5 Traits of People Who Always Get Promoted at Work

5 Traits of People Who Always Get Promoted at Work Ever feel like you’re doing quite a few things, however watching everybody around you advance while you’re still stuck wasting your time? Here are 5 things that effective human you know, the ones getting those advancements consistently appear to have in common.1. They Have a StrategyRather than proceeding to apply indiscriminately up the stepping stool, profoundly fruitful (and exceptionally advanced) individuals incline toward a progressively key methodology. They pick employments not founded on essentially getting to the following bar up, yet on their specific qualities. What's more, they have their best course of action as of now as a primary concern. They make sense of what their next activity ought to be before applying to another one. That way they’re continually pondering the aptitudes and encounters they ought to fabricate now to move consistently towards their next position.2. They Say NoYou may feel that you have to state â€Å"yes† to each propo sed venture so as to excel, however that’s false. Genuinely effective individuals (even Warren Buffett) realize that picking their undertakings and their fights and organizing what they have to never really up where they should be-is the better procedure. They’re merciless and segregating in their choices.3. They Know How to Handle The BossSuccessful individuals understand their managers hold the way in to their progression. In the event that a supervisor is disrupting everything, they discover a route around it. They work their supervisors, utilizing â€Å"we† language and engaging in what is important most to the individual responsible for their next vocation steps. They challenge their supervisors and increase their regard. Thusly, their supervisors realize when to advance them.4. They Keep Proof of Their SuccessesSuccessful experts realize they need bad-to-the-bone verification that they are proficient for their next activity. They’re continually plan ning how to take a shot at ventures that will dazzle their next supervisor as much as their present one. They don’t become complacent or seek after incidental instruction. They simply develop a clothing rundown of evidence that shows they are prepared to take things to the following level.5. They Make an ImpressionThe best individuals get that, so as to go anyplace in office life, they need to fabricate impact. This doesn’t mean manipulating or politicking. It just methods: comporting themselves with uprightness, trustworthiness, and demonstrable skill. They manufacture associations with the individuals and establishments that can have an enduring effect in their professions.

The Hand-in Assignments

Untitled Document 1 of 4 https://elearning. uol. ohecampus. com/bbcswebdav/foundation/UKL1/C†¦ WEEK 6 ASSIGNMENTS Print Page Use the connections underneath to hop legitimately to the related data. Turn in Assignment Individual Project HAND-IN ASSIGNMENT Hand-in Assignments are one route for you to show your learning. The Hand-in Assignments give a chance to apply ideas and techniques to a real setting. Regularly, Hand-in Assignments are composed papers or PC programs that are submitted to the Instructor.They expect you to arrange data from the week by week Learning Resources, the Discussion and your own encounters to address an issue from the point of view of a true circumstance. Except if in any case noticed, the papers you write in Hand-in Assignments must follow Harvard Referencing Style reference and reference rules. You should present your responses to the accompanying Hand-in Assignment (HA) inquiries before the finish of Day 7 (Wednesday). Answers will be submitted to the week by week Assignments zone, however are not to be posted in the module Discussion Board. Question 1 Activity Mean durationStd. dev. (days) A 11 0. 9 B 13 1. 1 C 7 0. 2 D 9 0. 8 E 6 1 F 7 1. 2 G 10 0. 7 H 9 0. 6 11/04/2013 9:52 AM Untitled Document 2 of 4 https://elearning. uol. ohecampus. com/bbcswebdav/foundation/UKL1/C†¦ I 8 0. 8 Table 1 Complete the accompanying: 1. Ascertain the venture fulfillment time. 2. Show the basic way exercises. 3. What is the likelihood of finishing this task somewhere in the range of 38 and 40 days? 4. What are the leeway esteems for exercises C and F? Decipher the significance of their leeway esteems? Question 2 An enrolled nurture is attempting to build up an eating routine arrangement for patients.The required healthful components are the absolute every day prerequisites of each wholesome component as showed in Table 2: Required dietary component aggregate and day by day necessities Calories Not in excess of 2,700 calories Carbohydrates Not in excess of 300 grams Protein Not under 250 grams Vitamins Not under 60 units Table 2 The medical attendant has four fundamental sorts to utilize when arranging the menus. The units of nourishing component per unit of food type are appeared in Table 3 underneath. Note that the expense related with a unit of fixing likewise shows up at the base of Table 3.Required healthful component and units of nourishing components per unit of food type Element Milk Chicken Bread Vegetables Calories 160 210 120 150 Carbohydrates 110 130 110 120 Protein 90 190 90 130 Vitamins 50 75 70 Cost for each unit ?0. 42 ?0. 68 ?0. 32 ?0. 17 Table 3 Moreover, because of dietary limitations, the accompanying perspectives ought to likewise be viewed as when building up the eating routine arrangement: 1. The chicken food type ought to contribute all things considered 25% of the complete caloric admission that will result from the eating routine arrangement. 2. The vegetable food type ought to give at any rate 30% of the base every day necessities for vitamins.Complete the accompanying: Provide a direct programming definition for the above case. (You don't have to tackle the issue. ) 11/04/2013 9:52 AM Untitled Document 3 of 4 https://elearning. uol. ohecampus. com/bbcswebdav/establishment/UKL1/C†¦ Save your Assignment as a . doc, . docx, or . rtf document and utilize the Turnitin interface underneath to submit it. Come back to top INDIVIDUAL PROJECT T he motivation behind this recreation venture is to give you a chance to utilize the POM-QM for Windows programming to tackle a direct programming issue and perform affectability analysis.POM-QM for Windows programming For this piece of this task, you should utilize the POM programming: 1. Peruse Appendix IV of the O perations Management (Heizer and Render, 2011) course book. 2. Introduce and dispatch the POM-QM for Windows programming and from the primary menu select Module, and afterward Linear Programming. Note: You can recover the P OM-QM for Windows programming from either the CD-ROM that went with your Heizer and Render (2011) course reading. 3. Program the straight programming detailing for the issue underneath and explain it with the utilization of POM. Allude to Appendix IV from the Heizer and Render (2011) course reading. ) Note: Do not program the non-cynicism requirement, as this is now accepted by the product. For extra help, if you don't mind reference the POM-QM for Windows manual gave in this week’s Learning Resources. Singular Project issue A firm uses three machines in the assembling of three items: Each unit of item 1 requires three hours on machine 1, two hours on machine 2 and one hour on machine 3.Each unit of item 2 requires four hours on machine 1, one hour on machine 2 and three hours on machine 3. Every unit of item 3 requires two hours on machine 1, two hours on machine 2 and two hours on machine 3. The commitment edge of the three items is ? 30, ? 40 and ? 35 for every unit, separ ately. Accessible for booking are: 90 hours of machine 1 time; 54 hours of machine 2 time; and 93 hours of machine 3 time. The direct programming plan of this issue is as per the following: Maximize Z = 30X1 + 40X2 + 35X3 3X1 + 4X2 + 2X3

The Number One Question You Must Ask for Collegeboard Sat Essay Samples

