Research & Discovery

Technical Notes

Gene therapy and protein therapy
by David Busch, Ph.D., M.D.

These two forms of therapy are commonly referred to when there is talk about finding a cure for XP.

XP patients may have a defect in any one of seven genes that normally provide the cell with information on how to make a protein that is important in repairing UV induced DNA damage. Gene therapy is a proposed method of changing the cell’s content of DNA so that the cell can make the missing protein, resulting in normal DNA repair and normal sun sensitivity. Protein therapy involves introducing the missing protein directly into the cell.

A form of protein therapy is in the process of undergoing clinical trials, while gene therapy for XP currently is science fiction but may become available within the next few years. Neither therapy will do anything to repair damage that has already occurred, such as freckling, still developing skin cancers, and neurological degeneration, although both could be helpful in minimizing future damage from future sun exposure. Here is a brief description of the techniques and their possible limitations.

Protein therapy is available to persons currently enrolled in Applied Genetics, Inc.’s T4N5 endonuclease study. In this study, XP patients are given a skin lotion containing a viral protein that simulates the action of the protein that XP patients lack. When the lotion is rubbed into the skin, the viral protein enters skin cells. It is hoped that this will result in the skin cells temporarily becoming more like normal cells, so that they repair UV damaged DNA better and there is less sunburning and skin cancer. The lotion method is analogous to giving insulin to diabetics; in that case, there is a problem with the body not making enough of an important protein (insulin), and the treatment is to give insulin injections to provide close to a normal level of protein.

It has not been proved that the lotion works, although it is likely to be useful; finding out if it works is the purpose of the clinical trials. The protein is not expected to persist in the skin indefinitely; it thus will be necessary to reapply it frequently, like a sunscreen. Thus, this really is intended as a treatment but not a cure. Protection is expected to exist only where the lotion is applied i.e., to the skin; other sun sensitive tissues such as eyes, nostrils, and front of mouth will not be protected if only the skin is treated; also, the protein does not enter the brain and thus should be of no value in preventing the development of neurological problems in the 20% (approximately) of XP patients with such problems.

A logical future development would be to instead administer a lotion containing the exact protein that the XP patient is missing, meaning that there would have to be seven different lotions for the seven different proteins defective in different XP patients with abnormal UV excision repair. This might work better than the viral protein. An advantage of the lotion is that if there are any undesired side effects, these should disappear immediately after use is discontinued.

Gene therapy may be attempted soon with XP patients in France. In this method, either an extra, normal copy of the defective gene is introduced into the cell so that there is an artificial normal gene in apition to the two abnormal genes the patient is born with; or else the abnormal genes are modified inside the cell so that they become normal genes. In either case, this means that the cell can now make the missing protein. Here are some of the many potential problems with gene therapy:

  1. It is very difficult to put genes into enough cells in a live person to make a difference in the person’s health.
  2. Even if the gene is introduced into the cell, it is possible that the cell may have difficulty using the gene to make the missing protein.
  3. The cell may lose the normal gene over time, in which case the therapy may need to be repeated periodically.
  4. It may be easier to treat some cells than other cells. It is likely that gene therapy will soon be available for the skin surface cells that produce most skin cancers, but it may take longer to invent effective therapy for the deeper skin cells that produce melanomas; and treating brain cells to prevent neurological problems may be much more difficult still.
  5. The therapy, if successful, will cause the body to make a protein that the body previously lacked, which could result in allergic reactions such as are seen in kidney rejection. If the allergic reactions are severe enough, it could be necessary to take toxic and dangerous immunosuppressive drugs to inhibit the allergic reaction.
  6. Depending on the method used, it is possible that the cellular DNA could be mutated extensively by the treatment, resulting in cancers from the therapy itself.

As more is learned about these techniques, it is likely that XP patients will benefit greatly from their use and lead much more normal, less restricted lives. However, it is important to realize that much still needs to be done in the way of developing and validating these techniques and in learning about their possible risks and limitations. XP patients should welcome the development of these techniques as a possible way of minimizing the consequences of their disease; but at the same time they must realize the limitiations of the techniques, and should be careful to learn of potential problems with the techniques and to weigh the potential risks against the possible benefits when considering their use, especially in the case of gene therapy.

July 1997

The XP Society is the international authority for XP family support and information in making intelligent decisions regarding the caregiving of XP family members.

Complementation group assignment
by David Busch, Ph.D., M.D.

