Seminário Internacional de Ciências Farmacêuticas — Método CRISPR

As manifestações associadas a Seminário Internacional de Ciências despertam importantes questionamentos clínicos e exigem orientação especializada para evitar os riscos da automedicação ou de restrições desnecessárias. Ao abordar o tema "Seminário Internacional de Ciências Farmacêuticas — Método CRISPR", este guia educativo reúne o conhecimento prático da equipe médica do Projeto Brasil Sem Alergia com a finalidade de esclarecer os fatores desencadeantes, os exames diagnósticos mais precisos e as estratégias terapêuticas modernas disponíveis atualmente.

Conteúdo Audiovisual Complementar: Clique aqui para assistir ao vídeo explicativo completo no canal oficial do Brasil Sem Alergia no YouTube.

Análise Clínica e Orientação Médica sobre Seminário Internacional de Ciências

Boa noite da espada jacksonville uh é uma grande honra ouvir o seu palavras sobre crispr este nascimento ii poderia dizer crispr é um capítulo intrigante da medicina é um capítulo intrigante da vida e ii tenho certeza que isso admite que você será bem sucedido e eu gostaria de agradecer a você e ao dr rabio lanot por aceitarem o convite agora vou compartilhar minha tela para começar minha palestra esqueceram um estrangeira uh é uma [Laughter] foreign is um start your speech welcome to the symposium thank you for the invitation let me start my presentation research work using the crispr cast 9 technology today's presentation will be divided into part i will first present the use of the crispr cas9 technology to treat iron steady disease and my graduate student gabrielle lamont will present all the crispr cas9 derived technology may be used to detect viral infection the word crispr stands for clustered regulatory interspace short penetrate repeats A tecnologia do sussurro cas9 é derivada de pesquisas sobre bactérias, foi descoberta inicialmente em 2005 que a sequência crispr continha sequência viral e ainda havia uma hipótese de que crispr cas9 é o sistema imunológico bacteriano foi só em 2013 que se descobriu que o crispr cas9 pode ser usado para induzir modificação específica do genoma humano e isso começou uma grande explosão de artificial na verdade, o sistema de fundição crispr é usado por bactérias para matar bacteriófago são extremamente abundantes são dez vezes mais abundantes em bactérias e há uma guerra em curso entre bactérias e bacteriófagos há milhões de anos it was initially discovered that they are in the genome area constant repeated sequence researchers were wondering what these repeated sequences were for and then they discovered that the sequence between a constant repeat were sequenced that were up from the genome of various bacteriophage in fact what normally happens is when a bacteriophage infected bacteria normally the bacterial ph wind bacteria is dead but sometimes you have a defective virus that will infect without killing the bacteria but they will then be able to acquire some part of the sequence of the virus and it will store that into its genome and it will be ready when it needs such bacteriophage again in the future from the dna in the crispr area bacteria will first express the pre-cr powder and then there will be a complex that will be made between tracker or crna and the cast iron protein this striker rna crna cas9 protein complex will then bind with the bacteria of hdna and induce a cut of the bacteriophage dna to essentially kill the virus to bind to the dna the cas9 protein requires the presence of the total spacer adjacent motif which is simply njj for these streptococcus pyrogene path then there is a crrna which contain a variable sequence of 20 nucleotides that will be complementary to the bacteriophage sequence and then there is also the tracker rna which is a constant rna sequence that forms a complex with the crm when the three parts are together cr rna tracker or a cast iron protein a cut will be induced at exactly three nuclear tight from the plant of course since this is a war there are contour measure that are taken by the phage they will mutate their genome so that they become resistant not recognized by the cr rna and will survive bacterial defense the crispr cas9 big bang started in 2012 when gen x and colleagues realized that the crisper cast 9 technology can permit to cut the genome of planets and animals they also fuse their cr rna and the tracker rna together to form a single guide rna this was done essentially because the single guide rna is something that can be patented whereas the crn in the tracker being natural they cannot be patented during the following year there was an explosion of article confirming that the crisper casino technology may be used to cut the genome of bacteria human cells carbon animal mouse drugs zebrafish plant flies nematodes monkeys many of these articles were published in prestigious journals such as nature science nature biotechnology molecular cell molecular cell there was a rapid explosion of article and as you can see as of november 26 2020 21 420 articles have been published where the word crispr is mentioned the main reason of this rapid explosion of article is that it costs much less to derive a sequence that will be able to cut a specific genome sequence compared to other pre-existing technology such as zinc finger protein and tailors as mentioned before the binding of the cas9 protein to the dna require the presence of a photo spacer adjacent onsite pam which is njj for the cas9 derived from streptococcus biology there is then the binding also of the single guide rna which will recognize the sequence of 20 nucleotides and when the complex is made between the cas9 protein the single guide rna and the dna there is a cut that will be made at exactly three nucleotides from the pan once inside the nucleus the resulting complex will lock onto a short sequence known as the pan the cast 9 will unzip the dna and match it to its target rna if the match is complete the cas9 will use two tiny molecular scissors to cut the dna when this happens the cell tries to repair the cut but the repair process is error-prone leading to mutations that can disable the gene allowing researchers to understand its function these mutations are random but sometimes researchers need to be more precise for example by replacing a mutant gene with a healthy copy this can be done by adding another piece of dna that carries the desired sequence once the crispr system has made a cut this dna template can pair up with the cut ends recombining and replacing the original sequence with the new version one of the reasons of the rapid explosion is that the crystal classic technology also used all what was developed by gene that have been the 30 years that preceded it we have methods to delivering the dna using aav and then associated virus cationic polymers liposome micro injection electro operation all of those techniques are used in the context of crispr when a double strand break is produced in the dna most of the breaks will be repaired by non-homologous adjoining that will lead to micro deletion or micro insertion this is frequently used to knock out the expression of a gene by changing the reading frame and it can also be repaired by homology directed repair which require the presence of the donor dna that contains sequence of homology with what is receding what is following the cut and then between the two homology sequence there is a sequence in blue which may be a single nucleotide or a whole gene that will be inserted at the side of the double strand break when we use the crispr class 9 technology we intend to produce a double strand break at a precise site in the human genome however the cast 9 may sometimes induce cut at other sites in the genome which are called buff target mutation computer software may predict these off-target mutation by using the whole human genome sequence however these softwares are not perfect and they will sometimes print it off target mutation at site where there is no cut and sometimes there will be a cut at the side which is not predicted by the computer there are or however some experimental techniques such as a guidestack method which permit to experimentally identify the sites of target mutation this guidesec technique used the introduction of a short 34 base pair oligonucleotide at the sides of the cuts and later by pcr the site of insertion may be identified by sequencing the presence of these off-target mutation is the main problem that delays the use of crispr cat9 technology for direct vehicle corrections one of the method to reduce of target mutation is simply to reduce by two or three the number of the single guide rna which is binding with the target dna another method is to use a mutated cas9 nuclease that cut only one strand of dna is then called the knee case there is variation of cas9 where the decays cut only the lower strand of dna and other mutation of class 9 which permit to cut only the upper strand of dna it is thus possible to induce more precise double strand break in the dna by using the cas9 nikkei and two single guide rna each detecting a sequence of 20 nucleotides close to one another it is also possible to use a non-functional cas9 cas9 nuclease fuse with the one nucleates then two guide rna are again needed to detect sequence which are close to another and the frog one nuclease need to form a dimer in order to be able to cut the dna finally some researchers have mutated the gene coding from cast 9.