<h1>The Number One Question You Must Ask for Collegeboard Sat Essay Samples </h1> <p>Jane additionally understood the hugeness of staying in the proper attitude when thinking of her absolute first draft. Realizing you have composed a decent exposition will offer you the positive force expected to assault the rest of the test. The absolute first bit of your framework should be a sentence that expresses your situation regarding the matter. </p> <p>Employing a few points of view exhibit a powerful idea practice. It is conceivable to likewise procure an expectation about what's to come. It is imperative to fathom why you miss or battle with an inquiry so you don't commit the very same errors later on. As it is beyond the realm of imagination to expect to know the exact inquiry you will be given, it is a lousy plan to enter the test without an all around considered procedure. </p> <h2> Who Else Wants to Learn About Collegeboard Sat Essay Samples? </h2> <p>People have numerous misinterpretations about the SAT paper and in this manner don't get the absolute best SAT score conceivable. There's no composite SAT Essay score (the 3 scores aren't included) and there are no percentiles. You will have the option to see scores online around 10-20 days following the test. The two tests incorporate discretionary articles whose scores are excluded from the last composite scores. </p> <p>Many circum stances the application article might be composed with the help of a school mentor. In the event that you locate a bizarre inquiry that don't have any thought what activities with, the cure is simply to ask a companion. 1 basic piece of exhortation is you have to figure if it's conceivable to dispose of at least one answer decisions. </p> <p>Make sure that your point sentence communicates the most significant thought of the human body passage as a definitive proclamation and isn't a subset of any critical detail inside the section. Close Strong The end might be the most basic piece of your paper. </p> <p>It would be justified, despite all the trouble to comprehend that SAT test graders assess your endeavors taking the entire of paper, and not just a bit of it. The SAT doesn't need to be this troublesome or upsetting inasmuch as you accept the open door to become familiar with the best technique to prepare for it. At test focuses outside the US, the SAT is rig ht now just controlled multiple times yearly versus multiple times for the ACT. </p> <p>Some schools may require you to complete the SAT Essay. For that, learn if your favored universities need such articles. </p> <p>If don't have any thought what the SAT article is about, you're ready to initially investigate our video that looks at the SAT exposition to the ACT paper and you may download a duplicate of a genuine SAT practice paper. So you wish to see how to beat the SAT Essay. You will comprehend what to focus on and expound on once you have to take the SAT paper so you get the most extreme score conceivable. Most SAT expositions incorporate an entry from a specific creator identified with a particular point. </p> <p>Looking for practically the entirety of the school composing structure will be provided on free! On the off chance that you own an enthusiasm, it's straightforward for your application to think of a topic, and afterward, at whatever point your confirmations instructor alludes to your application, they will be able to describe what it is you're about. The article starts with a composing brief or something to that affect. Regardless of whether it will be a dynami te article is subject to your own abilities and ability to compose. </p> <h2>The Hidden Treasure of Collegeboard Sat Essay Samples </h2> <p>All coaches are sufficiently prepared to flexibly the absolute best internet mentoring administrations. Schools don't need you to be balanced. They love to see that you are intrigued about something, and not exactly at a club level. The way that they will need to realize your score is valid, however their opinion of it isn't as clear. </p> <h2> The Number One Question You Must Ask for Collegeboard Sat Essay Samples</h2> <p>After the concise data is given, an inquiry will be posed. It's impractical to peruse every last one of them and score them dependent all in all substance. Every site recorded has entirely significant data that is all free. You're given models about how to intrigue the individual or people reviewing your SAT. </p> <h2> What You Can Do About Collegeboard Sat Essay Samp les Starting in the Next 2 Minutes</h2> <p>Each guide needs toward be clarified in another passage and stream sensibly between thoughts. When you are done, you're given various models with the score close to it. When you are done, go through the resulting 20 minutes speed composing. Remember, everything necessary I only two or three minutes to make a framework. </p>

A Guide to Writing Essays

<h1>A Guide to Writing Essays</h1><p>How would i be able to compose a paper theme for all the pretty ponies? You may have seen that when you go to the library or even your neighborhood book shop you frequently observe an assortment of titles for the subjects of articles. These subjects may have something to do with movement, creatures, sports, books, TV or even love. Regardless of what you pick, in the event that you need assistance composing a paper theme that is truly going to intrigue your peruser, at that point this article will assist you with getting started.</p><p></p><p>One of the reasons that you might be searching for extraordinary themes for expositions is on the grounds that you are not as acquainted with these subjects as you might want to be. You might have the option to think about some extraordinary points and not realize where to begin. There are a few locales online that will offer you tips and guidance on composing articles , however you should discover one that you are agreeable with.</p><p></p><p>Obviously, the overall population isn't the main crowd for your exposition. While your perusers are the individuals that will peruse your work, you will need to compose an exposition point that will speak to an expert crowd. That implies you will need to compose papers that have the correct degree of sentence structure and spelling.</p><p></p><p>Some schools and colleges will acknowledge articles that are not composed at a secondary school level. So in the event that you are not kidding about getting a degree in English and need to compose an exposition subject that has a portion of the things that the understudies will acknowledge, at that point you will need to utilize an article theme with the composing aptitudes that you are generally alright with. This can take a smidgen of research, yet it will assist you with starting to recognize the territories that yo u can improve.</p><p></p><p>If you are experiencing difficulty with specific zones of sentence structure or you have to improve your spelling, at that point you can generally go to online school paper subject indexes. These locales are anything but difficult to utilize and as a rule give everything that you have to ensure that you get an article theme that is straightforward. They likewise give you a rundown of famous themes and you can limit your hunt to locate the one that you are looking for.</p><p></p><p>One zone of intrigue that you might need to investigate when you are taking a gander at exposition points is the thing that the subject is about. On the off chance that you need to expound on movement, consider doing as such in the principal individual. That way, you will have the option to relate what you are doing as a component of your experience.</p><p></p><p>Another zone that you might need to consider when you are picking exposition points is the way you are introducing your feelings. You will need to consider how you present your contemplations with the goal that you can relate them to the point. You may likewise need to figure out how to fabricate your own proposition articulation and afterward incorporate that alongside your exposition topic.</p><p></p><p>Finally, a great point will be intriguing to peruse. Indeed, you might need to think about to how you are going to keep the peruser intrigued. You may find that the thought for an exposition point is anything but difficult to think of and once you make sense of how to do it, you can take the thoughts that you have and use them to compose your last bit of paper.</p>

USFA Application Essay Samples

<h1>USFA Application Essay Samples</h1><p>If you have to round out a USFA application exposition test, you should be quiet and devoted. While there are various exposition models you can use to assist you with showing signs of improvement handle on the rules utilized by USFA, it is significant that you go over them the same number of times as possible.</p><p></p><p>There are two sorts of USFA application article tests that are utilized to help with the procedure. One of the sort of test is fundamentally the same as what most different businesses require and the other is intended to enable the candidate to improve an association with the exposition. The kind of paper utilized for an exposition, as a rule depends on a particular subject that is extraordinary to the association or program you are applying to.</p><p></p><p>In the most broad sense, the USFA necessities for papers is to compose something that is about USFA re lated. They need candidates to put forth an attempt to assist them with assembling the exposition such that shows they have an energy for what they are attempting to state. Furthermore, the candidate ought to compose something that depends on what they think about USFA. This will assist the author with making the best contentions for their side of the contention and present it such that will have the option to be comprehended by an adjudicator and an affirmations officer.</p><p></p><p>The kinds of papers that the USFA requires are quite often dependent on issues identified with the strategic the USFA. A case of this would be the inquiries of what the reason for the USFA is, the statement of purpose, and the objectives of the USFA. When composing these sorts of articles, it is critical to consider the topic of why you need to apply for USFA in any case. This is the initial move towards building up a decent essay.