The impact of processing on the biophysical properties of synthetic DNA complexes for gene delivery

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THE IMPACT OF PROCESSING ON THE

BIOPHYSICAL PROPERTIES OF SYNTHETIC

DNA COMPLEXES FOR GENE DELIVERY

A thesis submitted for the degree of Doctor of Philosophy

by

Claire Nicole Mount

DEPARTMENT OF BIOCHEMICAL ENGINEERING

UNIVERSITY COLLEGE LONDON

January 2003

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A bstract

Abstract

Research is progressing fast to find safe and effective methods of delivering genes as

therapeutic agents. A technical barrier that remains relates to the need for large-scale

manufacturing process strategies and process optimisation. The present investigation

provides a means of assessing the impact of the formulation process and material

factors on the physical properties of plasmid-DNA vector. Response surface

methodology (RSM) is combined with experimental techniques to provide new insight

into scaleable processing of these systems.

To assess the influence of process and material factors on the physical stability of gene

complexes two techniques, photon correlation spectrophotometry (PCS) and laser

Doppler velocimetry (LDV) were used to characterise DNA complexes. A model

formulation was used consisting of ctDNA or pDNA MB 113 condensed by poly-L-

lysine (SOKDa) and a lipopolypeptide formulation, LID, prepared through

complexation of DNA with cationic lipid and peptide components.

Preliminary experiments showed several individual factors have important effects on

the physical stability of synthetic delivery systems; including the mean size and charge

of plasmid DNA molecules condensed by cationic agents. These factors include

physico-chemical properties of the buffer such as the pH and ionic strength as well as

conditions used to prepare the complexes, for example the method and intensity of

mixing the components of the complex. Using RSM to analyse experimental data it is

shown that the impact of these factors and the effects of their interactions on the

physical properties of the complexes are time-dependent. More specifically, for

plasmid DNA condensed by poly-L-lysine or cationic lipids, interactions between ionic

strength, pH and DNA concentration play a critical role. W hether poly-L-lysine should

be used as a condensing agent in the final delivery system or the LID formulation can

become clinically and commercially viable remains to be demonstrated. However, the

use of RSM combined with the scaleable experimental approach described here

represent a rapid and cost effect process tool that may be applied to any delivery

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Table o f Contents

List of Figures

Figure 1.1 Production of Plasmid DNA... 19

Figure 2.1 Gene Delivery...28

Figure 2.2 DNA Conformation...36

Figure 2.3 Non-viral DNA Delivery... 43

Figure 2.4 Synthetic Gene Delivery Systems... 50

Figure 2.5 Poly-L-lysine Condensed DNA...53

Figure 2.6 Formation of Lipopolypeptide Particles... 64

Figure 2.7 Antibody Fragments...65

Figure 2.8 Virosome Construction... 67

Figure 3.1 Poly-L-lysine Hydrobromide Chemical Structure...75

Figure 3.2 Hydrophilic Amino Acid Spacer... 76

Figure 3.3 DOTMA Chemical Structure... 76

Figure 3.4 DOPE Chemical Structure...76

Figure 3.5 Twin Syringe Pump Mixing Device... 78

Figure 3.6 Zetasizer 3000... 79

Figure 3.7 Variation in the Method of Collection... 81

Figure 4.1 A typical size distribution of stable DNA complexes... 92

Figure 4.2 Average particle size of ctDNA/PLL 29300 complexes as a function of DNA concentration...94

Figure 4.3 Size profile of ctDNA/PLL 29300 complexes as a function of DNA concentration 95 Figure 4.4 Size profile of pDNA MB 113/PLL 29300 complexes prepared in 20mM HEPES, as a function of pDNA concentration... 98

Figure 4.5 Size profile of pDNA MB 113/PLL 29300 complexes prepared in ultra-pure water, as a function of pDNA concentration...99

Figure 4.6 Typical size distribution profiles for pDNA complexes suspended in HEPES buffer, as a function of time...100

Figure 4.7 Typical size distribution profiles of pDNA complexes suspended in ultra-pure water, as a function of time...101

Figure 4.8 Average particle size of ctDNA/PLL 29300 complexes prepared at a DNA concentration of 50pgml ', as a function of storage temperature... 104

Figure 4.9 Average particle size of ctDNA/PLL 29300 complexes prepared at a DNA concentration of 200pgml ' as a function of storage temperature...105

Figure 4.10 Typical size distribution profiles for complexes prepared at a ctDNA concentration of 200p.gml ‘ and stored at room temperature, as a function of time...106

Figure 4.11 Typical size distribution profiles for complexes prepared at a ctDNA concentration of 200|Ligmr* and stored at 4°C, as a function of time... 107

Figure 5.1 Average particle size of ctDNA/PLL 29300 complexes as a function of collection method and volume...112

Figure 5.2 Average particle size of ctDNA/PLL 29300 complexes prepared using a tube internal diameter of 0.8mm, as a function of mixing flow rate...115

Figure 5.3 Typical size distribution profiles for complexes prepared using a tube internal diameter of 0.8mm as a function of mixing flow rate... 116

Figure 5.4 Average particle size of ctDNA/PLL 29300 complexes prepared using a tube internal diameter of 1.6mm, as a function of mixing flow rate...117

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List o f Figures

Figure 5.6 Average particle size of ctDNA/PLL 29300 complexes prepared using a tube internal

diameter of 3.2mm as a function of mixing flow rate... 118

Figure 5.7 Typical size distribution profiles for complexes prepared using a tube internal diameter

of 3.2mm, as a function of mixing flow rate... 118

Figure 5.8 Average zeta potential of ctDNA/PLL complexes prepared using a tube internal

diameter of 0.8mm as a function of mixing flow rate... 119

diameter of 1.6mm, as a function of mixing flow rate... 120

diameter of 3.2mm, as a function of mixing flow rate... 120

Figure 5.11 Size distribution profiles for DNA complexes prepared at a mixing flow rate of

60mlmin ' using a tube internal diameter of 1.6mm, as a function of mixing tube length... 122

40mlmin'' using a tube internal diameter of 1.6mm, as a function of mixing tube length... 123

