[Lecture Notes] Fundamentals of Biotechnology - L17: Inherited defects

Higher order animals are diploid or higher, meaning there is redundancy if one copy of a gene gets a mutation. Inbreeding makes genetic defects more likely as if rare copies of genes which are recessive occur in both the mother and the father, then the probability of the child inheriting both of these alleles (and subsequently expressing the defect) is increased.

Some defects arise from more than one gene. Gene defects such as cleft palate and diabetes result from the interactions between several genes whereas some other defects arise from additional chromosomes, such as down syndrome. Additional sex chromosomes can also exist in the configurations: XXX, XYY, and XXY.


In vary rare cases, both copies of a gene need to be mutation free and working for nothing to go wrong. In cases where even a single working gene is insufficient, we call this haploinsufficiency.

Cases where this happens: where enzymes or proteins need to be expressed in exact ratios, where structural proteins need to be expressed in large amounts (elastin leading to narrowing of aorta), or regulatory networks with stringent requirements on the amount of protein available.

Possessive dominant positive mutations

Also known as “gain of function” mutations, these are rare. For example, short limbed dwarfism is due to a mutation in the FGFR3 gene which encodes fibroblast growth factor (FGF). Rare mutations cause cause receptors to be active even when there is no FGF.

Possessive dominant negative mutations

In this case the mutant protein loses function, but also interacts with other proteins causing them to lose their function as well. Mutations which prevent protein production all together are usually recessive so these mutations are different. The protein is still expressed, but in a mutant form. Even if half the protein is non-functional, due to binding this can cause a 75% reduction in functional protein (fig 17.3).

Deleterious tandem repeats

Also known as CAG repeats, these can cause some disorders. CAG codes for glutamine, and so these CAG repeats cause polyglutamine repeats in proteins. Usually if the number is below 30, this isn’t a problem, however if this limit is passed, the number of CAG repeats can diverge (even over 100) and this can cause severe genetic defects such as neurodegenerative diseases. These proteins aggregate and form plaques which kill nerve cells. A well known example of this is Huntington’s disease. The expansion of CAG repeats occurs because DNA polymerase (in the germ-line) may slip during replication causing DNA to loop out, meaning additional CAG repeats are generated. Therefore successive breeding of people with these diseases can increase the polyglutamine length in proteins and accelerate the progression of the disease.

Some tandem repeats are unstable and may occur in regions of the genome which control the expression of the gene. One such example is fragile X. The replication of DNA causes the number of repeats to keep increasing and the site is eventually methylated, blocking transcription all together.

Imprinting defects

These are epigenetic changes. If the expression of a gene on an allele depends on whether it came from the mother or the father, then the expression may be silenced through imprinting. An example of a disease stemming from this is called Prader-Willi syndrome whereby imprinting patterns turn off certain genes while the paternal copies of the genes are deleted so there is no redundancy.

Mitochondrial defects

Even though mitochondrial DNA accounts for only 15.4kbp of the around 100Mbp of all human DNA, it can still lead to some defects, even though there are thousands of mitochondria (and subsequently thousands of copies of each gene) within each cell. Mutations in mtDNA can still spread due to its higher mutation rate because of the highly oxidative environment that the mtDNA finds itself in, fewer repair systems and the higher rate of replication.

Because sperm do not donate mitochondria to the zygote, any defects in mtDNA are passed down the maternal line. Therefore all the mtDNA that a person inherits exists within the oocyte, and there are ~100,000 copies of it. When the germ-cells start differentiating to form mature oocytes, few mitochondria divide which may lead to mutations. The proportion of the mitochondria carrying a defect affects the severity of the disease. Due to the stochastic nature of cell division, the ratio of partitioned mutated mitochondria to normal mitochondria is not constant, but as subsequent divsions happen in the germ-line, these differences get amplified and what started out as a few extra mutated mitochondria becomes many more mutated mitochondria. Diseases caused by this are things such as neuropathy or ataxia.


Candidate cloning is used to identify defective genes with very little success. This technique uses informed guesswork to test genes or proteins to investigate. The reason this hardly ever works is because the functions of so few genes in humans have been characterised. This is being remedied with KO mice where some studies are seeking to create full knockout libraries of all human genes in mice to test the phenotypic effects of every gene we have.

Functional cloning identifies the protein which causes the defect, and then using various techniques (such as mass spec) finds the coding sequence of the protein which is then found on the human genome for further study.

Positional cloning is used when the exact location of a gene isn’t known, but the approximate location is. When there is a deletion or insertion, certain markers may be able to make this visible on coiled chromosomes visible under a microscope. Narrowing the region down to a few genes reduces the workload and makes analysis easier.

Gene therapy

Gene therapy is a viable route to take if the disease in question is caused by only a single gene and is located to specific organs. Once the gene of interest has been identified, it must be cloned, and a vector to deliver it has to be chosen. Then the method of getting that vector into the body needs to be devised and the expression of the gene ensured.

Delivery can occur either by inhalation or by injection, or ex vivo growth of cells in culture and then transplant back into the patient. Common vectors are disarmed animal viruses which have been modified to include the healthy gene.


These are dsDNA, icosohedral viruses, made of 240 hexons with a genome of 36Kbp. Each vertex is at the meeting point of 5 faces and these vertices have a fibre with different subgroups attached. These fibers can bind to adenovirus receptors on cells causing the cell membrane to bulge inwards and take the virus into the cytoplasm. Then the virion breaks down and the DNA is incorporated into the genome.