</p><p></p><p>Another th ing that is required when composing a USFA application paper test is that the candidate ought to get thoughts regarding the points that are pertinent to USFA before composing the exposition. The reason for existing is to show that you comprehend the USFA strategic how that will help you as a candidate to have an effect on the USFA program. This incorporates knowing about USFA and being educated on the program.</p><p></p><p>When composing a paper, it is imperative to ensure that you get a few thoughts on USFA projects and a few thoughts on what your abilities are with regards to applying for USFA. Since USFA needs you to have the option to plainly communicate in your exposition, it is important that you don't attempt to pack a lot of data into your paper. It is additionally significant that you abstain from getting excessively specialized with your paper. This is on the grounds that USFA may take a gander at your exposition as being improper for an essay.</ p><p></p><p>One of the advantages to composing an article is that it is an extraordinary chance to learn new things and to improve your composing abilities. You will have the option to figure out how to utilize the structure of a paper to assist you with building up your own composing style and to ensure that your article streams. Probably the most serious issue with regards to article composing is that it isn't evident that you are composing a formal essay.</p><p></p><p>When it comes to paper composing, it is significant that you get a thought on the best way to set up your exposition, where to begin, and where to end your exposition. These expositions ought to be efficient and ready to convey a decent contention. Probably the greatest bit of leeway to composing an article is that it is generally the most effortless approach to figure out how to arrange your musings and to communicate unmistakably and effectively.</p>

Tips For Writing Informative Essays With Surprising Reversal Topics

<h1>Tips For Writing Informative Essays With Surprising Reversal Topics</h1><p>In request to make an educational paper with amazing inversion themes, you should know about the various techniques you can utilize. Useful papers with astonishing inversion themes are really the most helpful sorts of papers you can compose. In the event that you can work a very much idea out exposition on any subject, you will have the option to prevail with regards to composing a noteworthy and compelling article. You don't need to try sincerely or penance the nature of your composition, since you can increase a great deal by utilizing reverse points in your article.</p><p></p><p>For starters, you should realize certain information on switch themes so as to make it simpler for you to compose well. Above all else, you should comprehend the thought behind the entire converse theme idea. The thought behind opposite subjects is to compose an article so that it is con versational.</p><p></p><p>One of the most ideal approaches to accomplish this is to simply begin discussing yourself and afterward present the subject of the article at a later stage. Additionally, when you talk about yourself in the paper, it is significant that you use themes that are identified with the principle thought of the article. At the point when you have introduced the primary thought, you should talk about the reason and tone of the essay.</p><p></p><p>Here is a case of a fascinating converse subject. Let us expect that you need to compose an article on business morals. You may compose an exposition on business morals by discussing some disputable issues like vote based system and communism and the pertinence of the issues to business ethics.</p><p></p><p>These questionable points will be extremely intriguing themes to examine. Before the finish of the paper, you will have the option to introduce a few realities and you will have the option to legitimize your position on the issue.</p><p></p><p>In expansion to the presentation segment, you will likewise need to compose a prologue to the substance of the exposition on which you will talk about opposite points. By composing the presentation area, you will have the option to make a presentation for the proposal statement.</p><p></p><p>The theory proclamation is the place you will have the option to begin your contention on turn around points. The objective of the exposition is to give proof to help your theory proclamation. You will likewise need to introduce the subject of the article so that you will have the option to persuade your readers.</p><p></p><p>Another point to recollect when composing an exposition is that you ought to write in an organization that is generally gainful to you. The objective is to exploit the syntactic structures of your theme to manufacture the help of your contention. Notwithstanding that, you ought to likewise be clear and exact with your contention so you will have the option to persuade the reader.</p>

The Treatment of Cystic Fibrosis using gene therapy, in particular Adeno-Associated Viruses

In this dissertation we shall consider the field of gene therapy in specific relation to cystic fibrosis. We examine the different delivery vector mechanisms that have already been explored and concentrate primarily on the adeno-associated vectors. We examine the current state of research and consider the advantages and drawbacks of the various methods considered. We conclude with a discussion and analysis of our findings and make anumber of assumptions relating to the future direction of the researchin the field. The rate of progress in the field of gene therapy has been enormous. We must remind ourselves that the first clinical gene transfer experiment took place in 1989 when a patient with malignant melanoma received genetically modified auto logous T cells. (Rosenberg SA et al1990) Gene therapy encompasses two major areas. The in vivo field, where genes are incorporated into the target cells of the living body and the ex-vivo field where the target cells themselves are genetically modified outside the body and then re-implanted. Medical science has been using the basic techniques of gene transfer for a long time. The technique has been exploited when viral genes are introduced to human cells when a viral vaccine is administered. The key technologies that allowed the transition from vaccination to gene therapy were the evolution of methods that allowed the genes to be isolated and replicated (cloned) and manipulated (engineered) prior to transfer into human cells (Freeman SM et al 1996) The key principle in this process is the efficient transfer of the manipulated therapeutic genes into the nuclei of target cells usually be means of various vectors. This dissertation will be considering the utilisation of these vectors in some detail. In broad terms, the new or modified genetic material is able to produce new proteins which can restore deficient or abnormal functions of genetically diseased tissues, to generate tissues that have entirely new properties or to create transplantable tissues for the controlled release of therapeutic proteins. (Russell SJ1997) In terms of viral vectors, prior to 1996 science was dependent on the use of modified retroviral vectors (eg.MMLV) to effect gene transfer into the chromosomes of a target cell and the adenovirus vectors when such integration was not needed. Neither vector was particularly successful as the intact nuclear membrane (in then on-dividing state) was a major barrier for chromosomal gene integration. (Sikorski R et al 1998). A breakthrough came with the realisation that lentiviruses (e.g. HIV) have the same ability to transfer genetic coding into the cellular genome but could do this in the non-dividing or dormant phase cells. (Amado R G et al 1999) In vitro, lentiviruses have been shown to change the target cells expression of proteins for up to six months. importantly, they can be used for terminally differentiated cells such as respiratory epithelium. The only cells that the lentivirus cannot penetrate the nucleus are those in the quiescent (G0) state as this blocks the reverse transcription stages of protein synthesis. (Amado R G et al1999) Cystic fibrosis Cystic fibrosis is the most commonly lethal inherited recessive inthe caucasian population. It affects about 1 per 2,500 livebirths. Thetreatment of cystic fibrosis has improved enormously in the last fiftyyears with the life expectancy increasing from an average 10 years to30-40 years now. The prime cause of death in affected individuals is the repeatedcycle of infection, inflammation and fibrosis of the respiratory tractwhich eventually culminates in respiratory failure and death. The disease itself is caused by mutations in the single gene forthe cystic fibrosis transmembrane conductance regulator (CFTR) whichproduces a protein found in sweat and pancreatic ducts, gut,seminiferous tubules, lungs and