There are four laboratory tests that are particularly likely to be used in study of XP patients’ cells, of which the first two currently are in use in my laboratory, and the third is in the process of being introduced and is the main subject of this discussion .

The first method is the UV survival curve study, which measures how UV sensitive are the cultured cells i.e., how much UV is required to kill a given percentage of cells. XP patients may have anywhere from normal UV sensitivity (unusual) to up to about 10x normal UV sensitivity. Measuring their UV sensitivity this way provides one way of describing the severity of their defect.

The second method is the unscheduled DNA synthesis (UDS) assay, which measures proficiency of repair of UV induced DNA damage; this is the method that was provided by Dr. James Cleaver when he discovered the defect present in XP. Except for the occasionally encountered XP variants, who have the skin lesions of XP but normal UDS, XP patients may have anywhere from O% to about 50% of normal UDS. It is possible to have close to normal UV sensitivity with low UDS, or relatively high UDS but extreme UV sensitivity, so that UDS is another way of measuring the degree of the defect that is independent from UV sensitivity. The UDS assay is the most likely method to use when prenatal diagnosis is done, because results can be obtained quickly using relatively few cells,

The third method, complementation group assignment, determines what gene is affected in the patient. Seven different genes (XP group A through G genes) have been found to be affected in different XP patients. The severity and nature of the XP patient’s problems tend to correlate with what gene is affected, although it is important to realize that exceptions can occur due to differences in how the gene is mutated (see next paragraph). In general, XP group C, E, and F patients (and also XP variants) are spared the neurological problems that affect about a fifth of XP patients, while neurological problems may or may not occur in groups A, D, and G. Thus, if one is told one is in group C (the commonest group), one can generally rest easy from the standpoint of worrying about neurological problems; while if one is told one is in group A one is likely to worry a lot more, although there still is uncertainty about whether neurological problems will develop. Probably many would prefer to not be told their complementation group. Individuals in different complementation groups tend to have different degrees of deficiency in UV sensitivity and in UDS, so that it is possible usually to make an educated guess about what gene is affected if the first two tests have been done, realizing that this is only a guess and not reliable. For example, Katie Mahar’s lab tests indicate that she probably is in group C, and this certainly is consistent with her complete lack of any neurological problems, so that when we try to find out her complementation group we will first see if she is in group C before looking into other groups. However, it still is possible that she could be in another group, since her initial lab tests are no substitute for a complementation test. Different techniques exist for performing complementation group assignment, but generally they involve determining whether performing a laboratory procedure results in an improvement in the cell’s UDS. In my laboratory, the preferred method is DNA microinjection, in which the nucleus of a cell is injected with the normal version of an XP gene to see if the gene increases UDS, in which case the gene is proved to be same gene as is affected in the patient. Another popular method is somatic cell hybridization, in which the patient’s cell is fused with a known type of XP cell so that two cells are used to make one hybrid cell, and then the UDS test is performed on the hybrid cell. In that case, UDS will increase if the known XP cell is affected in a different gene than the patient’s cell. Knowing the complementation group will become very important in the future when gene therapy becomes available, since it will indicate what gene needs to be administered in the therapy.

The fourth method, allele identification, is used to determine in what way the patient’s affected gene is abnormal. There are thousands of ways (alleles) in which a gene can have an abnormal structure that could result in XP; and the way the gene is abnormal could greatly affect UV sensitivity, UDS level, and even the chances of developing neurological problems. Thus, XP group A patients tend to be the most UV sensitive, to have the lowest UDS levels, and to have the worst neurological problems with the earliest onset. However, if a certain region of the XP group A gene is affected, there may be no neurological problems, or at least greatly delayed problems. Information about the severity and progression of cases of XP is not available for all alleles, so it is possible that a patient could learn what is the allele but may have no way of knowing the health significance of the allele; indeed the same patient may eventually provide that information to researchers as the person ages and the allele’s significance becomes clearer. Thus, there is currently a need for a database in which clinical features are correlated with allele present; and it will take years to accumulate such a database for all naturally occurring XP gene alleles.

July 1997

The XP Society is the international authority for XP family support and information regarding making intelligent decisions in the caregiving of the XP family member.



Early Diagnosis of XP -
A Life Extending Endeavor

by Joseph Malak, M.D.; CHP Poughkeepsie, NY

Xeroderma pigmentosa (XP) is a very rare, heritable disorder whose distinguishing feature is an increased sensitivity to ultraviolet (UV) light. Early diagnosis and intervention can delay the onset of its many complications – including premature death.

