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Natural Gene Therapy for Precision and Safety

Spontaneous reversions of inherited disease mutations point the way to new approaches in gene therapy Dr. Mae-Wan Ho

Another example of natural versus artificial genetic modification

Natural gene therapy is the ability of cells in individuals with hereditary disease to back mutate the specific gene involved to regain lost function, thereby ameliorating the disease. This remarkable phenomenon is receiving increasing attention, thanks to cell sorting and DNA sequencing techniques that enable researchers to detect and analyse even rare populations of mutant cells.  It looks like another example of the precise natural genetic modification that cells and organisms carry out on a routine basis in order to better survive.

In a recent review article, “The new genetics and natural versus artificial genetic modification” [1], I contrasted the precision of natural process with the crude, artificial counterpart that inevitably damages the genome and interferes with natural genetic modification, which incidentally also explains [2] Why GMOs Can Never be Safe (SiS 59). Among the most exquisite examples of natural genetic modification is the ability of cells and organisms to activate or mutate just the right genes in order to overcome an obstacle to growth [3] (Non-Random Directed Mutations Confirmed, SiS 60). In microorganisms, such non-random mutations are obviously adaptive; though not so in multicellular organisms in which cells acquiring a mutation to multiply may mean cancer. But there are situations where such apparently non-random mutations can benefit the organism as a whole, and natural gene therapy is one of them.

Somatic mosaics and spontaneous reversions of inherited disease mutations

It has long been assumed that except for lymphocytes in the blood, which undergo genetic rearrangements and hypermutations to generate antibodies and other proteins of the immune system, all cells in the body carry the same genome. And even lymphocytes with genetic rearrangements in the immunoglubulins should have the same gene sequences in the constant subunits of those proteins and in any other gene in the genome. However, researchers are discovering to their surprise that most individuals are genetically multiple personalities; they have populations of somatic cells with different genomes, a condition known as somatic mosaicism. Of particular interest are those resulting from reversion to normal of disease mutations inherited from their parents [4-6].

Mosaicisms resulting from reversion to normal of an inherited mutation were discovered because of milder than expected clinical course and/or the presence of both phenotypically normal and abnormal cells in vivo and in vitro.

To-date, diseases for which spontaneous reversions have been identified include tyrosinaemia type 1, X-linked severe combined immunodeficiency (X-SCID), autosomal adenine deaminase (ADA) SCID, Wiskoff-Aldrich syndrome, Bloom’s syndrome, epidemolysis bullosa, Fanconi anaemia,  X-linked ectodermal dysplasia and immunodeficiency, leucocyte adhesion deficiency type 1 [6], Duchenne muscular dystrophy [7], Charcot-Marie-Tooth disease type 1A [8], and Lesch Nyhan syndrome [9].

There are no systematic data on the prevalence of spontaneous reversions, but they may be more common and involve a wider range of inherited diseases than reported so far. Spontaneous revertant lymphocytes are found in 20 % of Bloom syndrome patients [4, 5], up to a level of 75 %. In hereditary tyrosinemia type (IHT1), a severe disease affecting primarily the liver, reversion was observed in 88 % of patients; with reverted surfaces of the liver ranging from 0.1 % to 85 % [10].  And more than 1/3 of patients with epidemolysis bullosa, a condition involving blistering of the skin, have revertant skin patches [11].

We shall look at some examples in more detail.

X-linked severe combined immunodeficiency (X-SCID)

The latest report of natural gene therapy occurred in a patient suffering from X-SCID caused by mutations in the gene IL2RG coding for the gamma immunoglobulin chain (gc) common to the receptors for several cytokines (signalling molecules secreted by cells of the immune system): interleukin (IL)- 2, IL-4, IL-7, IL-9, IL-15 and IL-21, which signal T (thymus) and NK (natural killer) cell to multiply. Mutations in the gene abolish the function of all of these receptors, resulting in the absence or diminished numbers of T and NK cells critical to the innate immune system; while B cells that secrete antibodies into the blood stream are normal. Consequently, afflicted individuals often have infections very early in life, and usually die two years after birth. X-linked diseases usually appear in males, who have only one X chromosome, while females with two X chromosomes are less likely to have the disease, but can be heterozygous carriers with one X chromosome bearing a normal ‘wild type’ allele (form of a gene), and the other the mutant allele.