They have modified some sequence coding for amino acid to reduce the non-specific binding between the cas9 and the dna the sp cas9 gene is too big to be inserted with a single guide rna inside an adeno associated virus and thus researchers have identified cas9 from other bacteria that are smaller for example the staphylococcus it is smaller than the sp 9 but it has a different path which is more restrictive another type of crispr cas9 enzyme has been identified it's called cpf1 it produces sticky cancers it's another blood cut of the dna there is just a whole series of various cas9 containment values with kylia that require different tabs the crispr cas9 technology may be used not only to reduce cuts in the dna but it may also be used to induce the expression of a gene by fusing cas9 with bp 64 for example or choosing cast 9 with crab to redress the expression of a gene in my research group we are using the crispr cas9 technology to develop therapies for various disease transverse ataxia duchenne muscular dystrophy and alzheimer's disease i will present that now we have initially used the crispr cas9 technology to develop a treatment for fredrich ataxia it is an elixir disease due to the presence of a long try nucleotide with b gaa in intron one of the fataxin gene this long repeat reduce the expression of the fataxin gene leading to neurological problems and cardiac problems we have just used the crispr cas9 technology and generated single guide rna able to cut in intron 1 before and after the trinucleotide repeat leading to its removal and increasing the expression of ataxis indeed the removal of the trinocleotide repeat in cells of the yga sr mouse model of frederic ataxia doubled the expression of frataxin compared to the untreated cells and raised the expression of protection to almost the normal level the sp cas9 gene is too big to be delivered with two single guide rna by a single aav we are just currently using the smaller cg cas9 gene to remove the trinucleotide repeat in intron 1 of the fataxin gene using this smaller cg cas9 nuclease we are also able to remove the trinucleotide repeat in intron one of the frataxin gene we are also using the crispr cas9 technology to develop a treatment for duchenne muscular dystrophy duchenne muscular dystrophy is due to mutation in a gene coding for the dystrophin protein it is a large gene containing 79 exons some of these exons do not contain a multiple of three nucleotides and thus 70 percent of duchenne muscular dystrophy cases are due to deletion of one or several exams and the total number of coding nucleotides which are deleted is not a multiple of three for example in this presentation we have a deletion of exon 50 that does not contain a multiple of three nucleotides this lead to a frame shift and there will be a stop codon in exon 51.