20mlmin'^ using a tube internal diameter of 1.6mm, as a function of mixing tube length... 124

20mlmin * using a tube internal diameter of 0.8mm, as a function of mixing tube length... 125

60mlmin'* using a tube internal diameter of 3.2mm, as a function of mixing tube length... 126

Figure 5.16 Average particle size of ctDNA/PLL 29300 complexes prepared at varied mixing flow

rate and tube internal diameter, as a function of residence time... 127

Figure 6.1 Typical half normal plot generated using Design-Expert5®... 141

Figure 6.2. Effects graph showing the influence of factor E (pH) on the particle size of pDNA/PLL

complexes immediately after preparation... 142

Figure 6.3 Interaction graph showing the effect of an interaction between factors E, pH, and F,

pDNA concentration, on the particle size of pDNA/PLL complexes immediately after preparation...143

Figure 6.4 Effects graph for the effect of factor F, pDNA concentration, on the particle size of

pDNA/PLL complexes one week after preparation... 145

Figure 6.5 Interaction graph showing the effect of an interaction between factors A, mixing flow

rate, and factor B, mixing tube length on the particle size of pDNA/PLL complexes two weeks after preparation... 146

Figure 6.6 Interaction graph showing the effect of an interaction between factor A, mixing flow

rate and factor F, pDNA concentration on the size of pDNA/PLL complexes two weeks after preparation... 147

Figure 6.7 Effect graph showing the effect of pDNA concentration on the zeta potential of

pDNA/PLL complexes measured immediately after mixing... 148

Figure 6.8 Interaction graph demonstrating the effect of an interaction between factor A, mixing

flow rate and factor B, mixing tube length, on the zeta potential of pDNA/PLL

complexes immediately after preparation... 149

Figure 6.9 Interaction graph showing the effect of an interaction between factor D, tube material,

and factor F, pDNA concentration on the zeta potential of pDNA/PLL complexes one week after preparation...151

Figure 6.10 Effect graph showing the influence of factor F, pDNA concentration, on the zeta

potential of pDNA/PLL complexes after two weeks... 151

Figure 6.11 Interaction graph showing the effect of an interaction between factor D, tube material.

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List o f Figures

and factor F, pDNA concentration on the leta potential of pDNA/PLL complexes after two weeks... 152

Figure 6.12 Perturbation plots showing the effect of the three major factors, mixing flow rate (A),

pH (B) and pDNA concentration (C), on tie responses of pDNA/PLL complexes 155

Figure 6.13 Response surface plots illustrating the effect of varying mixing flow rate and pH on the

particle size of pDNA/PLL complexes immediately after preparation...158

Figure 6.14 Response surface plots illustrating the effect of varying pH and mixing flow rate on the

particle size of pDNA/PLL complexes one week after preparation... 160

Figure 6.15 Response surface plots illustrating the effect of varying the mixing flow rate and pDNA

concentration on the particle size of pDNA/PLL complexes two weeks after

preparation... 163

Figure 6.16 Response surface plots illustrating the effect of varying mixing flow rate and pH on the

zeta potential of pDNA/PLL complexes immediately after complex preparation 165

Figure 6.17 Response surface plots illustrating the effect of varying mixing flow rate and pDNA

concentration on the zeta potential of pDNA/PLL complexes one week after

preparation...167

Figure 6.18 Response surface plots illustrating the effect of varying pH and pDNA concentration on

the zeta potential of pDNA/PLL complexes two weeks after preparation...168

Figure 6.19 Experimental data versus predicted values for the particle size of pDNA/PLL complexes

at time zero as a function of pDNA concentration, pH and mixing flow rate...171

one week after preparation as a function of pDNA concentration, pH and mixing flow rate... 171

two weeks after preparation as a function of pDNA concentration, pH and mixing flow rate... 172

Figure 6.22 Experimental data versus predicted values for the zeta potential of pDNA/PLL

complexes at time zero as a function of pDNA concentration, pH and mixing flow rate... 172

complexes one week after preparation as a function of pDNA concentration, pH and mixing flow rate... 173

complexes two week after preparation as a function of pDNA concentration, pH and mixing flow rate... 173

Figure 6.25 Effects graph for the effect of factor G, NaCl concentration, on the particle size of

pDNA/PLL complexes immediately after preparation... 179

Figure 6.26 Interaction graph showing the effect of an interaction between factors A, pDNA

concentration and G, NaCl concentration on the particle size of pDNA/PLL complexes immediately after preparation...180

Figure 6.27 Effect graph showing the influence of factor A, pDNA concentration, on the particle

size of pDNA/PLL complexes thirty minutes after preparation... 181

Figure 6.28 Effect graph showing the influence of factor G, NaCl concentration, on the particle size

of pDNA/PLL complexes thirty minutes after preparation... 182

Figure 6.29 Interaction graph showing the effect of an interaction between factors A, pDNA

concentration and G, NaCl concentration on the zeta potential of pDNA/PLL

complexes... 184

Figure 6.30 Size profile of pDNA/PLL complexes prepared at a pDNA concentration of 200pgm l'\

in 20mM HEPES pH6.5, OmM NaCl, using a mixing flow rate of 60mlmin ', and PharMed tubing with an internal diameter o f 1.6mm and a mixing tube length of 4cm...187

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List o f Figures

in 20mM HEPES pH8.5, 150mM NaCl, using a mixing flow rate of 60m lm in'\ and PharMed tubing with an internal diameter of 1.6mm and a mixing tube length of 4cm...187

Figure 6.32 Size profile of pDNA/PLL complexes prepared at a pDNA concentration of 200pgml"\

in 20mM HEPES pH6.5, 150mM NaCl, using a mixing flow rate of 60mlmin ', and PharMed tubing with an internal diameter of 1.6mm and a mixing tube length of 4cm ...188

Figure 6.33 Perturbation plots showing the effect of the four major factors, pDNA concentration

(A), NaCl concentration (B), pH (C) and mixing tube length (D) on the responses of pDNA MB 113/PLL complexes...189