The patient, a boy, was diagnosed at 6 years of age with normal lymphocyte counts, but suffered from recurrent pneumonia and mollusca contagiosa (viral infection affecting the skin) [12]. As proliferative response of T cells and NK cells to the gc interleukins was poor, the researchers analysed the gene IL2RG. This turned up two forms of the gene, one mutant and the other normal, despite the fact that there was only one X chromosome. The normal version predominated in both naïve and mature CD8+ T cells, which increased over time. A fraction of gd+T cells (subpopulation of T cells abundant in gut mucosa) and differentiated effector memory T cells carried the reversion, while NK or B cells repeatedly tested negative. The patient has steadily improved over the past 7 years since diagnosis, only suffering once from an atypical pneumonia caused by Mycoplasma pneumonia; and after several years, his molluscum contagiosum started to disappear spontaneously as well.

The mutation inherited by the boy occurred in the extracellular part of the protein which sits in the cell membrane, resulting in the replacement of tyrosine 219 with asparagine. The reversion was a back mutation that restored tyrosine. Neither the mutation nor the reversion had been reported previously.

Other reversions of X-linked SCID mutations have been reported earlier. A patient with low to normal numbers of T cells and normal expression of gc chain in his T cells was diagnosed at one year of age [13].  At 6 month of age, the patient was treated for a large abscess containing bacilli Calmette-Guerin, probably resulting from a vaccination with bacilli Calmette-Guerin two weeks after birth. The abscess was successfully treated by a regimen of three antituberculosis drugs. Over the following two years, the patient had no further infections complications. He had been living at home for 12 months in good health, and continued to receive antibiotic prophylaxis as well as monthly infusions of immune globulins.

Direct genomic sequence analysis of the patient’s B-cell lines detected a single point mutation in the gc gene with a base change from T to C at position 343 in exon 3, corresponding to amino acid 115, replacing the normal cysteine codon with one coding for arginine. DNA analysis revealed that the patient’s mother was heterozygous for this mutation, so his normal T cells could have been derived from the mother. But T cell karyotyping ruled out this possibility. Like the more recent case of spontaneous reversion, neither NK cells nor B cells tested normal. So the reversion must have occurred after the T cell lineage separated from the B and NK progenitor cells in the bone marrow.

Both research teams favour an explanation for a reversion event conferring a distinct selective advantage in vivo. However, as the response of T cells to gc interleukins was poor, it is not clear what the selective advantage could be.

Adenosine deaminase deficiency severe immunodeficiency syndrome (ADA-SCID)

ADA-SCID is an autosomal recessive disorder, i.e., one depending on genes encoded on chromosomes other than the sex determining chromosomes, and two mutant copies are needed for the disease to appear. It is characterized by multiple viral, fungal and bacterial infections early in life and marked failure to thrive; and in the absence of therapy, death before age one. Two unrelated patients with ADA-SCID were presented early in life with apparently life-threatening disease [4]. But instead of dying as expected during infancy or early childhood, they had improved over time, and were alive 12 and 18 years later respectively. The older received no bone marrow transplant because a matched sibling donor was lacking, he received partial exchange transfusions intermittently, but had not had any therapy for several years. The younger had a sibling who died of the disease before 2 years of age. He had not received any therapy for religious reasons. In both patients, somatic mosaicism was identified as the probable basis for the unusually mild clinical course.

In the older child, a missense mutation was identified in a B-lymphoid cell line and in fibroblasts, but the mutation in the second allele could not be identified. Fourteen years later, however, fresh B lymphoid cell line was obtained from the patient. This enabled the researchers to identify the second, splice-site mutation that resulted in an unstable mRNA. But in the B cell line established 14 years later, the splice site mutation was gone, and 50 % of expressed ADA mRNA was normal while 50 % carried the previously described missense mutation.

In the second unrelated younger patient, blood samples were obtained from both the parents as well as the patient. In the patient the maternally transmitted missense mutation was found in 13/15 B cell lines and in only 17% of single alleles cloned from blood DNA. The maternal missense mutation was only 11 bp upstream of the splice site mutation from the father. Although the results suggested site specific reversion, they could not rule out intragenic recombination (exchange of parts in the same gene) or gene conversion of a short tract, where one allele acts as template for converting the other. The patient had greater residual immune function and lower concentrations of toxic metabolites compared with the other family members carrying the same inherited mutations. He also had substantial ADA activity and enzyme protein in both T and B cell lines and was relatively healthy. Enzyme replacement, while further lowering toxic metabolites was accompanied by diminution in the number of revertant cells, which should be a strong counter-indication for the treatment.