Thus the beginning of this the dystrophin protein is expressed but not the end of the dystrophin protein this situation may be corrected by inducing the skipping that is a removal of exon 51 in the messenger rna this is done using antisense polygon nucleotide this exam 51 may also be deleted by inducing cuts in intron 50 and in front 51 to completely remove that exon this results in the expression of the beginning of the dystrophin protein and of the end of the dystrophin protein but there is however a small part of the protein which is missing in the center of the protein the removal of one or several complete exam may thus restore the reading frame and convert a duchene patient into a becker patient however some backer patients have severe symptoms and are bound to a wheelchair at the age of 11.

Therefore the improvement of some duchenne muscular dystrophy may not be significant this is because the dystrophin protein has a complex structure indeed in its central part the distro fin protein contains 24 spectrum-like repeats each spectrum like repeat is made of three alpha helix a lxb and lxc note that lxa is starting on the left side and elixi is ending on the right side and normally there is a succession of abc the main problem of deleting complete exon to restore the expression of dystrophin protein is that the beginning and the end of the spectrine like repeat indicated in this scheme by the black arrow do not correspond with the beginning and the end of exiles and thus when removing complete exhaust the resulting of the spectrum-like repeat structure may not be normal vector muscular dystrophy patient have a deletion of one or several exams but the total number of coding nucleotides which are deleted is a multiple of three nucleotides and thus there is no frame shift this is the case for example for becker patient having a deletion of exam 45 to 47 or having a deletion of 45 to 49 in both case there's no frame shift the beginning and the end of the dystrophin protein are expressed however at the junction site between the remaining codons there is an abnormal structure of the dystrophin protein and due to this abnormal structure of the dystrophin protein these vector patients are bound to a wheelchair at an early age dusk in my research group instead of trying to restore the normal reading frame by deleting complete exons we have instead aim at producing enabling exam 5054 which not only restored a normal reading frame but codes for a dystopian protein with a normal structure we have done our initial experiments using the myoblast of a duchenne patient having a deletion of exam 51 52 and 53.

Thus because the number of coding nucleotides was not a multiple of three there was a step codon in exon 54 due to the frame shift we have just used the crisper cast 9 technology to induce a cut in exon 50 and a cut in exon 54 to create the ebrate exon 5054 to produce this implicated example we initially identify what are the possible sp cas9 pan or what are the site where we can cut in exon 50 and in exon 54 we identify 10 times sites in exon 50 and 14 different pan size in exon 53 this table predicts what are the results of inducing cuts in exam 50 and in exam 54.

When we have a blue square it means that the cut has been produced in exon 50 right after the three nucleotide and calls for an amino acid and the cut has been produced in exon 54 just before the three nucleotide that codes for an amino acid thus at the junction point we have amino acids that are one escorted by exon 54 followed immediately by an amino acid coded by example there's no frame shift and there is no new amino acid however when the square is clean that means that we have at the junction site a new amino acid which is produced because we are caught in exon 50 right after one of the nucleotides of a codon and the two nucleotides that will complete these codons are from example before we have cut right after one nucleotide of the codon in exon 54 and thus first two nucleotides left to create a new codon at the junction site there is thus a new amino acid at the junction site but there is no frame shift and all the other amino acids are the correct ones it is also possible in this square in red to have a new codon produced at the junction site but this new codon is a stop codon this is not real we want to do all the squares in wide because there is a frame shift we're cutting after one nucleotide of a codon in exon 15 and we're cutting before the last nucleotide of a cooldown in exon 54 and thus add the junction side there is a new codon which is done because there is a frame shift and all the amino acids that follows are not the correct one we have dust testers various pair of guide irony one guide irony cutting in at zone 50 and the other guide rna cutting in exon 54 just produced an ibrit exam 1554 that had the predicted size of this hybrid exam note that when we are doing cuts in exam 50 and in exam 54 in a normal dystrophin g we are deleting 160 000 base pair and despite that the junction between exon 50 and 54 is of the size predicted we have sequenced this hybrid exam that was produced by cutting in exon 50 and in exon 54.