many other tissues. The mutationsresult in an abnormal protein which, when expressed in the lungs,produces thick viscous and dehydrated secretions. This does not allow for the efficient expulsion of bacterial pathogensfrom the lungs and a number of highly resistant forms of bacteria arecommonly found in cystic fibrosis (viz pseudomonas aeruginosa)(Porteous DJ et al 1997). An individual must receive a defective copy of the cystic fibrosisgene from each parent in order to develop the clinical picture ofcystic fibrosis. Following normal genetic principles, if two carriersconceive a child, there is a 25% chance that it will have cysticfibrosis, a 50% chance that it will be a carrier and a 25% chance thatit will have two normal cystic fibrosis genes. Viral and non viral vectors Viruses have an ability to enter a host cell and combine their owngenetic material with that of the host cell. This is the basicrationale behind the science of gene transfer therapy. As we shalldiscuss in some detail in this dissertation, there are a number ofpotential viral vectors that have been explored, evaluated andexploited in the search for an efficient and safe form of therapy.Viruses are not the only vector that can be utilised . Simply placingDNA in the nasal mucosa will produce some incorporation into theepithelial cells (Knowles MR et al 1998). This absorption can bedemonstrably enhanced further by the combination of the DNA withvarious plasmid or lipid complexes(Zabner et al 1997) The advantages of lipid or plasmid assisted transfer mechanisms arethat they do not appear to generate the immunological responses thatare seen with the viral vectors. They can also be used to facilitatethe transport of much larger pieces of DNA which would otherwise belimited by the packaging consideration incumbent on the viral vectors.(Felgner P 1997). The use of retroviral vectors is far from straightforward. The heavilypublicised case in April 2000 brought some of the problems to theattention of the media. A retroviral manipulation of a case of X-SCID(X linked severe combined immunodeficiency) was treated by gene therapywith an apparent degree of success (BBC 2002). This particular diseaseprocess is caused by a mutation on the gene which codes for the C chainof the cytokine receptors which is situated on the X chromosome andvital for the functional development of T Killer lymphocytes which aretherefore completely absent in the condition A multinational team used a retroviral vector to insert a functionalcopy of the gene into bone marrow stem cells which were thenre-transfused back into the patient. (Cavazzana-Calvo M et al 2000).This particular case resulted in a return to normal levels of T cellsin a comparatively short period of time. This was hailed in both thepopular media and the peer reviewed journals as a major success and itcan indeed be considered a landmark as it pioneered the successful useof an ex-vivo procedure that avoided direct in vivo transfer of thevector. The reason for specifically highlighting this particular case isthat following the initial optimism of the clinical team, two of thefirst ten patients with this condition who were treated in the same waysubsequently developed a leukaemia-like illness. Genetic analysis ofthe malignant cells suggested that the retroviral vector used in thetransfer had also activated an oncogene LIM-only2 (LMO2) which is knownto be associated with some forms of leukaemia. The clinicians reviewingthe situation felt that, although it was not the only cause of themalignancy it was one of the events that triggered it. Similar concernshave been raised in respect of other clinical trials. (Lehrman S 1999) The prime reason for presenting these events is to demonstrate thefact that there is both a theoretical and practical risk of insertionalmutagenesis. Reduction of the risk requires greater specificity of thetargeting of the genetic deficit perhaps by directing the expressionof the therapeutic genes to various specific tissues utilising bothtransductional and transcriptional targeting. Relph K et al 2004), In terms of specific considerations of the arguments in favour of theuse of retroviral vectors, one can cite the fact that they have ahighly efficient mechanism of gene transfer together with lowimmunogenicity. It is a well researched and well studied system and isknown to selectively infect actively dividing cells. The conversearguments reflect their disadvantages including their ability todisturb or activate oncogenes, the fact that they are difficulty tospecifically target and it is difficult to obtain high titres in theclinical situation (after Olsen, J. C. 1998). In broad terms, the principles behind the use of retroviral vectorsare that they must be modified in order not to be able to transmit anyovertly pathological coding. This involves the deletion of viral helpergenes such as gag, pol and env to render the replication processinvalid. This is done by utilisation of a producer or packaging cellline. (Nichols, E. K 1998). An example of a commonly utilised and extensively researched vector isthe MoMuLV. It is an engineered vector which can store 8 kb of RNAwithout compromising packaging efficiency. It is a hybrid cell lineeasily grown in mouse fibroblast cells There is a subdivision of the retroviral vectors known as thelentivirus, which is the only retroviral vector capable of integratinginto the chromosomes of non-dividing cells. This has been effectivelydemonstrated in vitro (Naldini L et al 1996). The biggest problem with the lentivirus vectors is that theyappeared to only produce very low titres. Some recent researchsuggested that a modification to a amphotropic envelope protien wascapable of allowing higher titre levels. (Rolls M et al 1999) At about the same time that the scientific press was learning aboutthe problems with retroviral transfer (see above) other investigatorswere working with adeno-associated viruses (AAVs). A similar processwas invoked using adeno-associated viruses to correct a genetic defectinvolving coagulation factor IX. The adeno-associated viruses were usedas they were considered to be amongst the safest candidates for genetransfer. They do not naturally cause disease processes in humans andhave only rarely been found to incorporate in a random fashion into thehuman genome. Although it is noted that adenoviruses do cause oncogeneactivation in rodents although it has not been found in humans(Blacklow NR 1988). The trial had a very positive outcome. (Kay MA et al 2000), but thetrial author (in later research work) published a study which suggestedthat, in study mice, the vector used in the trials actually integrateditself into gene containing regions of DNA more frequently that it didinto non-coding regions (Kay MA et al 2003). The findings were reportedas the fact that new genetic material was randomly distributed amongstall of the chromosomes particularly at sites of gene activity. On thisbasis, there appears to be at least a theoretical basis for thepossibility of similar cellular defects such as occurred in the X-SCIDpatients. Adenoviruses are comparatively simple structures. They arecategorised as double stranded DNA viruses. They have icosahedralcapsids with twelve vertices and seven surface proteins. The virionitself is spherical and non-enveloped and in the region of 70-90 nm insize. Their natural history is that they are spread easily in the naturalstate by the faeco-oral route and also by respiratory inhalation whichclearly has great implications for the treatment of cystic fibrosis. A theoretical analysis would immediately suggest that the adenovirus should be a suitable candidate for gene therapy as they can codefor specific proteins and they do not produce infection pathogenicviral offspring. The early trials into this particular area were reviewed by Griesenbach(Griesenbach U et al 2002) who pointed out that the cystic fibrosisgene was first cloned in 1989 and in the subsequent years, 18different trials were carried out, all with rather low degrees ofsuccess. They collectively trialed three different vectors, namelyadenoviruses, adeno-associated viruses 2 and cationic liposomes, andalmost universally found that each vector had a very low rate ofclinically significant gene transfer and none was sufficient to achieveclinical benefit Plasmid Complexes At its most basic level, a plasmid is a small accessory collection ofDNA which is found in the cytoplasm outside of the nucleus. They arecapable of independent replication and can be manipulated with rathermore ease than nuclear DNA. Early investigations into the field of gene transfer explored thepossibility of plasmid vectors and demonstrated the feasibility of themethod to effect CFTR gene transfer in vitro (Alton EW 1993). Otherteams had demonstrated the fact that, in clinical use theplasmid-liposome is both nontoxic and non-immunogenic (Hyde,SC et al1993). This appeared to raise the possibility that many of theimmunological problems encountered by teams working with viral mediatedgene transfer mechanisms might be circumvented. In vivo work (Yoshimura,K et al. 1992) had demonstrated that genescould be transferred into the cytoplasm by this method and Stribling, R(et al 1992) demonstrated that, once there, they would then replicatenormally. Alton experimented with a CFTR-plasmid preparation in miceand demonstrated that it was capable of correcting the chloride levelsin cystic fibrosis mice back to normal levels (Alton EW 1993) Although the initial results were encouraging, clinical trials weredisappointing as the plasmid complex could not easily penetrate thethick mucous residues in the diseased lungs of patients with cysticfibrosis. (Erickson,R 1993) The plasmids typically have a positively charged head-group which isable to bind to the DNA strand and a hydrophobic tail group whichfacilitates the transfer of the complex across the cellular membranes.Initial studies suggest that between 100-1000 times more DNA isrequired to effect successful gene transfer when this method iscompared to viral vectors. (Santis,G et al 1994). One alternative adaptation has been reported by Stern M (et al 2003)who points out that one of the solution of delivery is to ensure thatthe respiratory epithelium is exposed to the DNA over a long period.Their solution was to encapsulate the CFTR-plasmid in a slow releasebiocompatible polymer. Clinical trials are underway but not yetreported. The adeno-associated vectors appear to have (at least on atheoretical basis) a number of advantages over the vectors that we havealready discussed. They are based on a virus vector that is alreadynon-pathogenic (Berns, KI et al 1995) and has a mechanism that allowsit to be a long-term persistent entity in human cells (Blacklow, NR etal 1989). The adeno-associated vectors are particularly useful indealing with disease process that involve single gene mutations. This,therefore makes it particularly suitable for single gene disorderssuch as cystic fibrosis and alpha 1 antitrypsin deficiency. (Flotte, TRet al 1998). In addition, some workers have also developed vectors which are capableof producing either inducible or constitutive expression of thecytokine, interlukin-10 (IL-10) which is an importantanti-inflammatory protein which, on a theoretical basis, could beuseful not only in cystic fibrosis but in other disease process whichhave chronic inflammation as their prime manifestation (viz Type Idiabetes mellitus or inflammatory bowel disease) (Egan, M et al 1992).These manifestations have been studied and have now reached the stageof early clinical trials (Wagner J et al 2002). With specific reference to the implications of cystic fibrosis, wecan point to trials which have resulted in the expression of cysticfibrosis transmembrane conductance regulator (CFTR) from rAAV(recombinant adeno-associated vectors) in cell cultures (Flotte, TR etal 1993), in animal models (primates) (Afione, SA et al 1996), andagain in early phase I clinical trials (Wagner, J et al 1998) The rAAV-IL-10 model has been studied in bronchial cell culturesfrom cystic fibrosis patients, to determine the functional consequencesof CFTR complementation. This has not yet been demonstrated in vivowith humans but in both mice (Song, S et al 1998), and monkeys (Conrad,CK et al 1996) The overall results of these (and other) studies have shown that itis possible to achieve long term gene transfer and functionalexpression of the replaced gene (some studies for as long as 18 months)without any overt pathological findings. The histological findings are something of a surprise however, as,at least in both primate and mouse studies, the vector-introduced DNAin this form does not appear to be assimilated into the geneticmaterial of the chromosome, but persists in log strings or concatemersthat are episomal, which is in complete contrast to what happens whenthe naturally occurring agent infects the cell. There is some evidenceto suggest that host cell intrinsic factors such as DNA-dependentprotein kinase play some role in this process (Song, S 2001). The significance of this finding could be that the exclusion of thefunctional, newly introduced DNA from the rest of the nuclear gene poolmay be less likely to produce effects that could be either potentiallydisruptive to the host cell and less likely to activate oncogenes.Phase I trials have demonstrated significant rises of CFTR levels inboth sinus and lung tissue with no evidence of vector-related toxicity.(Wagner, JA et al 1999) The adeno-associated vectors are constructed from proviraladeno-associated vectors plasmids, which have the Rep and Cap proteinsdeleted and substituting the appropriate gene (CFTR or equivalent)between the rAAV2 inverted terminal repeats together with other signalsequences such as promoter and polyadenylation sequences (Flotte, TR etal 1994) The packaging processes allows for about 5 kb of rAAV genomes to becarried in the vectors which are prepared using a cotransfectiontechnique utilising human embryonic kidney cells (HEK-293) where thevector plasmid is cotransfected into the cells with helper agents(plasmid pDG) being used to encode the rAAV2-rep and -cap genestogether with the adenovirus helper functions (Grimm, D et al 1998).These are incubated for between 48 and 72 hrs. The cells are then lysedand the resultant agents are then separated by ultracentrifugationagainst a density gradient and affinity chromatography (Zolotukhin, Set al 1999). The vectors are thereby amenable to being separated by both theirphysical characteristics and also their biological characteristics(infectious units). They are carefully screened to ensure the absenceof any possible contamination from non-modified (replication competentAAVs) prior to clinical usage. (Muzyczka N 1994) The comparatively small payload of the adeno-associated vectorsis proving to be a significant problem. The vector itself is small whencompared to the comparatively large size of the CFTR gene. (Flotte TRet al 1993) It does not leave any room to manoeuvre to manipulate thevector-specific sequences in the way that we have described with theretroviral and adenoviral groups. (Flotte TR et al 2001). A number of authors have characterised the problem with theobservation that the rAAV is typically about 20 nm across which allowspackaging of about 4.7 kb (kilobases) of transferable modified gene(exogenous DNA). (Dong JY et al 1996), If it is combined with otherenhancers such as the promoter, the polyadenylation signal, thisclearly reduces the capacity for the DNA component. (Duan D et al2000). The Yan paper (Yan Z et al 2000) has outlined a novelexploitation of the unique ability of the rAAV genomes to link togetherin strings which appears to have the ability to bypass this particularlimitation.( Flotte TR 2000). The mechanism itself is the capacity of two distinct rAAV genomes thathappen to simultaneously infect the same target cell to undergo anintermolecular recombination insider the transduced nucleus of thetarget cell. This was a chance finding which arose from work involvingrAAV-derived episomes (Kearns WG et al 1996) in primate airways. It wasfound that some of these episomes were configured as circular head totail concatemers (Duan D et al 1999). This could have been either froma rolling circle replication from a single vector or alternatively,from an intermolecular recombination of material from multiple cellularpenetrations which combined within the palindromic inverted terminalrepeat sequences that are an intrinsic part of the AAV genomestructure. The authors were of the opinion that it was likely to be thelatter eventuality (Duan D et al 1998) It was a logical progression to try to exploit this phenomenon andthereby bypass the limitations imposed by the relatively smallpackaging capacity of rAAV. The adeno-associated vectors capsid onlyhas a capacity of about 5 kb. If we consider that the 145 nucleotidestretch of the AAV-ITR (inverted terminal repeat) sequence has to be inplace at both ends of the single-strand DNA for the vector DNA to beboth replicated and packaged, this only leaves in the region of 4.7 kbof genetically active material in each rAAV particle. As we have cited earlier in relation to the Dong paper (Dong JY et al1996) the CFTR gene accounts for about 4.5 kb which leaves very littlespace for other enhancing material. Because of this, the actual CFTRvector that has been used in the clinical trials to date uses only theminimal promoter activity of the AAVs-ITR itself to actually activateand drive the CFTR expression (Flotte TR et al. 1993). To look at this potentially important development in a little moredetail we can consider Duans original paper (et al 2000) and theauthors describe what they call a superenhancer. They describe acombination of a potent simian virus (SV40) and CMV immediate earlyenhancer elements as being packaged in one rAAV vector and a luciferasegene assisted by a small minima;l promoter in another rAAV vector. Invitro experiments suggested that either the SV40 or the intrinsicpromoter activity of the AAV-ITR was sufficient for this purpose. Theintermolecular recombination described above, was found to occur