

22 | P a g e

pre-treatment. This is due to these models demonstrating effective long-term expression at

elevated levels without incurring adverse effects. Therefore future studies should aim at

developing these further and pushing forward into developing them for clinical use so their

effectiveness and safety can be assessed in human subjects. Also, evidence suggests that

further research into targeting progenitor cells could prove very beneficial in life-long

sustained expression. Success in these areas could witness the development of the first

successful and sustainable curative therapy for Cystic Fibrosis.

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Appendix

Year Findings Paper

1989 CFTR gene first cloned. Roirdan et al, 1989

1990 Correction of cAMP-mediated chloride

conductance in vitro shows proof-of-principle

for retrovirus-mediated gene transfer.

Drumm et al, 1990

1992 Ad-mediated CFTR cDNA transferred into

cotton rats showed evidence of successful

CFTR mRNA and protein expression.

Rosenfeld et al, 1992

1993 Partial correction of chloride transport in

tracheal epithelium achieved from non-viral

CFTR cDNA transfer in CF knockout mice.

Hyde et al, 1993

1993 First CF gene therapy trial conducted in small

no placebo controlled test with three patients.

Zaber et al 1993

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Partial restoration of cAMP-mediated chloride

transport found from first-generation adenoviral

vector administered to nasal epithelium.

1994 First Phase I dose-escalation gene therapy study

conducted. Aimed at evaluating safety,

inflammatory response was witnessed at 5x109

plaque forming units (PFU/patient).

Crystal et al, 1994

1995 First evidence of partial correction of cAMP-

mediated chloride transport achieved from non-

viral GTA complexed with CFTR cDNA in

nasal epithelium of CF patients.

Caplen et al, 1995

1999 Non-viral GA (GL^&A) complex with CFTR

cDNA demonstrated partial correction of

cAMP-mediated chloride transport in the lungs.

This study remains the only study to assess

CFTR transfer and function in the lower lungs.

Alton et al, 1999

Table 1. Important miles stones which have occurred since the discovery of the CFTR gene

and feature studies which have contributed to the development of gene therapy treatment for

CF between 1993 and 1999.

Country Incidence at birth Common mutations

Ireland 1 in 1,800 F508del, G551D, R117H

France 1 in 2,350 F508del, G542X, 711+1G-

>T

Italy 1 in 2,438 F508del, N1303K, R1162X

Chez Republic 1 in 2,833 F508del, CFTRdel2,3,

G551D

Germany 1 in 3,300 F508del, R553X, N1303K,

G542X

Spain 1 in 3,500 F508del, G542X,

1811+1.6Kb A->G

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Greece 1 in 3,500 F508del, G542X, L346P

Netherlands 1 in 3,650 F508del, A455E, 1717-1G-

>A

Denmark 1 in 4,700 F508del, 394delTT,

N1303K

Russian federation 1 in 4,900 F508del, CFTRlele2,3,

N1303K

UK and northern island 1 in 5,350 F508del, G551D, R117H

Sweden 1 in 7,300 F508del, 394dellT,

3659delC

Finland 1 in 25,000 F508del, 394dellT, G542X

Table 2. European countries with known CF incidence rate at birth with corresponding

common mutations within each country. (WHO Report: The molecular genetic epidemiology

of cystic fibrosis (2004)).

Class Effect on CFTR

protein

Molecular defect Example of mutation % CF

patients

(Europe)

I Shortened protein,

no CFTR protein

expressed

Nonsense, mutations or

deletions cause

premature stop codons

(PTC),

W1282X instead of

inserting the amino

acid tryptophan (W),

the protein sequence is

prematurely stopped

(indicated by an X)

7

II Protein fails to

reach cell

membrane

Some missense

mutations and in-frame

deletions disrupt CFTR

∆F508. A

phenylalanine amino

acid (F) is deleted;

85

34 | P a g e

protein folding and

trafficking to the surface

N1303K

III Channel cannot be

regulated properly,

‘gating defect’.

Missense mutations

result in disrupted

regulation so CFTR

channel no longer opens

in response to channel

agonist

G551D: a ‘missense’

mutation: instead of a

glycine amino acid

(G), aspartate (D) is

added.

<3

IV Reduced chloride

conductance,

‘conductance

defect.

Missense mutations

result in changes to

CFTR protein structure

that forms the channel

pore.

R117H, R347P,

R334W.

<3

V Reduced amounts

of normal CFTR

protein being

synthesized and

therefore less on

cell surface.

Missense mutations that

result in alternative

splicing, disrupting

mRNA processing by

originating both

alternative and normal

transcripts, albeit the

latter in minor amounts.

3120+1G>: a splice-

site mutation in gene

intron 16.

<3

Table 3. Class I, II and III mutations generally lead to complete loss of function and a more

severe disease. Class IV and V cause a reduction in function and have milder effect. Two

major groups, those which reduce or prevent CFTR production and those which reduce or

prevent CFTR function. (Adapted from table found at:

http://www.nchpeg.org/nutrition/index.php?

option=com_content&view=article&id=462&Itemid=564&limitstart=4).

35 | P a g e

Appendix 2: Transcript of an interview conducted on the 06/01/2015 at Nottingham

Children’s Hospital. The interview was between Elizabeth Dodd and Dr. Jayesh Bhatt,

MBBS, MD, DCH, FRCPCH. Also present was the mother of a CF sufferer who attends

regular check-ups at Nottingham Children’s Hospital, and shall remain anonymous, referred

to in this transcript as mother x and child x. Interview transcript:

Elizabeth: Are there any mutations you personally see more often, or which stand out

significantly?