The appearance of the first symptoms of XP is typically between 1 and 2 years of age. About half of the children present with a history of severe sun burns on minimal exposure to sunlight. The others present with numerous freckle-like spots on sun-exposed parts of the body. Later on, other symptoms – including premature aging and cancers of the skin, eye problems, and neurologic abnormalities – can develop.

The physician seeing a small child with numerous pigmented lesions on sun-exposed areas will probably suspect XP quickly, even if the child has a fair complexion. The small child with a history of sunburn may be a bit more problematic. If the doctor suspects that more than simply too much sun exposure was involved, the child will likely first be tested for porphyria, a disorder of red cell metabolism. This condition occurs more often than does XP. If the testing for porphyria is normal, and the child has an otherwise normal appearance (this excludes some other rare disorders also showing sun sensitivity), the child should be tested for XP. This involves measuring the activity of DNA repair enzymes in a skin biopsy specimen. Only a tiny piece of skin is needed and is usually obtained from the upper arm.

The workup of XP can take several months to complete. From the moment that XP is considered a reasonable possibility, it is important for the doctor to advise parents to carefully reduce, if not totally avoid, UV light exposure. Experience has shown that early avoidance can delay the onset of complications and lead to a healthier – and happier – patient.

The XP Society is the international authority for XP family support and information regarding making intelligent decisions in the caregiving of an XP family member.



Reconstruction of DNA repair-deficient xeroderma pigmentosum skin in the laboratory

This breakthrough development, published in July 2001, should enable research to be conducted without direct experimentation on XP patients. The full title of this work is:
“Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro” by Françoise Bernerd, Daniel Asselineau, Corinne Vioux, Odile Chevallier-Lagente, Bakar Bouadjar, Alain Sarasin, and Thierry Magnaldo

T4N5 Liposome Lotion Study Findings Published

AGI Dermatics has published the T4N5 study results.

This DNA repair lotion reduced the occurrence of the most common form of skin cancer, basal cell carcinoma, by 30% and a pre-cancerous form of skin cancer, actinic keratosis, was reduced by 68%.

The study was conducted among patients with xeroderma pigmentosum because they lack DNA repair and develop skin cancer in childhood.

The new drug, T4N5 Liposome Lotion – trade name DimericineT – was developed by Applied Genetics Inc. Dermatics. It delivers a purified DNA repair enzyme in liposomes to the skin to reverse sun damage to DNA and thereby prevent skin cancer. The drug supplements the missing enzyme in this disease and reduces the rate at which cancers develop.

Daniel Yarosh, Ph.D., president of AGI Dermatics, presented his findings at the March American Academy of Dermatology Meeting.

The XP Society is the international authority for XP family support and information in making intelligent decisions regarding the caregiving of the XP family member.


Functional Retroviral Vector for Gene Therapy of Xeroderma Pigmentosum Group D Patients

by M. Carreau, X. Quillet, E.Eveno, A. Salvetti, O. Danos, J-M. Heard, Alain Sarasin, et al.
Human Gene Therapy 6:1307-1315 (Oct. 1995)
Mary Ann Liebert, Inc.

Overview Summary
The first step toward gene therapy for the DNA repair disease xeroderma pigmentosum (XP) has been established by Carreau et al. They report the construction of a retroviral vector containing one of the DNA repair genes, the XP complementation group D previously known as ERCC2. Gene transfer was carried out on fibroblasts from two XP complementation group D patients referred to their laboratory. Complete phenotypic correction of the DNA repair defect in cells from both patients was achieved after the introduction of a functional copy of the XPD gene. This vector will then be used to modify keratinocytes to produce repair proficient reconstituted skin for engraftment at the most exposed areas. In this view, this study represents the first step toward long-term skin cancer therapy for XP patients.

The XP Society is the international authority for XP family support and information in making decisions in the caregiving of the XP family member. - database of allelic variations of XP genes
This Website, curated by Dr. James E. Cleaver and sponsored by the XP Society, hosts an interactive repository of mutations and other allelic variations of the genes involved in the DNA repair disorders, Xeroderma Pigmentosum (XP), Cockayne Syndrome (CS), Trichothiodystrophy (TTD), and other UV-sensitivity disorders.

Reconstruction of DNA repair-deficient xeroderma pigmentosum skin in the laboratory

Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomised study

The Ad Hoc Group for Medical Research Funding

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