inboth vitro and in vivo experiments and was found to be sufficient tohave a greater than additive effect. Initial results from these varying methods are encouraging insofaras they are producing results of transgene expression which are 100-600times greater than with the unenhanced vector alone. (Yan, Z et al2000) Although not directly referable to our considerations of cysticfibrosis, we should note that Yans group and other workers have doneexperimental work which has culminated in the long term expression offunctional levels of erythropoetin with this two vector method in micein vivo. (Naffakh N et al 1995), This basic principle has been further enhanced by Sun (Sun L et al2000) with an ingenious manipulation of the system. They triedinserting the promoter and the first half of the coding sequence in onerAAV vector, immediately followed by a splice donor and then theupstream half of an intron. In the other rAAV vector was the downstreamhalf of the intron, the splice acceptor, the second half of the geneand the polyadenylation signal. To quote the author verbatim: This strategy is efficient enough to mediate high-level expressionand the intermolecular junctions are apparently stable enough tomediate expression for several months in vivo. Although this is clearly an ingenious augmentation of the sameprinciple , we should note that there are both advantages anddisadvantages to both pathways. The strategy that adopts the superenhancer takes its strengths fromthe fact that the recombination mechanisms optimise theposition-independent and orientation-independent functions of theenhancers. Consideration of the options would suggest that there arefour potential recombination outcomes from the process described.Either of the two vectors could be on the 5 end of the heterodimericmolecule and clearly either molecule could be in either orientation. With the superenhancer option, all four of these possibleintermolecular recombination outcomes should be functional fortransgene expression whereas if compared to the split intron strategy,by using the same reasoning, it is clear that only one out of the fourcould work. On the other side of the argument, the superenhancer option has thedisadvantage that the actual coding sequence of the gene to betransferred must still fall within the packaging capacity of the vectoritself whereas the split intron allows for a greater functionalexpansion of the packaging capacity. (after Flotte TR et al 2000) In either event it can be seen that these ingenious modificationseffectively eliminate the main size limitation of the rAAV deliverysystem. Although initial pre-clinical work is encouraging it appearsthat there is still some potential for a degree of immune responseparticularly if the host organism has not experienced the newlyproduced protein before. A number of studies have been done on animal (vertebrate andprimate) with only minimal success. Different administration methodshave been studied including direct administration into the lung (WagnerJ et al 1999), IM injection (Song, S et al 2001 B) and hepatic portalvein infusion (Song, S et al 2001 A) Human clinical trials have taken place with these vectors (Flotte T etal 1996)(Wagner J et al 1998) (Virella-Lowell, I et al 2000). Thestudies were done on adult male and female patients (18-47 yrs) whowere pseudomonas free and had recently been hospitalised for IVantibiotic infusions The disappointing results were probably a reflection of the factthat the CFTR defect is also interconnected in some way with aproinflammatory phenotype which appears to be triggered by the abnormalprotein via an unfolded protein response. The authors were able to showevidence that the rAAV-CFTR mechanism was able to correct the proteinproduction defect, they found it clinically difficult to transduce asufficient number of cells in the airway to reverse the inflammatoryresponse. It is proposed to run further experimental work which combines theCFTR expression with an anti inflammatory gene such as the IL-10.There is some in vitro work to suggest that this may be a possibleworkable approach (Teramoto, S et al 1998). Other work on ways ofenhancing the phenotypic expression of the modified genotype hassuggested that the use of various promoters and the rAAV-CMV/beta-actinhybrid promoter (CB-AAT) was found to be tone of the most efficient, atleast when it was compared to the other tested options such as the CMV,E1, U1a and U1b promoter constructs (Teramoto, S et al 1998) Overall, the initial results appear to be encouraging. A singleinjection of an rAAV-CB-AAT vector in animal studies has resulted inhigh level, stable transgene expression which has persisted over thelife span of the experimental animals and that there was no detectableinflammatory response in the animals who had received this form oftreatment (Flotte TR 2002) Flotte (et al 2002) reports that four human clinical trials at bothPhase I and Phase II level are currently underway examining the effectsof the rAAV-CFTR vector. They had an entry cohort of seven patientswith the vector being applied to the nasal lining, the maxillary sinusand the bronchus. The authors report no adverse effects being found andthat they have observed transgene expression at doses of 6 x 108 drp inthe sinus or 1 x 1013 drp in the lung. There are no reported interimfindings from the Phase II trials as yet. There is clearly a potential for clinical benefit on the basis of theresults found to date if one can extrapolate from in vitro and animalexperiments. The authors comment that, in contrast to the adenovirusvectors there is a marked lack of inflammatory toxicity with the rAAVvectors. Despite these positive comments, we should not, however, overlook thepotential limitations of this particular delivery system. These havebeen identified by various authors as: The inhibitory effect of preexisting airway inflammation on rAAV transduction in the lungs (Virella-Lowell, I et al 2000) A relative paucity of receptors on the apical surface of airway epithelial cells (Summerford, C et al 1998), The relatively weak nature of the minimal promoters used in the first-generation rAAV-CFTR vectors(Flotte, TR et al 1993), The potential for adverse long-term effects from rAAV vector DNA persistence. (Wu, P et al 2000) The Flotte group are currently investigating this problem by examiningthe hypothesis that the barriers in the airways of the cystic fibrosissufferer are primarily due to the neutrophil-derived -defensins (HNP1and HNP2) and are actually reversible by the mechanism of AAT proteindelivery (Virella-Lowell, I 2000) Wu and his co-workers have been looking at ways of manipulating thegenetic make up of the rAAV2 capsid and thereby trying to enhance thetargeting ability so that the vector specifically targets the serpinenzyme complex receptor on IB31 cells which is virtually specificfor the Cystic fibrosis bronchial cells Zabner, J (et al 2000), have considered alternative rAAV serotypesin the hope of finding one that will bind more specifically to thebronchial cells Other peripheral adjuncts have also been explored includingpromoters to enhance the effects of complementation and superenhancerswhich have been shown to improve the ability of the rAAV toconcatermerise with the help of smaller amounts of promoter agents(Duan, D et al 2000). Perhaps it is appropriate to conclude this section on considerationof adeno-associated vectors with a critical analysis of a very recentmulticentre, double-blind, placebo-controlled trial (Moss RB et al2004) This was a well constructed, fully statistically significant anddouble blinded trial which considered both the safety and thetolerability of repeated doses of adeno-associated serotype 2 vectorrepeatedly given by aerosol inhalation. The vector contained cysticfibrosis transmembrane conductance regulator (CFTR) complementary DNA(cDNA) [tgAAVCF], an adeno-associated virus (AAV) vector encoding thecomplete human CFTR cDNA. The entry cohort was comparatively small with 42 patients, of whom20 received the active agent. A number of indices of airway functionwere measured. Of particular interest to our considerations in thisdissertation was the fact that vector shedding was found in alltreated subjects up to 90 days after inoculation. And that all subjectswho received the active agent exhibited at least a fourfold increase inthe serum AAV2 neutralising antibody levels. Of the 20 treated patients, six subsequently underwent bronchoscopy.Of those six, gene transfer but not gene expression was demonstrated inall of them. On this basis, it would appear that the actual transfermechanism is effective, but there are other factors present whichappear to interfere with the subsequent expression of the gene in termsof protein production. The study did not comment on the possiblereasons for this. The authors were able to conclude that the delivery system workedwell with no evidence of adverse effects and that treated patientsdemonstrated an encouraging trend in improvement in pulmonary functionin patients with CF and mild lung disease. Lipid 67 We have