Figure 6.34 Response surface plot illustrating the effect of varying pDNA and NaCl concentration

on the particle size of pDNA/PLL complexes immediately after preparation... 191

Figure 6.35 Response surface plots illustrating the effect of varying mixing tube length and pH on

the particle size of pDNA/PLL complexes immediately after preparation...192

the particle size of pDNA/PLL complexes immediately after preparation...194

on the particle size of pDNA/PLL complexes thirty minutes after preparation... 195

the particle size of pDNA/PLL complexes thirty minutes after preparation...197

on the zeta potential of pDNA/PLL complexes... 198

the zeta potential of pDNA/PLL complexes... 200

at time zero as a function of NaCl concentration, pDNA concentration, pH and mixing tube length...202

thirty minutes after preparation as a function of NaCl concentration, pDNA

concentration, pH and mixing tube length... 202

complexes as a function of NaCl concentration, pDNA concentration, pH and mixing tube length...203

Figure 7.1 Typical half normal plot generated using Design-Fxpert5®...221

Figure 7.2 Effects graph for the effect of factor G, NaCl concentration, on the size of LID

complexes immediately after preparation at time zero... 222

Figure 7.3 Effects graph for the effect of factor F, Tube Internal Diameter (ID), on the size of LID

complexes thirty minutes after preparation... 223

Figure 7.4 Effects graph for the effect of factor G, NaCl concentration, on the size of LID

complexes thirty minutes after preparation... 224

Figure 7.5 Effects graph for the effect of factor A, pDNA concentration, on the zeta potential of

LID complexes... 225

Figure 7.6 Effects graph for the effect of factor B, pH, on the zeta potential of LID

complexes...225

Figure 7.7 Effects graph for the effect of factor G, NaCl concentration on the zeta potential of LID

complexes...226

Figure 7.8 Size profile of LID complexes prepared at a pDNA concentration of 10^gm l'\ in 20mM

HEPES pH6, OmM NaCl, using tubing with an internal diameter of 0.8mm... 228

Figure 7.9 Size profile of LID complexes prepared at a pDNA concentration of I50|lgml ', in

20mM HEPES pH6, I50mM NaCl, using tubing with an internal diameter of

3.2mm... 229

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List o f Figures

Figure 7.10 Perturbation plots showing the effect of the four major factors, pDNA concentration

(A), NaCl concentration (B), pH (C) and tube internal diameter (D) on the responses of LID complexes...231

on the particle size of LID complexes immediately after preparation at time zero 233

on the particle size of LID complexes thirty minutes after preparation...234

on the zeta potential of LID complexes... 236

Figure 7.14 Experimental data versus predicted values for the particle size of LID complexes at time

zero... 237

Figure 7.15 Experimental data versus predicted values for the particle size of LID complexes thirty

minutes after preparation...238

Figure 7.16 Experimental data versus predicted values for the zeta potential of LID

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List o f Tables

List of Tables

Table 3.1 Comparison of Tubing Material... 82

Table 3.2 Six Factor Half-Factorial Experiment: Factors and Levels...86

Table 3.3 Three Factor RSM Experiment: Factors and Levels...86

Table 3.4 Seven Factor Quarter-Factorial Experiment: Factors and Levels... 87

Table 3.5 Four Factor RSM Experiment: Factors and Levels...87

Table 3.6 Seven Factor 1/16-Factorial Experiment: Factors and Levels... 88

Table 3.7 Four Factor RSM Experiment: Factors and Levels...88

Table 6.1 Design-Expert 5® Statistical Analysis Tables... 138

Table 6.2 Three-Factor RSM: Numerical Optimisation... 175

Table 6.3 Four-Factor RSM: Numerical Optimisation... 205

Table 7.1 Design-Expert 5® Statistical Analysis Tables...219

Table 7.2 Four-Factor RSM: Numerical Optimisation... 239

Table A l.l Some Physical Properties of Formulation Materials... 250

Table AlO.l Factorial Experiment 1 - Screen: Six-Factors...268

Table A10.2 Factorial Experiment 2 - D-Optimal RSM Design: Three-Factors...269

Table A10.3 Factorial Experiment 3 - Screen: Seven-Factors... 270

Table A 10.4 Factorial Experiment 4 - D-Optimal RSM Design: Four-Factors...271

Table A10.5 Factorial Experiment 5 - Screen: Seven-Factors... 272

Table A10.6 Factorial Experiment 6 - D-Optimal RSM Design: Four-Factors... 272

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A bstract

Acknowledgments

I would like to thank my supervisor Professor Parviz Shamlou for his invaluable

guidance and advice and for the great enthusiasm for the development of the project.

Additionally my supervisor Dr. Ahmed Yasin of GlaxoSmithKline (GSK) has

provided important help and advice for which I am grateful.

I gratefully acknowledge the support of GlaxoSmithKline and the Biotechnology and

Biological Sciences Research Council.

I also wish to thank Andrew Scott (GSK) and Professor Tom Feam (UCL) for their

assistance with statistical design techniques.

I wish to acknowledge LiKim Lee for all the help and endless patience she gave

throughout my time at UCL. Also, I would like to thank Supti Sarkar for her friendship

and help.

I would like to thank all the people who have made the past few years enjoyable

including everyone in the department of Biochemical Engineering, particularly those in

the Colonnades Office and Foster Court. But the biggest thanks goes to Jobin, Nicky

and Tone for the many entertaining lunch and coffee breaks and essential after school

activities.

Finally a very big and very special thank you to the people who have helped me

personally, including Kirstie, my brother Jon, Jobin, Nicky, Tone and Raju, without

whom I would not have made it through. Most of all I would like to thank my parents,

Julie and Len, who, without exception, have been there with huge amounts of love and

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Chapter 1. Aims and Objectives

This project has focused on two main areas of research, processing and physical

stability of plasmid DNA complexes, in order to aid the development of clinically

effective non-viral gene delivery products. The genomics revolution is providing an

increasing range of information on genes and their relationship to diseases rendering

more and more disease amenable to gene delivery (Mountain, 2000). In response gene

delivery research is advancing rapidly from concept to clinic. However a great deal of

work is still required to overcome the real concerns over the ability of DNA to be

delivered intact to the nuclei of target cells (BBSRC business, July 2000). It is believed

that colloidal properties of DNA complexes plays a key role in the capacity to deliver

genes to cells and has an impact on gene expression. It is therefore important to

understand the complex interaction of factors that control these properties in order that

they may be manipulated to create the gene delivery system with desired

characteristics (Smyth Templeton and Lasic, 1999). The aim of this investigation is to

study the impact of formulation processing and scale-up on the physical stability of

gene complexes using statistical design techniques.