Reversions have been reported in three additional ADA-SCID patients. Two compound heterozygous patients exhibited site specific single nucleotide reversions. Again recombination between the two alleles (intragenic recombination) or gene conversion cannot be ruled out.

More recently, an ADA-SCID patient with a single allele reversion of a mutation in T cells was given enzyme placement therapy; this led to the disappearance of the revertent T cells after three months, development of a germinal cell tumour, and death at the age of 67 months from sepsis [14]. The boy was diagnosed at age of 1 month. By 23 months, he suffered several moderate to severe infections and failure to thrive. But he showed increased lymphocyte counts that were mostly T cells though still below normal for age. In contrast, B cell counts had remained unchanged while NK cell counts improved slightly. By age 50 months, the patient already exhibited normal numbers of total lymphocytes but suffered multiple infections and chronic lung damage despite the continued use of prophylactic antibiotics and intravenous immunoglobuliins. He was placed on enzyme replacement therapy with PEG-ADA (polyethylene-glycol modified bovine ADA).

This highlights the lack of understanding on the physiology of spontaneous reversions, and the treatment appropriate for such cases. All the signs are that spontaneous reversions can ameliorate severe disease symptoms and may even restore health. So it is important to understand the precise mechanisms that bring about such spontaneous reversions, and the environmental interventions most appropriate for promoting what amounts to natural gene therapy. 

Frequent mutation reversions in hereditary tyrosinemia type 1 (HT1)

Spontaneous reversions are not restricted to mutations affecting haemopoietic cells. HT1 is a severe liver disease affecting also the kidneys and nervous system, caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme in the catabolic pathway of tyrosine. HT1 is clinically heterogeneous, with no correlation between genotype and phenotype. There are two main forms. The acute form is characterized by liver failure in the first months of life and death within the first year if untreated. The chronic form is milder with chronic liver disease, renal tubular dysfunction and hypophosphatemia with rickets. Chronic patients are at high risks of developing hepatocellular carcinoma later in life. Both forms often present with extensive liver injury and regeneration of liver cells. Initially, the primary effective therapy was liver transplant, but since 1992, drug therapy with 2-(2-nitro-4-trifluoro-methylbenzoyl)-1, 3-cyclohexane-dione (NTBC) has been used to reduce the accumulation of toxic metabolites and ameliorate the several clinical symptoms. There are 41 known mutations in the FAH gene but no clear relationship exists between a particular mutation and clinical manifestation. Different mutations have been found to revert to normal in patients.

In a study carried out on the livers of 26 French Canadian HT1 patients who underwent liver transplant, spontaneous reversion was found in 88 % of the patients with reverted surfaces ranging from 0.1 % to 85 % [9]. The most common mutation in the sample was found in all but two of the patients, and in homozygous form in 21 of them. Three other rare mutations were identified.  The reverted cells were found as nodules of regeneration, and they have feature of normal liver cells. Reversion was correlated with amelioration of disease. The surface of reversion was 1.6 % of the liver in the group of acute patients who had severe hepatic crises. In contrast, the average surface of reversion was 36 % in the chronic group who did not have hepatic crises, with 1 exception. In the subacute group, the average surface of reversion was 2.8 % in patients with symptoms similar to those of the acute group, whereas a higher surface of reversion (22 %) was found in those showing more chronic symptoms.

One patient with no hepatic or neurological crises was in good health, and had discontinued her restrictive diet without any reported deterioration. Transplant was done at age 10 years and 9 months solely to avoid eventual development of hepatocellular carcinoma. Her liver had an almost normal appearance with little fibrosis and a surface of reversion of 85%, the highest observed.

The unusually high reversion rates found in this condition has been attributed to the mutagenicity of the accumulating metabolite fumarylacetoacetate, and the selective advantage of normal cells. However, there was no report that mutations in other genes had increased, or that normal cells can invariably outcompete mutant cells. Mostly, normal and mutant cells tended to co-exist.              

