## In vivo gene therapy

Some cells of the human body cannot be easily removed and reinserted after modification, at least not without serious consequences. In addition, specialized fat, muscle, or nerve cells have lost their ability to divide, so if their genetic information is to be altered, they must be targeted directly in their place, i.e., in the patient's body. An example of in vivo gene therapy is a drug targeting a rare recessive disease caused by the absence of the enzyme lipoprotein lipase (lipoprotein lipase deficiency – LPLD). The disease is characterized by the presence of high concentrations of triacylglycerides and lipoproteins in blood plasma, leading to other clinical complications such as diabetes, pancreatitis and pancreatic cancer. This drug uses an adeno-associated virus 1 (AAV1) that carries a copy of the human LPL gene, which encodes a functional enzyme. As mentioned earlier, the viral DNA carrying the human gene is not incorporated into the patient's DNA in this case, but remains in the form of an episome. Multiple injections are administered into the thigh muscles. This drug was the first approved gene therapy in Europe; it was launched in 2012. Another prime place was its price: at $ 1 million per therapy, it was the most expensive drug in the world at the time. Although it proved effective, it was administered to only 31 people in total. The high price and low interest meant that the license was not renewed and the drug was thus withdrawn from the market.

Gene therapy has shown promise for treating various forms of vision loss. A specific example is age-related macular degeneration (AMD). Unlike the previous examples, AMD is a multifactorial disease whose cause is not precisely known. The environment, the genetic background of the individual as well as age play a very important role in its occurrence. It is a chronic disease associated with irreversible loss of vision with an onset of symptoms at the age of 50, being most common in people over 70. One of the most important factors in AMD is likely our complement immune system (the non-specific immune response thaht complements the immune system), which can cause inflammation of the eye or permanent damage to vision if it overreacts. A drug with the gene CFI (human complement factor I), whose product is able to alleviate and slow retinal atrophy by regulating the immune response, is in the clinical trial phase. Another example is the treatment of hereditary retinal dystrophy (retinitis pigmentosa), which is caused by the loss of functional photoreceptors. This is a collective term for diseases with similar symptoms but with different causes. In the past, several dozen genes have been identified whose mutation can lead to the manifestation of this disease – one of them is RPE65 (the product of this gene is a key enzyme for the light cycle, in which the light signal in the retinal cells is converted into an electrical signal leading to the brain). There is an approved gene therapy with a healthy copy of the RPE65 gene using adeno-associated virus 2 (AAV2). However, the application of this therapy is difficult as it must be delivered directly to the retinal area. This is achieved by injecting the drug into the space between the retina and choroid (Figure 10.6). Since the damage to the light-sensitive cells of the retina is irreversible, the therapy must be administered as soon as possible. Furthermore, the aim of the two aforementioned therapies is not to restore vision to its original state, but to stop or slow down the progression of the disease.

Another inherited disease whose treatment has attracted much attention is spinal muscular atrophy (SMA). It is one of the most common autosomal recessive diseases with an approximate incidence of 1:10,000 in the population. It affects the central nervous system, motor neurons, and skeletal muscles. The mutations responsible for this disease were mapped to the SMN1 gene. In addition to the SMN1, the human genome also contains its copy – the SMN2 gene. SMN2 can be present in varying numbers, from one to multiple (> 4) copies, and the SMN2 protein, which is the product of this gene, can compensate to some extent for the loss of SMN1. The more copies present, the milder the disease symptoms in the case of the SMN1 mutation (Table 10.1).

Table 10.1 SMA patient prognosis related to the presence of different numbers of SMN2 gene copies.

type | Age of symptoms onset | Artificial ventilation in birth | Ability to sit | Ability to stand up | Ability to walk | Life expectancy | # SMA2 copies
0 | prenatal age | yes | no | no | no | < 6 months | 1
1 | < 6 months | no | no | no | no | < 2 years | 2
2 | 6-18 months | no | yes | no | no | 10-40 years | 3
3 | > 18 months | no | yes | yes | assisted | adulthood | 3 - 4
4 | > 5 years | no | yes | yes | yes | adulthood | > 4

An important difference between the two genes is that exon 7 of the SMN2 gene is excised in some cases during mRNA maturation (Figure 10.7). The genetic information contained in the sequence of this exon, which is important for the folding and function of the resulting protein, is thus lost. It has also been shown that a single nucleotide substitution in this exon can lead to the pathological condition. Splice modification of the exon 7 of the SMN2 gene has therefore become the target of antisense RNA gene therapy. By binding the antisense RNA (complementary to a natural mRNA), exon 7 is not excised during the maturation of the mRNA. However, the drug does not have a great longevity after the administration. In addition, the blood-brain barrier must be bypassed for the therapeutic RNA to reach its destination. Therefore, the therapy must be administered intrathecally (into the space between the brain and its soft membrane (pia mater) or between the spinal cord and its soft membrane) an average of three times per year, which is painful and technically challenging.

An alternative in SMA gene therapy is a drug that uses adeno-associated virus 9 (AAV9), which carries a healthy copy of the SMN1 gene. A major advantage is that the AAV9 viral vector has the ability to reach the brain and thus cross the blood-brain barrier. After entering the neurons, the genetic information of the virus moves to the nucleus, where it exists in the form of an episome. Due to the one-time and less painful administration, this drug seems to be a better choice for patients suffering from SMA.