discussed the various shortcomings of the virus-associatedvectors and this has prompted researchers to explore and consider otheroptimising options for facilitating gene transfer. Zabner (J et al1997) considered the use of cationic lipids in this process and foundone GL-67:DOPE (colloquially known as lipid 67) which appeared to beparticularly helpful in the process. Cationic lipids appear to show a degree of promise as possible vectorsfor CFTR cDNA transfer into respiratory epithelial cells of cysticfibrosis patients. Zabners group developed a preparation of plasmidencoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) whichappeared to facilitate the gene transfer to a significantly greaterextent than previously tested lipid complexes. They performed in vivostudies which compared the gene transfer rate to the epithelial cellsof the nasal mucosa of DNA-lipid complex and DNA alone. In generalterms, their findings indicated that the DNA-lipid complex was far moreeffective in achieving gene transfer than was simply giving DNA. Theauthors felt able to conclude that: These results indicate that nonviral vectors can transfer CFTR cDNAto airway epithelia and at least partially restore the Cl- transportdefect characteristic of CF. However, improvements in the overallefficacy of gene transfer are required to develop a treatment for CF. In this dissertation we are primarily considering the issues ofgene therapy in direct relation to cystic fibrosis. Inevitably, thishas meant considering the issues on a wider front, as many areasoverlap on a theoretical or practical basis. The prime biochemical cellular defect in cystic fibrosis is anabnormality in the cystic fibrosis transmembrane conductance regulator(CFTR). From a theoretical perspective it should be obvious thatreplacement of the defective gene with a working alternative would bebest achieved in the neonatal period before the body had time todevelop substantial fibrotic changes in the lungs that were secondaryto repeated episodes of infection (Dark J et al 1996). If successful, this could be expected to reduce both morbidity andmortality for cystic fibrosis. We have been able to cite evidence thatgene transfer has been accomplished both in vitro and in vivo. We havediscussed the results of a number of research groups who haveinvestigated various delivery systems which, to varying extents, haveproved able to deliver at least a small quantity of functional respiteto the cystic fibrosis sufferer. It is also important to be fully aware of the possibility ofinadvertent side effects in the field of gene manipulation. We havehighlighted the problems with oncogene activation. But this appears tobe associated with some vectors more than others. In short, it wouldappear that the problems and limitations that appear with this type ofprocedure are a function of the parent virus. The initial work with adenoviruses appeared promising as genetransfer could be accomplished but the major drawback was the doselimiting inflammatory effects which arose primarily as a result of thelarge amount of viral protein which was required to achieve atherapeutic dose. The subsequent modifications which had a greaternumber of coding sequence deletions appeared to be more effective inanimal experiments as they generated a lesser response from the cellmediated immunity mechanisms and therefore had a greater duration ofaction. (Caplen NJ et al 1995). It seemed a logical step from there to produce vectors that had noviral genes at all. This did not produce any significant benefits orimprovements from the previous agents. A number of research teamsacross the world tried different subsidiary strategies including druginduced immunosuppression or modifications of various immunogenicepitopes. The plasmid-lipid complexes appeared to have a number of clinicallyimportant advantages insofar as they did not appear to generate anyimmunological response which is in distinct contrast to many viralvectors. Initial optimism did not appear to be translated intopractical application as the delivery systems explored appeared to beunable to deliver sufficiently large quantities through thepathological mucous layer that is the main feature of the cysticfibrosis patient. (Crystal RG 1992). The adeno-associated vectors have received a large amount of attentionwhen it became clear that alternative vectors were needed to optimisethe therapeutic effect. They have now reached the stage where animaltesting has lead to human Phase I and II clinical trials. As a group,they appear to have the advantage that they dont trigger theinflammatory reaction in the same way, or to the same extent as theadenovirus group. The major practical difficulty with this grouphowever, is the fact that because they are so small compared to thecomparatively large size of the CFTR gene it leaves no space forvector-specific sequences on which to base assays to help todistinguish the endogenous RNA from the vector-expressed RNA. (FlotteTR et al 2001) All the evidence that we have seen appears to suggest thatadeno-associated vectors have a satisfactory safety profile andcertainly appear to produce a longer duration of clinical effect thanthe other modalities. Another variable, and indeed challenge, in the field of genetherapy, is to find the optimum delivery vehicle. We have cited studiesthat have tried direct insufflation to the bronchial epithelium. Thisappears to be a superior mode of delivery to the aerosol which appearsto have the ability to cause agent specific reactions in the alveolarmembranes. There is continuing work which is currently looking at therelative merits of nebuliser delivery mechanisms as compared toconventional aerosol delivery systems. Others that have tried avoidingthe bronchial tree and utilising the respiratory epithelium byintroduction to the maxillary sinuses through intranasal antrostomies. Conclusion including future of gene therapy. In this dissertation we have presented evidence of from a number ofdifferent approaches to the problem of gene therapy in tacklingmonogenic conditions such as cystic fibrosis. As with most areas ofscientific exploration many blind alleys have to be explored beforean appropriate avenue of research becomes apparent. The initial enthusiasm the greeted the exploration of theplasmidDNA vector did not appear to be well founded although it isclear that further exploratory work is continuing in the field. The area of adeno-associated vectors appears to be currently themost promising with, at least in vitro, suggestions that many of thecurrent limiting problems may be on the verge of being solved. The major stumbling blocks at the moment are the difficulties ofproducing a high vector titre in the clinical situation and the longterm safety considerations, particularly those relating to mutagenesisof ongogenes. On this point the Flotte group are optimistic and feelable to make the comment: The data from our laboratory strongly indicate that the bulk of rAAVDNA in the lung, muscle, and liver is episomal and that rAAV genomesinteract with host cell proteins such as the DNA-dependent proteinkinase in the formation of stable high-molecular weight concatemers. It is the episomal situation of the gene that is currently thoughtto be the best insurance against inadvertent iatrogenic oncogenesis(Flotte et al 2002) but this is clearly no substitute for long termcareful and rigorous safety studies. It is often assumed, quite incorrectly, that the field of genetherapy is a discrete and academically isolated field. Progress in thisarea, as in so many other areas of research, is completely dependent ofdiscoveries and improvements in other areas of science. The future direction of research will be determined, to a degree,by improvements in our ability to manipulate cell types and cell linesoutside of the body as this will inevitably aid our ability to implantgenetically engineered agents. Reflection over the advances inknowledge from just the last decade indicates that new and innovativedelivery mechanisms will be developed, explored and evaluated. It islikely that the known short term problems of immunogenicity, low titredelivery and inefficient packaging will be addressed, very possiblywith new delivery vectors. It goes without saying that these investigations are hugelyexpensive in terms, not only of money, but of time, expertise andinvestment generally and therefore it is likely that the limitingfactor in terms of development will be the availability of resources(Russell S 1997) References Afione, SA, Conrad, CK, Kearns, WG, et al (1996) In vivo model of adeno-associated virus vector persistence and rescue. J Virol 70,3235-3241 Alton,EW et al. 1993 Nature Genetics. vol 5. 1993 Amado R G Chen YJS 1999 Lentiviral Vectorsthe Promise of Gene Therapy Within Reach? Science. 285 (5428): 674-76. BBC Online News. Bubble boy saved by gene therapy. 