1.1 Vector Production

If non-viral gene delivery proves to be clinically effective the enormous potential of

non-viral vectors will only be realised if they can be manufactured in a commercially

acceptable form that is amenable to scale up (Anchordoquy et a l, 2001, Bally et a l.

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Chapter 1.________________________________________________________________ Aim s and Obiectives

1999; Davis, 2002; Marquet et a l, 1995). Biophysical characteristics of non-viral

DNA formulations, such as particle size and zeta potential are known to affect gene

delivery efficiency but attempts to relate attributes of the vector formulations has

proved very difficult as changes in one component often effects more than one

biophysical attribute of the resultant vector (Anchordoquy et a l, 2001; Bally et a l,

1999). For gene delivery products to be successful the vectors will need to retain

constant physical and chemical properties when manufactured at scale. The interaction

of formulation variables complicates manufacturing scale up and current non-viral

vectors are therefore made in relatively small batches by mixing a solution of DNA

with a suspension of complexing agent (lipid or polymer). This small-scale method of

preparation results in highly heterogeneous suspensions of particles that possess a wide

range of charge ratios and particle sizes (Anchordoquy et a l, 2001). For the long term

success of non-viral gene delivery techniques it is essential that scalable techniques for

the manufacture of stable and predictable products be developed.

The investigation detailed in this report examined a scaleable technique of

manufacturing stable non-viral gene delivery products. The whole process of

producing a gene delivery product involves many steps and this investigation focuses

on formulation, the final stage of the process (Figure 1.1). To circumvent the problems

created when using the small-scale laboratory based method of preparation, which

employs simple manual techniques of mixing, and avoid the concerns with respect to

the order of addition of components a semi-automated twin syringe pump mixing

device was used to prepare the DNA complexes (Figure 3.5) (Lee and Huang, 1997;

Zelphati et a l, 1998). The impact of scale up of the automated mixing method to

produce industrial scale quantities of DNA complexes was studied by varying key

processing variables such as flow rate, flow characteristics and residence time. The

results of this investigation demonstrated the capacity of a scaleable automated method

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Chapter 1._________________________________________________________________Aim s and Obiectives

1.2 Physical Stability

Following manufacture it is imperative that the biological properties and gene delivery

efficiency of non-viral vectors can be preserved. This requires a thorough

understanding of the factors affecting these properties (Anchordoquy et a l, 2001;

Florence and Hussain, 2001). For in vivo studies and clinical trials it is essential that

the complexes are homogeneous and do not aggregate at high concentrations, but to

achieve this has proved problematic (Davis, 2002). As yet no non-viral gene delivery

system has demonstrated therapeutic levels of gene expression in human clinical trials

and significant variations in product quality and gene delivery efficiency has

highlighted the need for further research. The main focus has been on the improvement

of transfection efficiency, however, this has meant that crucial pharmaceutical aspects,

such as stability, have been ignored. If preparations become unstable after production

the quality control and the transfection rates, which are known to plague clinical gene

therapy trials, will be adversely affected. It is well known that aqueous suspensions of

non-viral vectors have a tendency to aggregate over time, particularly under conditions

of interest such as physiological conditions, making the ideal vector characteristics

very difficult to achieve (Anchordoquy et a l, 2001; Lee et a l, 2001). Post

administration aggregation in the patient may also be a problem due to the interaction

of complexes, particularly cationic lipid/DNA particles, with blood components such

as albumin. Such interactions may reduce the zeta potential of the complexes, hence

reducing the charge repulsion between particles (Li et a l, 1999; Pouton and Seymour,

1998). There are a number of formulation variables known to aggravate the problem of

instability including the type of cationic lipid used, the type and quality of additional

lipids used to prepare the formulation, composition of diluents and buffer used, method

and rate of mixing, temperature at mixing, DNA purity and the length of time after

complex formation (Bally et a l, 1999).

The aggregation problems clearly affects the quality of gene delivery formulations and

the significant variations in product quality and gene delivery efficiency have

stimulated interest in developing stable synthetic vectors that resist aggregation

(Anchordoquy et a l, 2001). Although recent formulations claim to have improved

stability the shelf life is typically measured in hours or days rather than the eighteen

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Chapter 1.________________________________________________________________ Aim s and Obiectives

months to two-year time frame required for marketable pharaiaceutical products

(Anchordoquy et a l, 2001). To circumvent the aggregation problem a number of

solutions have been under development but aggregation is extremely difficult to

control and as yet no method has provided DNA complexes with the level of

consistency required to achieve long term stability (Bally et a l, 1999). Surfactants are

often added to enhance the stability and prevent aggregation of DNA complexes.

However the addition of excipients can complicate the characterisation, toxicity and

manufacture of the delivery system (Zelphati et a l, 1998). A number of methods have

been developed to prevent the interactions among the components within a suspension

that lead to aggregation. For example Hong and Colleagues and Eastman and co

workers used PEG-lipid conjugates to sterically stabilise the particles in aqueous

suspensions thereby preventing aggregation (Barratt, 1999). If done properly, sterically

stabilised particles can be formed at concentrations exceeding milligram quantities of

DNA per millilitre (Davis, 2002). While aggregation was reduced the technique has

not been widely employed due to the fact that steric stability is also known to lower

transfection activity as it curtails interactions with cells and interferes with cellular

processing. Finally, the free uncomplexed liposomes and DNA typically contained in a

bulk suspension of DNA complexes also have a negative impact on the stability of the

complexes. The stability of non-viral DNA complexes can therefore be increased if the

complexes are isolated from the bulk suspension. However, the present separation

techniques, such as sucrose gradient electrophoresis, are non-seal able (Anchordoquy et

a l, 2001). The current methods available to prevent aggregation clearly have

disadvantages and alternative methods of minimising aggregation with minimal

interference with the constituents of the complex itself are therefore being sought.