These viruses are advantageous because they are somewhat harmless causing only minor infections, of the epithelial cells, and they are not tumour causing viruses. They can be cultured easily and many of their gene functions are known making them easier to engineer, and the complete sequence is available to researchers for modification.

The E1A protein gene is deleted in adenovirus for therapeutics to prevent it from replicating. One needs to make sure that engineered virus genome is not more than 5% shorter or longer than the wild-type or packaging of the virus particles fails.

Retrovirus gene therapy

Retroviruses require dividing cells to properly infect. Retroviruses have been successful in treating humans with gene therapy. The retrovirus RNA genome contains 3 genes: gag, pol and env, flanked by long terminal repeats (LTRs). There is a packaging signal between the gag gene and its LTR. Murine leukaemia virus (MuLV) has been modified to remove all the genes except the packaging signal and the LTRs. The gene of interest can be cloned in but because the three native genes have been deleted, the virus cannot package itself. This is why a packaging construct which is a defective provirus is used to help build the virus particles. This creates particles which contain only the gene of interest and no other viral DNA. These particles can then be used to infect patients. These virus particles cannot self replicate and therefore raise no immune response.

Adeno-associated virus (AAV)

AAV is a satellite virus that needs adenovirus to function. AAV is advantageous because it’s harmless and produces no inflammation, it does not raise an immune response, and therefore no immunity is built up because no antibodies are produced. This means it can be used multiple times. It can target a broad range of dividing and non-dividing tissue types and because it’s small it can penetrate the tissue well. We know that the AAV always integrates into a specific location on chromosome number 19, meaining it is a permanent form of treatment. Also one can produce different serotypes of the virus which have infection affinities for different types of tissue. However because the virus is small, only short sequences can be cloned in.

Preparing the AAV uses a mixture of particles; half of them contain (+)ssDNA and the other half with (-)ssDNA, these are then converted into the replicative form.

Recombinant AAV (rAAV) uses again two virus constructs, like with retrovirus therapy. One carries the gene to be inserted and the other carries the genetic information for making and packaging new virus particles. The rAAV vector contains a transgene with a promoter but this time flanked with inverted repeats. The packaging system recognises these inverted repeats and supplies the materials to make the virus particles (REP (for replication) and CAP (capsid) proteins)


Lipofection is the process of taking a hollow liposome (made of phospholipid bilayer) and inserting in the gene or protein to be delivered, and then transferring these liposomes to the patient. They will merge with the cell membrane and insert their contents into the cell. It is a non-specific technique as liposomes do not discriminate which cell they merge with.

If injected directly into cancers, liposomes carrying TNF proteins could prove promising anti-cancer treatments.

Gene therapy for cancer

Cancer is a genetic disease of somatic cells, not passed down through the germ-line, hence it can still be treated with gene therapy.

Gene replacement therapy: the cancer is first analysed and the defective genes are identified. The wild-type can then be inserted (usually wild-type P53) by either adenovirus or liposomes.

TNF is released by tumour infiltrating lymphocytes which can cure small cancers, however white blood cells can be treated with multiple TNF genes, or modified TNF genes with enhanced activity to better fight the cancer. Putting the TNF gene next to an inducible promoter can allow the TNF only to be expressed at the cancer site rather than everywhere in the body, causing toxic effects.

A gene for an enzyme which converts a benign molecule into one which kills cells can be inserted into cancer cells using gene therapy. The patient then can take this drug which doesn’t affect other cells in the body, only the cancer cells which are expressing this enzyme.

Cancers often do not appear foreign to the body’s immune system, allowing the cancer to survive. HLA proteins on the surface of cells aid in the recognition of foreign and native cells. If non-native HLA genes are inserted into cancer cells, making them appear to be foreign cells in the body, then the body’s natural immune system can begin destroying the tumour.

RNA gene therapy

RNA can be used in the same way as the RNAi techniques discussed before. But RNA is taken up poorly and is quite unstable. Liposomes and replacing DNA with RNA are one way around this. One can also provide a gene which codes for the full length anti-sense RNA so that when it is transcribed it anneals to the mRNA and blocks its translation.


RNA oligonucleotides can be designed to fold up and bind to the active sites of a target enzyme which we wish to block the activity of. These are called aptamers and they are 50-60 bp long and made using solid-state methods. A random pool of oligos is made and passed through a column which has the enzyme of interest bound to the stationary phase. A few sequences will bind, these can be amplified and used, or they can be subject to a round of mutagenic PCR to generate some variability and keep selecting to find better aptamers.

Ribozymes can also be made which can recognise specific sequences on an mRNA. These ribozymes have a recognition domain and and a catalytic domain with nuclease activity. This is still experimental but if successful, one could treat a patient with these ribozymes which selectively find and cleave disease causing mRNAs - these are not degraded like antisense mRNA is by DICER and RISC, so it has a more prolonged effect.


One can use bacterial vectors which are able to synthesise the RNA needed for RNAi. Whole bacteria can enter the target cell and synthesise short hairpin RNA (shRNA). As the liposome degrades (the bacteria has been transformed with a gene coding for listeriolysin which breaks down liposomes), the bacteria will be destroyed (a non-pathogenic strain has to be used) and the RNA will go into the cytoplasm and act.

Typically the bacteria are transformed with T7 RNA polymerase genes to produce large amounts of the shRNAs, they are also given the inv gene to produce an invasive protein allowing them to enter animal cells, and the hlyA gene which allows them to break open the liposome and release the shRNA.

References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.