Duchenne muscular dystrophy (DMD)

DMD is a recessive X-linked form of muscular dystrophy which results in muscle degeneration and eventual death. It is caused by a mutation in the very large dystrophin gene coding for an important component within muscle tissue that provides structural stability to the dystroglycan complex of the cell membrane [15]. Dystrophin positive fibres have been found in as many as 40 % of DMD patients [7]. Such dystrophin positive fibres also occur in cardiac muscle and in skeletal muscle of the mouse disease model. A study carried out in the 1990s investigated the prevalence of dystrophin-positive fibres in muscles of MDM patients by direct counting on immunostained sections in biopsy specimens from 85 patients. Dystrophin positive fibres were found in 22 specimens (26 %) at frequencies ranging from 0.01 to 6.81 %. Frequencies over 1 % were found only in patients older than 6 years. Of the 42 DMD cases screened by cDNA probes, 32 had an intragenic deletion, and dystrophin-positive myofibres were found in 14 (33 %) of them. In 9 patients, the deletions involved 1 to 3 exons and in only 1 patient 9 exons. All deletions were located between exons 44 and 53.

The prevalence of dystrophin-positive fibres by counting the total number of observed positive clusters was 3.7 x 10-3 (which estimates the reversion rate per progenitor cell).

Another way to estimate the rate of reversion is from the observation that one third of all possible deletions may restore the correct reading frame, the reversion rate in these patients would be one-third of the actual rate at which deletions are produced, which is about 1 %.

Leukocyte adhesion deficiency type 1 (LAD-1)

LAD-1 is an autosomal recessive immunodeficiency caused by mutations in the b2-integrin CD18, leading to the inability of white blood cells, particularly neutrophils to adhere to the endothelium and migrate to sites of infection. Three LAD-1 patients with markedly reduced Cd18 expression in neutrophils each had a small population of lymphocytes, predominantly T cells, with normal C18 expression, and a memory/effector phenotype [6]. Microsatellite (short repeated sequences used as genetic fingerprinting) analyses proved that these originated from the patient. Sequencing showed that in each patient, one of the CD18 alleles had undergone further mutation. All three survived to adulthood without bone marrow transplants, and in all three, reversions were heterozygous.

Patient 1 was homozygous for a missense mutation of A to C at nucleotide 392, and all DNA clones derived from his CD18- lymphocytes carried the mutation. In contrast, 4/10 of CD18+ clones showed A to T mutation at nt 392, which replaced serine (codon TCT) with phenylalanine (codon TTT). The remaining 6 of 10 CD18+ clones had reversed the original missense mutation from C back to A at nt 392 for the amino acid tyrosine.  Patient 2 was homozygous for the missense G to A mutation at nt 850, which was found in all DNA clones from her CD18- cells. Her CD18+ T cells showed 3 different sequences. Five of 10 clones carried the original G to A mutation in the codon GGC (glycine) to AGC (serine), 3 of 10 clones had G to C missense mutation at nt850 leading to arginine. Two of the 1o clones had a second site mutation C to G at nt 852 in addition to the original mutation G to A at nt 850, also leading to arginine at amino acid position 284, but using two different codons. Thus, in both patients, reversion was not to the wild type but to a third amino acid that restored function.

Patient 3 was a compound heterozygote. His mutation at A to G at nt 1052 was not detected in either parent, indicating it was a de novo mutation. He also carried a splice mutation caused by a 12-bp addition to the transcript, resulting in an in-frame addition of 4 amino acids (PSSQ, proline-serine-serine-glutamine) between proline 247 and glutamic acid 248 (247 _ PSSQ). This splice mutation arose by C to A substitution in the 3’ end of intron 6, generating an aberrant splice acceptor site. Only the splice mutant had undergone reversion to wild type.

In all patients, the reverted CD18 molecules supported the proliferation response of cells to stimulation by the superantigen and IL-2 better than the mutant molecule. (Superantigens are a class of antigens causing non-specific activation of T-cells, resulting in polyclonal T cell activation and massive cytokine release.) Reversion events were not identified in progenitor cells, neutrophils or monocytes, indicating that the events did not take place at a pluripotent stem cell level, but occurred in lineage committed progenitors

Patient 1 was a white male diagnosed at 1o weeks after birth and had no matched related donors. He had sepsis at 10 months of age, followed by other problems including chronic infections, recurrent ulcers, inflammatory bowel disease recurrent, nonhealling groin and perianal ulcers requiring multiple skin grafts. Matched unrelated bone marrow transplant at age 21 years was complicated by severe graft-versus-host disease and death.

Patient 2 was a white female with omphalitis (infection of umbilical cord stump) at 10 days after birth, and diagnosed for LAD-1 at 6 years of age. After a succession of infections and inflammatory conditions followed by extensive gingivitis (gum disease) which was resolved by the removal of all permanent teeth, her gingivitis resolved at 22 years. At 28 years old, she was doing very well.