3 April 2002, Berns, KI, Linden, RM (1995) The cryptic life style of adeno-associated virus. Bioessays 17,237-245 Blacklow NR. 1988 Adeno-associated viruses of humans. In: Pattison JR, ed. Parvoviruses and human disease. Boca Raton, FL: CRC Press; 1988; 165174 Blacklow, NR, Hoggan, MD, Kapikian, AZ, et al (1989) Epidemiology of adenovirus-associated virus infection in a nursery population. Am J Epidemiol 88,368-378 Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, Durham SR, Jeffery PK, Hodson ME, Coutelle C, et al.1995 Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995 Jan;1(1):39-46.. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al.2000 Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288: 669-72: Conrad, CK, Allen, SS, Afione, SA, et al (1996) Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther 3,658-668 Crystal RG. 1992 Gene therapy strategies for pulmonary disease. Am J Med 1992; 92:44S-52S Dark, J., et al. 1996 Transplantation. Shale, D. J., ed. Cystic Fibrosis. BMJ Publishing Group. 1996. pp120-133. Dong JY, Fan PD, Frizzell RA: 1996 Quantitative analysis of the packaging capacity of recombinant adeno- associated virus. Hum Gene Ther 1996, 7:2101-2112. Duan D, Sharma P, Yang J, Yue Y, Dudus L, Zhang Y, Fisher KJ, Engelhardt JF: 1998 Circularintermediates of recombinant adeno-associated virus have definedstructural characteristics responsible for long-term episomalpersistence in muscle tissue. J Virol 1998, 72:8568-8577. Duan D, Yan Z, Yue Y, Engelhardt JF: 1999 Structural analysis of adeno-associated virus transduction circular intermediates. Virology 1999, 261:8-14 Duan, D, Yue, Y, Yan, Z, et al (2000) A new dual-vector approach to enhance recombinant adeno-associatedvirus-mediated gene expression through intermolecular cis activation. Nat Med 6,595-598 Egan, M, Flotte, T, Afione, S, et al (1992) Defective regulation of outwardly rectifying Cl-channels by protein kinase A corrected by insertion of CFTR. Nature 358,581-584 Erickson,R 1993 New Scientist. 2 Sep 1993. Felgner P. 1997 Nonviral strategies for gene therapy. Sci Am 1997;276:86-91. Flotte TR, Afione SA, Solow R, Drumm ML, Markakis D, Guggino WB, Zeitlin PL, Carter BJ:1993 Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem 1993, 268:3781-3790. Flotte, TR, Afione, SA, Zeitlin, PL (1994) Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol 11,517-521 Flotte, T, Carter, B, Conrad, C, et al (1996) A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease. Hum Gene Ther 7,1145-1159 Flotte, TR, Carter, BJ (1998) Adeno-associated virus vectors for gene therapy of cystic fibrosis. Methods Enzymol 292,717-732 Flotte TR 2000 Size does matter: overcoming the adeno-associated virus packaging limit Respiratory Research 2000, 1:16-18 Flotte TR Laube BL 2001 Gene Therapy in Cystic Fibrosis* Chest. 2001;120:124S-131S. Flotte TR 2002 Recombinant Adeno-Associated Virus Gene Therapy for Cystic Fibrosis and 1-Antitrypsin Deficiency Chest. 2002;121:98S-102S. Freeman SM, Whartenby KA, Freeman JL, Abboud CN, Marrogi AJ. 1996 In situ use of suicide genes for cancer therapy. Semin Oncol1996;23:31-45. Zhang J, Russell SJ. Vectors for cancer gene therapy. Cancer Metastasis Rev 1996;15:385-401. Griesenbach U, Ferrari S, Geddes D Alton EW 2002 Gene therapy progress and prospects: cystic fibrosis. Gene. Ther. 2002 Oct;9(20):1344-50. Grimm, D, Kern, A, Rittner, K, et al (1998) Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum Gene Ther 9,2745-2760 Hyde,SC et al. 1993 Nature. vol 362. 1993. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, et al. 2000 Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nature Genet 2000;24: 257-61. Kay MA, Nakai H. 2003 Looking into the safety of AAV vectors. Nature 2003;424: 251. Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR:1996 Recombinant adeno-associated virus (AAV-CFTR) vectors do not integratein a site-specific fashion in an immortalized epithelial cell line. Gene Ther 1996, 3:748-755. Knowles MR, Noone PG, Hohneker K, Johnstone LG, Boucher RC, Efthimoiou J, Crawford C, Brown R, Schwartzbach C, pearlman R 1998 A double-blind, placebo controlled, dose ranging study to evaluate thesafety and biological efficacy of the lipid-DNA complex GR213487B inthe nasal epithelium of adult patients with cystic fibrosis. Hum Gene Ther. 1998 Jan 20;9(2):249-69. Lehrman S. 1999 Virus treatment questioned after gene therapy death. Nature 1999;401: 517-8 Moss RB, Rodman D, Spencer LT, Aitken M, Zeitlin P, Waltz D, Milla C, Brody AS, Clancy JP, Ramsay B, Hamblett N, Heald A, 2004 Repeated adeno-associated virus serotype 2 aerosol-mediated cysticfibrosis transmembrane regulator gene transfer to the lungs of patientswith cystic fibrosis : a multicenter, double-blind, placebo-controlledtrial clinical investigations Chest Feb 2004 Muzyczka N: 1994 Adeno-associated virus (AAV) vectors: will they work? J Clin Invest 1994, 94:1351. Naffakh N, Henri A, Villeval JL, Rouyer-Fessard P, Moullier P, Blumenfeld N, et al. 1995 Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts. Proc Natl Acad Sci U S A 1995;92:3194-8. Naldini, L., et al. 1996 In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996. 272: 263-267. Nichols, E. K. 1998 Human Gene Therapy. Cambridge, Massachusettes: Harvard University Press, 1998. Olsen, J. C. 1998. Gene Transfer Vectors Derived From Equine Infectious Anemia Virus. Gene Therapy. 5: 1481-1487. Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, et al. 1997 Evidence for safety and efficacy of DOTAP cationic liposome mediatedCFTR gene transfer to the nasal epithelium of patients with cysticfibrosis. Gene Ther 1997;4:210-18. Relph K, Kevin Harrington, and Hardev Pandha 2004 Recent developments and current status of gene therapy using viral vectors in the United Kingdom BMJ, Oct 2004; 329: 839 842 ; Rolls, M. M., et al. 1999 Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self replication RNA. Cell. 1999. 79: 497-506. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan AA, Moen R, et al. 1990 Gene transfer into humans: immunotherapy of patients with advancedmelanoma using tumor infiltrating lymphocytes modified by retroviralgene transduction. N Engl J Med 1990;323:570-8. Russell SJ 1997 Science, medicine, and the future : Gene therapy BMJ, Nov 1997; 315: 1289 1292 Santis,G. Geddes,D. 1994 Post Grad Med J. vol 70. 1994. Sikorski R, Peters R 1998 Gene Therapy: Treating with HIV. Science. 282 (5393): 1438a. Song, S, Morgan, M, Ellis, T, et al (1998) Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc Natl Acad Sci U S A 95,14384-14388 Song, S, Embury, J, Laipis, P, et al (2001 A) Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT)after portal vein injection of recombinant adeno-associated virus(rAAV) vectors. Gene Ther 8,1299-1306 Song, S, Laipis, PJ, Berns, KI, et al (2001 B) Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle. Proc Natl Acad Sci U S A 98,4084-4088 Stern M, K Ulrich, D M Geddes and E W F W Alton 2003 Poly (D,L-lactide-co-glycolide)/DNA microspheres to facilitate prolongedtransgene expression in airway epithelium in vitro, ex vivo and in vivo Gene Therapy (2003) 10, 1282-1288. Stribling,R et al. 1992 Proc Nat Acad Sci. vol 89. 1992. Summerford, C, Samulski, RJ (1998) Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 72,1438-1445 Sun L, Li J, Xiao X: 2000 Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat Med 2000, 6:599-602. Teramoto, S, Bartlett, JS, McCarty, D, et al (1998) Factors influencing adeno-associated virus-mediated gene transfer tohuman cystic fibrosis airway epithelial cells: comparison withadenovirus vectors. J Virol 72,8904-8912 Virella-Lowell, I, Poirier, A, Chesnut, KA, et al (2000) Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients. Gene Ther 7,1783-1789 Wagner, JA, Moran, ML, Messner, AH, et al (1998) A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis. Hum Gene Ther 9,889-909 Wagner, JA, Messner, AH, Moran, ML, et al (1999) Safety and biological efficacy of an adeno-associated virusvector-cystic fibrosis transmembrane conductance regulator (AAV-CFTR)in the cystic fibrosis maxillary sinus. Laryngoscope 109,266-274 Wagner JA, Nepomuceno IB, Messner AH, et al. 2002 A phase II, double-blind, randomized, placebo-controlled clinical trialof tgAAVCF using maxillary sinus delivery in CF patients withantrostomies. Hum Gene Ther 9,889-909 2002 Wu, P, Xiao, W, Conlon, T, et al (2000) Mutational analysis of the adeno-associated virus type2 (AAV2) capsidgene and construction of AAV2 vectors with altered tropism. J Virol 74,8635-8647 Yan, Z, Zhang, Y, Duan, D, et al (2000) From the cover: trans-splicing vectors expand the utility of adeno- associated virus for gene therapy. Proc Natl Acad Sci U S A 97,6716-6721 Yoshimura,K et al. 1992 Nuclei Acids Res. vol 20. 1992. Zabner J, Cheng SH, Meeker D, Launspach J, Balfour R, Perricone MA,Morris JE, marshall J, Fasbender A, Smith AE Department, Welsh MJ1997 Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J,Clin. Invest. 1997 Sep 15;100(6):1529-37. Zabner, J, Seiler, M, Walters, R, et al (2000) Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apicalsurfaces of airway epithelia and facilitates gene transfer. J Virol 74,3852-3858 Zolotukhin, S, Byrne, BJ, Mason, E, et al (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6,973-985