The physical stability of nanoparticle suspensions is crucial to the success of the

synthetic gene delivery approach (Florence and Hussain, 2001). The numerous factors

affecting stability of DNA delivery products compounds the problem of isolating the

key factors that could be used to control or circumvent the aggregation problem and

the aim of this investigation was to identify factors and assess their interactions. Of the

formulation variables previously investigated, the method of mixing the plasmid DNA

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Chapter 1._________________________________________________________________Aim s and Objectives

expression in vitro and also affects lipoplex structure and stability (Ferrari et al.,

2001). Alternative methods of forming lipid-based DNA carriers in a more controlled

manner are therefore currently under development and have been used in this

investigation (Wasan et a l, 1999). A semi-automated method of preparing non-viral

DNA complexes was employed in the present investigation whilst varying both

processing variables, such as flow rate and mixing tube dimensions, and formulation

variables, such as buffer pH and DNA concentration. The degree of interaction

between the variables and the affect on the aggregative characteristics of the DNA

complexes were assessed on the basis of particle size and zeta potentials. The results of

the investigation highlighted the factors that have a significant effect on the stability of

non-viral DNA complexes, the level of interaction between the factors and allow the

identification of the optimum conditions required for the production of stable and

defined complexes.

1.3 Experimental Design

The aim of this investigation, the definition of critical parameters involved in the

production of physically stable gene delivery complexes, was achieved through the

variation of a number of factors involved in the formulation and processing of non-

viral gene delivery complexes followed by the characterisation of the resultant

complexes. The investigation utilised both classical and statistical experimental design

techniques. The classical “one factor at a tim e” (OFaT) techniques were used to gain a

basic understanding of the potentially influential factors. Subsequently this process

knowledge was used in the construction of the more complex factorial and response

surface designs. The statistical techniques were used in order to provide guidelines as

to the reliability and validity of the results and thereby add objectivity to the decision

making procedure involved in process optimisation.

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Chapter 1. Aims and Obiectives

Figure 1.1 - Position o f this investigation in relation to other aspects o f the process fo r the production o f plasmid DNA fo r gene therapy

Plate - slock culture o f E. co ll

transformed with plasmid

IS

Shake

flask Fermenter C entrifuge

N on-viral drug delivery system preparation:

prepare D N A and co m p lex in g agent solu tion s, set charge ratio, set pH environm ent, control m ixin g using syringe pump d evice

Ultra-pure Plasm id

C hrom atography C entrifuge

R

_W

Lysis

I

0

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Chapter 2. Literature Survey

-Gene Delivery: A Historical Perspective

To date very little published information on key issues related to the bioprocessing and

manufacture of gene delivery products exists. At present much research is

concentrating on scientific issues related to gene therapy. W hat follows is therefore a

historical overview of gene delivery.

2.1 Gene Delivery

Gene delivery, one of the most exciting, controversial and highly publicised areas in

biotechnology has a remarkable history and potentially an important future. The

techniques involve the transfer of foreign genes into cells of a recipient organism in

order to induce a therapeutic effect (FDA, 2000). As long ago as the mid 1950’s gene

delivery was demonstrated when cells were shown to be capable of taking up foreign

nucleic acid extracted from viruses, and express it as if it were their own (Feigner,

1997). Since, these findings have provided the incentive for scientists to harness the

potential of this technology. In the last decade gene delivery has become a major field

of research, aimed at improving the efficiency of delivery in order that it may be used

for medical and therapeutic purposes. The future of gene delivery lies in two main

areas of research, gene therapy and gene vaccines, both of which hold great promise

for patients worldwide (Feigner, 1997). The use of genetic material for treatment is

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Chapter!.____________________________________________________________________Literature Survey

theoretically a relatively simple concept and with current research, building on the vast

research that has taken place over more than a decade, gene delivery techniques have

the potential to revolutionise the management of inherited and acquired disease thereby

becoming valuable medical products for the future (Geddes and Alton, 1995; Tseng

and Huang, 1998).

2.1.1 Gene Therapy

After a decade of research gene therapy, whilst still in its infancy, has become a robust

scientific discipline, which, once reaching the market place promises to revolutionise

and reshape medicine in the same way as antibiotics have (Anchordoquy et a i, 2001;

Geddes and Alton, 1998; http://ims-global/insight, 2000; http://southflorida, 1998).

While traditional treatments for intractable genetic diseases prolong life and alleviate

suffering by curing the symptoms gene therapy aims to reduce or completely eliminate

a disease from the patient (Bally et a i 1999; FDA, 2000). The treatment relies on the

introduction of a ‘drug’ in the form of genetic material into human cells in order to

correct the deficiencies at the molecular level of DNA (Bally et a l, 1999; Brown,

1995; Deshmukh and Huang, 1995; Marquet et a l, 1995; Pappas, 1996; Pack et a l,

2000). The genetic material can be delivered to restore a defective biological function

or homeostatic mechanism. Alternatively, antisense DNA can be applied, which could

inactivate a gene or other peptides and nucleotides to treat diseases such as cancer.

The technology of DNA delivery has the advantage of being a broad platform

technology that has the potential to impact all diseases presently affecting humans,

ranging from inherited genetic diseases such as cystic fibrosis (CF), inflammatory

diseases, vascular disease, neurological disorders and cancer to acquired diseases such

as AIDS (Anchordoquy et a l, 2001; Bally et a l, 1999; Deshmukh and Huang, 1995;

Pappas, 1996; Pack et a l, 2000). The technology therefore offers hope of substantial

improvements in the therapeutic ratio and cure rates for diseases presently considered

untreatable or poorly managed (Mountain, 2000).