Patient 3 was a white male presented at 10 years of age with poor wound healing after tracheostromy for complicated infection of the voice box and windpipe. At 18, he developed crohn’s disease and recurrent poorly healing ulcers on the legs and thighs and severe gum disease. At 28, he had emergency surgery that resulted in removal of part of his large intestine. At 35 he started on the drug infliximab for colitis, to which he has responded well.

Bloom syndrome (BS)

BS is an autosomal recessive disorder characterized by instability of DNA. About one fifth of patients have mosaicism in lymphoid cells with a small percentage of cells exhibiting low sister chromatid exchange. (Sister chromatid exchange is a sign of DNA instability in BS.) Virtually all such individuals with mosaicism in the rate of sister chromatid exchange have been heterozygous for two different mutations, and the mosacism can be shown to result from intragenic recombination [4]. However 2/7 of the reversion patients were homozygous for the mutations, and could not have revertant cells by intragenic recombination. One patient was homozygous for the mutation 1544insA (insertion of base A at position 1544) and the other for the mutation 2702GtoA. The revertant cells were heterozygous for each of the mutations that had reverted back to the wild type, indicating that true back mutations can also occur [6].

Reversion of somatic cells in patients with inherited disorders was first detected in Bloom syndrome patients in 1977. Although the authors considered selection of the revertant phenotype as an explanation, they admitted that the role of selection in phenotype reversion in humans has been difficult to assess.

Wiskott-Aldrich syndrome (WAS)

WAS is a rare X-linked disorder characterized by thrombocytopenia (decrease in blood platelets), eczema, and immunodeficiency. It is caused by mutations in the WAS protein gene WASP. A research team identified 3 members of a single family who were WAS revertants. The first patient had a spontaneous reversion of a 6bp insertion and was described previously (see [4]). The team analysed samples from 3 additional WAS patients from the same kindred and found 2 of them also had revertant cells [16]. Molecular analysis confirmed that the same true back mutation had occurred in two of the three additional patients.  One of them had died at age 33 of renal failure, the other age 16 is in good health and on prophylactic antibiotic treatment. No WASP expressing cells was detected in the youngest patient (1 year of age). It is not known if reversion is age related.

As in the previous patient, WASP expressing cells were only detectable among T lymphocytes, but not in B cells or NK cells, suggesting that the reversion event occurred in a T-cell progenitor. Evidence for a selective advantage of WASP expressing cells is that in both patients, the percentage of WASP expressing cells was markedly higher among T memory cells than in naïve T lymphocytes.

Fanconi anaemia (FA)

FA is an autosomal recessive condition characterized by congenital abnormalities, progressive bone marrow failure, fragile chromosomes and susceptibility to cancer. There are at least 11 groups and 8 FA genes identified: FANCA, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, and FANCL/PHF9. The genes are involved in an integrated cell response to DNA damage during the stationary phase, especially from DNA interstrand crosslinks. A key step in the response is the addition of one ubiquitin molecule to the FANCD2 protein. There is very high clinical variability among FA patients, only partially attributed to different genotypes. Revertant cells may also account for the clinical variability. However, neither the frequency nor the consequence of different reversions is known. The large number of genes and possible mutations make molecular analysis difficult. Diagnosis of FA is based on the chromosome breakage and cell cycle tests, which depend on the hypersensitivity of FA cells to DNA cross-linking agents [17 and reference therein].

A study carried out in 53 FA patients in France [17] characterized by FANCD2 and chromosome breakage tests detected ubiquitinated FANCD2 (reversion) in peripheral blood lymphocytes in 8 (15%) of the patients. FA reversion was further confirmed in these patients by comparing primary fibroblasts with peripheral blood lymphocytes. Reversion was associated with higher blood counts and clinical stability or improvement.

Epidermolysis bullosa (EB)

EB is a recessive autosomal disorder typically caused by mutations in the type XVII collagen gene COL17A1. A special form of EB, dystrophic EB involves recessive autosomal mutations in the type VII collagen gene COL7A1.  A 21 year old man carrying homozygous nonsense mutation in the gene had an unaffected skin patch on his neck where blisters never occurred. The patch stained normally for collagen VII immunologically, whereas the protein was strongly reduced in affected skin. In the unaffected skin, the somatic nucleotide substitution G to T at nt6510 reverted the nonsense codon to tyrosine, thereby restoring functional protein production [18]. Reversion mosaicism is considered rare in dystrophic EB. But it might be more common than previously thought. The patient was the third identified by the authors in a short period of time, and the mechanism of reversion differed from those previously reported. The first dystrophic EB reversion mosaicism identified was due to intragenic recombination, and the second due to nucleotide deletion. The authors stated: “This is important because the natural gene therapy phenomenon may provide opportunities for revertant cell therapy.”