Dr.Bhatt: More than 10500 people in this country have cystic fibrosis and more than 90%

have a copy of the common one a deletion at 508. 70% will have 2 copies of it, so that is the

commonest.

Elizabeth: Have you been involved in or know of any research studies that appear to be

particularly promising?

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Dr.Bhatt: We are quite active in research as a unit and there is one study we are currently

involved in which is looking at the deletion effect of the commonest mutation, 508, and

whether you can improve the function of the protein CFTR by use of these two drugs. So you

get more CFTR up to the top of the cell and keep it open for longer. That study has happened

and we are waiting to see if it will get funded, and because Child x has one copy of the 508

and one copy which is not that common. And it’s a gene defect which is only effecting

around 5 in 100 people with CF in this country. We’ve been using this drug for a good year

and a half and it is amazing, it’s superb the response we’ve had. So there a few steps to take

before we look at those subjects for the other gene effects but it’s very promising.

Mother x: If you have something that works on quite an unusual gene defect, how difficult is

it to make it work for the more common one?

Dr.Bhatt: So the one that already works they’ve been using for about 18 months or so and it

is the type with the faulty gene which gets the protein to the top of the cell but doesn’t keep it

open for long, so in that way it works very well for the common one, in which not enough

protein gets properly formed and is dropped down before it gets to the top of the cell. So this

combination treatment is trying to get more to the surface. The principle can be applied to

any gene defect but it takes a long time for the research.

Elizabeth: Do you have any thoughts on gene therapy as a treatment?

Dr.Bhatt: We keep hearing that the research has happened. The thing about gene therapy is

that is that there is something that you need to inhale, the other treatment I was talking about

that’s tablet intake. Gene therapy you have to take every month, this tablet is every day. So

gene therapy they’ve given to people who are over 12 and in that instance it doesn’t matter

which gene defect you have, common or uncommon. You have gene therapy every month;

they’ve done 12 doses and are waiting to hear the results of it. So the work being done shows

that you can give it and it appears to be well tolerated. Now this will tell us whether it works

or not. If it works then obviously it needs to go through licencing and processing.

Elizabeth: Do you think drug therapy is more effective and more promising then?

Dr.Bhatt: Well what I would say is that it is the more advanced in stages than gene therapy

but until we hear the results from gene therapy then we can’t really say.

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Elizabeth: Some of the most significant gene therapy studies in the past have used HIV-

based vectors. Do you think the public may have any negative attitudes towards this and have

you ever encountered any opposition?

Dr.Bhatt: I think you’d need to ask the public. In terms of virus, and Americans not using

viral vectors but liposomes, so the gene is packaged in fat and inhaled. As naturally with viral

vectors being inhaled the body will fight so it is much easier than the viruses they were using.

There’s a new type of virus they were using that they’re looking at called lentivirus, so

hopefully the body doesn’t fight the virus as much and you can still deliver the gene.

Mother x: So your research here is that mainly drug based?

Dr.Bhatt: Yes

Mother x: Is that financial; is it cheaper than gene therapy?

Dr.Bhatt: Well we don’t know what gene therapy is going to cost in the long run. But the

type of drug we’ve been using, at the moment on about 400 people in the country, the cost of

that is about £500 a day. That’s indefinite, lifelong. That’s only 400 people in the country so

imagine if you start using it in the common gene defect which is looking at about 5,000 with

that gene defect but you can only use that drug on people above 6. So that’s about 3,500. If

the two drugs were to become available at the price that the one drug is, then the budget

would be £9 billion for the NHS. So there has to be negotiation. So that’s what stage it’s at,

it’s called ivacaftor. With gene therapy we simply don’t know, we don’t know how effective

it is. Secondly, if it is effective it has to go through the licencing process and then the pricing

mechanism, though it’s likely to be high.

Mother x: I’ve been to a couple of seminars at stoke and they are really please that their

adult clinic is the same size as their children’s clinic. Which is only testament to how well the

treatment is going, because Daniel was diagnosed with heel prick tests; are you finding with

cascade screening that you’re getting less children coming through? Are we screening out,

are people deciding not to have children with CF or are there the same numbers coming

through?

Dr.Bhatt: What we don’t screen for is carrier status. In Italy they do, and what they’ve found

in Italy is that population screening of carriers has led to decline in the prevalence of cf, but

that is not the case in the UK because we don’t screen for carriers we screen for disease.

38 | P a g e

Mother x: But you could have cascade screening if you wanted to?

Dr.Bhatt: Yes but that’s different and is decided as an adult. It may pick up some carriers but

it is by chance. But when looking at cf statistics for every year for some reason, maybe a

statistical quirk. Every year, roughly there is about 150 babies diagnosed by heel prick

screening and in 2013 there was only 150. But I think we need to wait and see because it may

be a statistical quirk, for the first time in 2007 there were more adults with cf than children,

so as paediatricians we need to pat ourselves on the back that the change happened at that

time.

Elizabeth: When it comes to research, how do you pick which patients go forward?

Dr.Bhatt: So obviously if it is gene related illnesses then ultimately you have to have the

gene type. Then there is a long list of inclusion and exclusion criteria to meet. Majority of the

time there is age, age is important because you need to be able to measure an improvement

and that is done by lung function. For cf it is people of 12 that we start recruiting, that’s what

gene therapy did as well. Can you measure? In babies you need to put them to sleep while

with adults you can cough up sputum to analyse.

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