There are two types of gene therapy, somatic and germ line, however, due to ethical

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therapy involves the manipulation of an individual’s genetic material, adding

therapeutic copies of the defective gene to the bodies’ somatic cells. Following

expression of the inserted wild type gene the correct function of the cell restored

(Pappa et a l, 1996; Stribley et a l, 2002). However, somatic cell therapy does not cure

the disease from the population. Germ line therapy involves the same principal of

manipulation as somatic cell therapy although the target cells are those of the germ line

i.e. the cells that eventually produces gametes. Progeny of the treated individual will

therefore inherit any correction that results from the treatment thereby preventing

propagation of the disease. Germ line gene therapy has shown success in both plant

and animal models, however, at present the manipulation of germ line cells in this way

is illegal in many countries due to unresolved ethical issues regarding the unknown

consequences and potential risks of genetic engineering to future generations (Stribley

et a l, 2002). Germ line therapy, while having the potential to eradicate genetic disease

from the population is not feasible at present and somatic cell therapy is therefore the

focus of current research.

Once gene therapy technology is finalised it will provide doctors with powerful tools

with which to fight a range of conditions. The worldwide gene therapy market is

estimated to be worth $3.6 Billion by 2005 (http://southflorida, 1998). The gene

therapy industry has however suffered many setbacks during its history, which have

hindered its success. The most significant of these setbacks has been the struggle to

improve the effectiveness of the technology in order to efficiently introduce and ensure

expression of the genetic material in the targeted cells (Bally et a l, 1999;

http://www.imms-globa.com/insight, 2000). The majority of clinical trials have been

small phase FIX studies aimed at demonstrating safety, transfer of genes plus

information to guide dose selection for phase II and III efficacy studies (Mountain,

2000). Some trials are ongoing such as those for chronic granulomatous disease, brain

cancer and non-inherited diseases such as AIDS (Pappas, 1996). At present however

most of the research has not reached clinical trials and many that do tend to uncover

additional problems mainly concerning the delivery of the genetic material into the

defective cells (Crystal, 1995; Deshmukh and Huang, 1995; W olfert et a l, 1996).

While great progress has been made gene therapy research has a long way to go before

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the technology is fully developed and any manufacturers of gene therapy products will

be required to test their products extensively to meet regulatory agency requirements

for safety, purity and potency before they will be put on the market (FDA, 2000; Smith

and Doherty, 2000).

2.1.2 Gene Vaccines

Vaccination is one of the great success stories of m odem medicine and vaccine

development, just entering the third generation of technology, has a long history

(Babiuk, 1999; Brower, 1998; Spack and Sorgi, 2001). The original vaccines,

consisting of live attenuated pathogens, were invented as long ago as AD 900. These

vaccines, however, were only capable of mobilising the Cytotoxic T-Lymphocytes

(CTL’s) and carried risk of infection. Further research led to the development of

vaccines produced from killed (attenuated) pathogens or purified polymeric

components. While many of these vaccines are still in use worldwide there remain

many pathogens that are still resistant to current vaccine approaches and an increasing

number of once treatable pathogens are now becoming resistant (Brower, 1998; Spack

and Sorgi, 2001). The main reason is that although these second generation vaccines

are able to produce an antibody response many are not capable of provoking the

cellular immunity that is often required for effective protection against some pathogens

(Smith and Klinman, 2001; Spack and Sorgi, 2001; Wahren, 1996). Genetic vaccines,

the third and most recent generation of technology, were discovered during

experiments using replication defective retroviral vectors when it was realised that

through the direct injection of antigen expressing DNA a small number of infected

cells (10^^ to 10^) could raise a protective immune response (Robinson, 2000). The

discovery led to testing of proviral DNA used to produce infectious retroviral vectors

in the influenza model for the antibody to act as vaccine in chickens and resulted in the

first demonstration of protective immunisation by a DNA vaccine (Robinson, 2000;

Scheerlink, 2001). The field of DNA vaccines was then established in the 1980’s and

early 1990’s surrounded by hype and optimism (Page and Cudmore, 2001). Since,

numerous other animal and human studies have been carried out to show that DNA

immunisation does induce potent cell mediated protective immune responses and in

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2002; Cohen, 1998; Perrie et a l, 2001; Robinson, 2000). The clinical studies also

suggest DNA vaccines are well tolerated reporting only mild to moderate local adverse

effects in human volunteers (Smith and Klinman, 2001). Since the early reports that

demonstrated the potential feasibility of the approach there has been an explosion of

research on DNA vaccines for both humans and species of veterinary interest and is

expected to provide significant improvements in the technology (Krishnan, 2000).

The DNA vaccination technology involves the inoculation of a purified preparation of

plasmid DNA expression vector alone or with a delivery system. The bacterial plasmid

construct contains a pathogen gene(s) encoding immunogenic protein(s) plus gene(s)

to aid expression of foreign DNA within the host. Following vaccination with a gene

vaccine, the plasmid DNA is taken up by the host cells and pathogen proteins are

expressed and processed. After processing the pathogen proteins are then presented in

the context of self M ajor Histocompatibility Complex (MHC) class I and class II

molecules eliding an immune response thereby generating immunity to one or more

diseases (Gurunathan et a l , 2000; Krishnan, 2000; Prazeres et a l, 1999; Robinson,

2000; Smith and Klinman, 2001; Tacket et a l, 1999; Wahren and Brytting, 1997;

Zoon, 1996).

Genetic vaccination, although a relatively new technology, has already demonstrated

superiority over conventional vaccination in several respects including improved

safety, customisable, wider applicability and cost effective (Prud’homme et a l, 2001;

Robinson, 2000; Spack and Sorgi, 2001). Although DNA vaccines share many of the

advantages of live or attenuated viral and bacterial vaccines they do not have the safety

concerns. For instance plasmid DNA vaccines are not able to revert to a virulent form

and cause severe infection and the antigen is produced within the patients cells, which

provides the advantage of presenting the proteins in the same way as in a natural

infection but without the vaccine being infectious (Babiuk, 1999; Caldwell, 1997;

Doepel, 2001; Fomsgaard, 1999; Spack and Sorgi, 2001; Wahren, 1996; Wahren and

Brytting, 1997). Plasmid DNA constructs can be tailored to include only essential

DNA in order to produce an immune response focused only on the antigens necessary

for generating a safe, effective and specific immune response (Caldwell, 1997;

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Chapter2.____________________________________________________________________Literature Survey

Fomsgaard, 1999; Page and Cudmore, 2001; Robinson, 2000; Wahren and Brytting,

1997). The ease with which pDNA constructs can be made should also permit tailoring

of the plasmid to provide the possibility for multi disease targeting, addressing several

diseases in one vaccine shot, which is particularly attractive to developing countries.