More than 1/3 of patients with the typical EB involving mutations in COL17A1 display revertant skin patches; correction mechanisms are highly diverse, and include back mutations, additional nucleotide changes, insertions or deletions and gene conversions, sometimes within the same patient. Reversion mosaicism has been described also for patients with EB simplex due to mutations in the keratin gene KRT14.       

Epidermal stem cells populate only small skin areas, so it seems likely that the correction mutation arose in earlier stages of embryonic epidermal stem cell development.

The authors highlighted promising therapeutic opportunities, such as revertant cell therapy, i.e., grafting ex vivo–grown reverted keratinocytes onto affected skin. Already,  keratinocytes and fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into keratinocytes, providing an unlimited source of autologous keratinocytes (see [19] The Promise of Induced Pluripotent Stem Cells, SiS 51), although such transplants with iPSCs are not without risks, and the preferred option is to encourage the patient’s own stem cells to replace lost cells in situ and in vivo.

A team at University of Groningen in the Netherlands and Free University Brussesls in Belgium had carried out a trial on a patient with typical EB [20]. The patient’s own revertant cells taken from skin biopsies were expanded in culture into sheets for transplant to replace diseased skin. The team also devised a new technique using adhesive tape to remove the diseased epidermis over an area for receiving the transplant, which is simple, effective, and almost painless. The transplant was very successful, and the acceptor site healed without scarring. Unfortunately, the replacement skin had too few revertant cells to prevent blistering.

It is likely that appropriate culture conditions are needed to promote the growth of revertant cells, again highlighting the need for investigations into the environmental/ physiological factors that may encourage progenitor or stem cells to revert to normal function and to multiply.

Directed mutation a good working hypothesis

This review makes no claim to being exhaustive. Nevertheless it already indicates that many individuals with inherited single gene disorders (20 to 30 % or more) regain the lost gene function in a variable proportion of their somatic cells, which is correlated with amelioration of clinical symptoms and/or an improvement in health. The mechanisms for such reversions are genetically diverse, ranging from true back mutation to the wild type involving single nucleotide substitution, deletion, or insertion, or deletion of multiple nucleotides, to intragenic recombination, gene conversion, and compensatory mutation in the same gene.

Many researchers have commented that the phenomenon amounts to natural gene therapy; though the common assumption that reversion events are purely random appeared to have discouraged any further investigations into possible environmental and physiological factors involved.

The phenomenon is most reminiscent of directed mutations that have now been confirmed in bacteria [3]. Some researchers have pointed to common mechanisms for directed mutations that might apply to E. coli, yeast and human cells alike. For example, the secondary stem-loop structure of single stranded DNA exposed during transcription leaves unpaired G and C bases in loops intrinsically mutable, and these are where mutation hotspots tend to be located [20]. However, we still need a mechanism to direct mutagenesis to just the right genes.

There is evidence suggesting that molecules intercommunicate by electromagnetic signals and molecules that interact attract one another by resonating to the same frequencies (see [21] The Real Bioinformatics Revolution, SiS 33). That may be how cells know which genes to turn on, and how sequential enzymes in metabolic pathways get organized into multi-enzyme complexes within the cytoplasm (see Chapter 18 of [22] Living Rainbow H20, ISIS publication). In the same way, the accumulation of specific metabolites or ‘signaling’ molecules may emit signals to direct the transcription and mutation of specific genes expressing the appropriate enzyme or target protein involved in the biological function blocked by the inherited germline mutation. The end result is back mutation to the wild type or the recovery of a functional protein by another mutational/recombination event. Directed mutation is a reasonable working hypothesis for further investigations on the frequent reversion of inherited disorders. The immediate prediction of directed mutation is that the rate of mutation in other (functionally irrelevant) genes are not increased, which can be readily confirmed or falsified. Another prediction is that there should be specific electromagnetic frequencies, most likely at subtle or subliminal levels (see [23] The Principle of Minimal Stimulus in the Dynamics of the Living Organism, SiS 60) that might enhance the rates of reversion events. Directed mutation is part and parcel of the new genetics and natural genetic modification that we have come to expect [1], which demands a complete overhaul of reductionist biology and medicine still largely based on the old Mendelian genetics.

Article first published 02/12/13


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