Such techniques should also permit the enhancement of the vaccine capabilities by the

conjunction of genes that encode various cytokines including chemokines or co

stimulatory molecules that will enhance the action of the therapeutic genes (Brower,

1998; Babiuk, 1999; Prud’homme et a l , 2001; W ahren, 1996; Wahren and Brytting,

1997). DNA vaccines also have major immunological advantages over previous

vaccine generations and can therefore be applied to a wider selection of pathogens.

Most currently available vaccines induce antibody responses capable of mediating

long-term protection, however, some diseases such as HIV, leishmaniasis and

tuberculosis require vaccines that are capable of also inducing cell mediated immunity.

In the past 5-lOyears DNA vaccination has been shown to be eapable of producing

long-lived humoral and cellular immune responses in vivo in a variety of models

(Brower, 1998; Caldwell, 1997; Feigner, 1997; Gurunathan et a l, 2000; Page and

Cudmore, 2001; Packet et a i, 1999). DNA vaccines, by mimicking viral infection, are

able to access the MHC class I and class II processing and presentation pathway

(Robinson, 2000; Spack and Sorgi, 2001). The ability to use generic methods for

vaecine production in terms of the construction of plasmid DNA using recombinant

DNA technology as well as growth and purification of plasmid DNA also makes

manufacturing relatively easy and cost effective compared to vaeeines containing

attenuated virus, recombinant subunits or peptides (Babiuk, 1999; Cohen, 1998;

Robinson, 2000; Spaek and Sorgi, 2001; Wahren, 1996). The stability of DNA over a

wide range of temperatures and the fact that some formulations ean be lyophilised,

eliminating the need for continuous cold storage also reduees the cost through storage

and increasing the acceptable storage time thereby also making the vaccines more

accessible for developing countries (Evans et a l, 2000; Robinson, 2000; Spack and

Sorgi, 2001; Wahren, 1996).

With all of the potential advantages o f genetic vaccines seientists have begun working

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gene vaccines against many other conditions using both prophylactic and therapeutic

approaches. Current targets include vaccination against dangerous diseases such as

AIDS, tuberculosis, rabies, herpes and malaria, m ost of which are entering clinical

trials, as well as autoimmune conditions such as multiple sclerosis and juvenile

diabetes and even some cancers (Caldwell, 1997; Cohen, 1998; Collins, 2001; Doepel,

2001; Fomsgaard, 1999; Girard et a i, 1999; Hawkins et a i, 2002; Smith and Klinman,

2001; Spack and Sorgi, 2001; Wahren and Brytting, 1997; Zoon, 1996).

Although gene vaccines are advantageous compared with previous vaccine

generations, and have proven to be effective in many clinical trials, research is not yet

complete due to issues that need to be addressed concerning vaccine potency and

safety issues (Tollefsen et a l, 2002). Originally gene vaccines were developed as

naked DNA preparations, however, researchers have shown this would require an

excessive quantity of genetic material to achieve a significant response. Consequently

vaccine research is now focusing on a combination of DNA with adjuvants, lipids or

polymers in order to boost the effect of the DNA, protect it from degradation and

therefore reduce the quantity of DNA required (Brower, 1998; Oregonadis et a l, 1997;

Ferrie et a l, 2001). A novel technology, expression libraries, has also been designed to

facilitate the rapid identification of microbe antigens that induce protective immunity

when delivered as DNA vaccines whilst avoiding those that might cause unwanted

effects. This is expected to increase the pace of new candidate antigens that can be

tested (Krishnan, 2000; Wahren, 1996). Despite these improvements the present

potency of DNA vaccines is an issue with several phase I human clinical studies

reporting little or no immune responses (Selby et a l, 2000). Emerging infections such

as those caused by immunodeficiency viruses and remerging diseases such as drug

resistant forms of tuberculosis, pose new threats for vaccine development in first world

countries which are even more pronounced in the third world where effective vaccines

are often unavailable or cost-prohibitive (Caldwell, 1997; Fomsgaard, 1999; Robinson,

2000). A number of safety considerations will also need to be resolved before the use

of DNA vaccines becomes wide spread. For instance if combination of genes from one

virus or parasite or with mixtures involving genes from different microbes are used it

will be mandatory that they do not interfere with each other in an unpredictable way.

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There are also concerns with respect to integration into the patients genome and the

preferred approach is to keep the genetic content other than the gene itself to a

minimum in order to diminish the potential risks of integration (Wahren, 1996). The

direct introduction of DNA into animal tissues has many of possibilities for

immunotherapeutic interventions, and while significant progress has been achieved, it

is clear that the potency of DNA vaccines must be increased in order to harness the

advantages these vaccines clearly posses and to achieve successful application of this

technology to humans (Scheerlink, 2001; Selby et a l , 2000; Wahren and Brytting,

1997).

2.2 Types of Treatment

A gene delivery treatment requires the therapeutic gene(s) to be inserted into the target

cells of the patient and can occur by one of two modes, ex vivo or in vivo. The ex vivo

technique involves the removal of cells from the patient followed by transfection of the

cells before réintroduction (Figure 2.1) (Marquet et a l, 1997a). This technique results

in very efficient gene transfer and created the opportunity for cell propagation

generating higher cell densities. However ex vivo delivery is largely patient specific

due to cell immunogenicity, the effectiveness depends on permanent introduction of

the recombinant genes into the target cell and cell manipulation adds manufacturing

and quality control problems thereby making the technique costly. In vivo delivery

involves administration of genetic material directly into the patients body. Delivery is

therefore not patient specific, which makes it less efficient, but reduces costs, logistics

and infrastructure requirements compared to ex vivo delivery (Figure 2.1) (Marquet et

a l, 1997a; Mountain, 2000; Stribley et a l, 2002). The use of either technique must be

decided on a case-by-case basis depending on the purpose of the recombinant gene,

nature of the target cell or organ and the choice of vector (Stribley et a l, 2002).

Overall, for healthcare purposes the significance of the disadvantages associated with

the ex vivo delivery mode often outweighs those of the in vivo technique and has

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Cliapter2. Literature Sun>ev

Figure 2.1 Gene Delivery

Modification o f the genetic material o f living cells, either:

ex vivo (indirect) in vivo (direct)

Non-viral

Viral

gene

0

Non-viral

2.3 Socio-economic Issues

T he success o f gen e delivery relies not only on the d ev e lo p m e n t o f efficient, none

toxic delivery system s but also on public perception. In recent years the public

backlash against industries, such as that a im ed at the d ev e lo p m e n t o f genetically

m o dified foods by the agricultural industry, has d em o n stra te d the strong public interest

in genetic en g in ee rin g and highlighted the nee d to ensure the public are kept well

inform ed o f potential benefits, risks and protection m easu res that are in place for all

future d ev e lo p m e n ts in areas o f genetic research (h ttp ://w w w .im s-g lo b al.c o m /in sig h t,

2000). A lthough the perceived advantages o f gene delivery therapeutics o ver c u n e n t

pharm a ceutica ls are undisputable, so far gene delivery has not d elivered the prom ised

results (F riedm ann, 1996; M o u n tain , 2000).

T he perceiv ed advantages o f gene therapy o v e r conventional small m olecules or

biologies include the c o n e c tio n o f the genetic ca u se o f the disease, selective treatment

o f affected (diseased) cells and tissues and the long term treatm ent after a single

application. As public ac ceptance o f gene thera py is a large d e te im in a n t to its success

the p ro m in en t co verage of the first death o f a patient in gene perception clinical trials

C la ir e N i c o l e M o u n t

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came as a huge blow to public support, giving disparity between theory and practice,

and heightened public concern as to the safety of such treatments (http://www.ims-

global.com/insight, 2000; Rubanyi, 2001). The patient died on a clinical trial in 1998

due to an adverse immune reaction to a high dose of adenoviral vectors used to

administer genes encoding ornithine transcarbamaylase (http://www.ims-

global.com/insight, 2000; Stribley et a l, 2002). Fatalities such as this underscore the

risks involved in pioneering new approaches in medicine and highlight the requirement

for comprehensive new approaches in medicine and the requirement for

comprehensive regulatory guidelines and stringent review procedures for clinical

protocols (Rubanyi, 2001). To keep the faith of the public it is important to clearly

demonstrate the successful use of gene delivery techniques. A number of qualities

related to the application of gene delivery, however, also merit concern (Rubanyi,

2001). The distribution and persistence of DNA sequences contained within gene

transfer products create concern due to the potential for the sequences in the vector to

be expressed in non-target cells/tissues and in particular the potential for inadvertent

gonadal distribution and germ line integration (Sims, 2001). The fear of sequence

integration also creates concern due to the potential for induction of cancer mutation if

sequences integrate within oncosupressor genes (Robinson, 2000). Additional concerns

related to gene vaccines inparticular include the potential to induce immune tolerance

to vaccine antigens, the induction of antibodies to vaccine DNA or autoimmunity. The

success of Alain Fischer early in 2000 finally demonstrated the tremendous potential

of gene therapy when two infants with SCID-X were successfully treated. Bone

marrow stem cells with a normal copy of the cytokine receptor y chain were

transfected. Following treatment the patients were finally able to leave protective

isolation, discontinue treatment and grow and develop normally (Page and Cudmore,

2001; Stribley et a i, 2002). The major outcome of studies including thousands of

patients who have received gene therapy to date was that gene transfer was safe in the

vast majority of cases. Any side effects observed in these studies have, in most cases,

been manageable, with the exception of the one well publicised case, and it is hoped

that the examination of DNA sequences identified in the Human Genome Project

(HOP) will uncover further genes involved in diseases that could form the basis of

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Chapter2.____________________________________________________________________Literature Survey

et a l, 2001). However at present the overwhelming technical obstacles remaining from

the last decade of research hinders success.

With hundreds of clinical studies involving more than 3000 patients much controversy

exists regarding how many patients have benefited from gene therapy (Mhashilkar et

a l, 2001). The current sentiment is that we now merely understand the critical

strengths and weaknesses of a first generation technology. It seems that too many

enthusiasts in the field of gene delivery promised too much in the early studies and

when they failed to deliver scepticism has set in (Friedmann, 1996). It has become

clear that a credible partnership between the public and scientists is needed not only to

deal with the current issues of the field but also to establish a basis on which to face

those of the future when gene therapy experiments move into uncharted and

contentious areas (Mhashilkar et a l, 2001). To date, however, few of the concerns

have been realised in preclinical or clinical trials (Robinson, 2000).

2.4 Genetic Material and Disease

DNA, the genetic material of 99% of natural entities, excluding some RNA viruses, is

the building block from which each individual is created. Each individual possesses a

unique genetic code or genotype consisting of DNA sequences from the gene pool and

codes for the proteins that drive the bodies biochemical reactions and in combination

with the environment dictate the phenotype of the individual (http://www.southflorida,

1998). The unique genetic code of certain individuals can however have detrimental

effects on the phenotype when key sequences are defective or mutated as a result of

inheritance or spontaneous/induced mutation in the individual lifetime and result in

either incorrect processing or a complete lack of protein production, disease and even

death. In man alone there are more than 40,000 recognised diseases that involve a

genetic component, more than 3000 of these are now known to result from a single

gene defect and many more caused by multiple gene defects, including inherited/non

inherited cancer and inherited illnesses such as cystic fibrosis, diabetes, haemophilia

and muscular dystrophy (Hug and Sleight, 1991; Lee et a l, 1997; Pack et a l, 2000;

Stribley et a l , 2002). As the understanding of the biochemical nature of the human

population has developed, with the aid of the Human Genome Project, it has led to a

The impact of processing on the biophysical properties of synthetic DNA complexes for gene delivery