Not only were the eblid exam exactly the size as predicted but the sequence were also exactly as predictive for example we had a junction site the fusions of exon 50 in exam 54 producing exactly the predicted amino acid we also obtain at the junction site a new codon coding for the predicted amino acid or coding for a stop codon and sometime as predicted by the white square we had a frame shift and when there was a frame shift there were stop codons that were meant in the resulting liberal exiles as mentioned before the dystrophin protein contained in its central part 24 spectrum like repeat each one being made of three alpha helix a b and c we have just used a guide rna to induce a cut in exon 50 in a sequence coding for lxc and another guide rna inducing a cut in exon 54 also in a sequence coding for helix c the resulting evident exon 5054 calls for an ibrim elixi where the beginning of nxc is coded by exon 50 and the end of elixi is coded by exon 54.

This is a structure that has been computer predicted using the sequence of the resulting protein when the bioglass of this duchenne patient having a deletion of exon 51-53 are fused together to form some small muscle fibers and culture called myotubes in fact these muscle fibers do not express dystrophin because this is a duchenne patient however the myoblast of a healthy human when they fuse together they form a small biotube and they do express this topic as can be seen here in this western blood when we use the bioblast of the duchenne patient and we induce the formation of the ignitive zone 5054 we have expression of the dystopian protein in culture and as you can see this dystrophin protein has a lower molecular weight than the normal dystrophin because there is deletion of exon 51 to 53 and an additional deletion of part of exon 50 and part of the exam 54.

Thus there is a part of the dystrophin protein which is absent this is why the protein is smaller but it is nevertheless expressed for our initial individual experiments we have used the html mouse model this is a transgenic mouse that express the human dystrophin gene all introns and all exons we have electroporated in the muscle of this mouse plasmid coating for the sp cas9 fuels with the fluorescent green protein and two single guide rnas this a month later when we took the muscle there was expression of the green fluorescent protein confirming that the plasmid has been correctly electroporated in the muscle fibers leading to the expression of the fluorescent green protein and probably also leading to the expression of the sp cast iron and after two guide ironing we then extracted the dna from these muscle and we first confirmed using a test called the surveyor enzyme but indeed they were constantly being produced in exon 50 and 54.

We then use pcr to amplify the eblatex zone 5054. Note that in the muscles that are not treated with the crystal class 9 technology there is no amplification of example 5054 because there are 160 000 base pair between these two exiles and therefore the two primers for the pcr are too much separated from one another we then sequence this exon 1554 that was produced in vivo in the mouse muscle expressing the human dystrophin gene and as predicted we had the sequence of exon 50 and a new codon at the junction side followed by all the correct codon coding for the normal amino acid in exon 54.

This is exactly as predicted in vivo however for delivery of the cast 9 gene in evil to several muscles we need to use an adeno associated virus as mentioned before the sp cas9 gene is too big to be delivered with two guide rna by a single aav and we therefore use for our next experiments the cast knife of the staphylococcus moleus which is a smaller cas9 which permit delivery with two guide irony by a single avp in this case we have used uh guide rna that induced cuts in exam 47 in a sequence coding for lxb and in exon 58 in a sequence again coding for elix b this resulted in the formation of an ebola exam 4758 coding for an ibrigid lxb which has a normal structure the beginning of the exp be encoded by exon 47 and the end of that evil philips be being coded by exon 58 we then use for our imbibo experiment a new mouse model called hdmd delta 52 it's the same mouse model as previously that contains the dystrophin gene with all the exons and all the intron except that this mouse has a deletion of exon 52 and thus there is no expression of the human dysphoric gene we have just used the crispr class 9 technology to induce a cut in exot 47 and a cut in exam 58 producing the ibrine exam 4758 resulting in the production of a dystrophin protein containing an evil exam every lxp we have injected to these hdmd delta 52 mouse aevs coding for sc 9 and 2 single guide rna a month later we observe in the muscle the expression of the human dystrophin gene including in the heart we are just proposing a treatment of duchenne muscular dystrophy which would be the systemic delivery by an adeno associated virus of the class 9 gene and of two guide rna to form any bread exam in contrast with exon skipping which is a treatment done at the level of the messenger rna the treatment that we propose at the level of the dna would be permanent the crispr cas9 technology is evolving rapidly and new technologies derived from the crispr cas9 permits now the modification of a single nucleotide more than 32 000 single nucleotide modifications are responsible for early steady disease and thus the capacity to correct a single nucleotide would provide treatment for most of these healing steady disease the first treatment which permits the modification of a single nucleotide was developed by kamal in 2017 which use a cas9 nikkei that is the modified cast 9 which is able to cut only one strand of dna and this cast 90 case is fused with acetaminophen this technology is permanent to clinically modify city bean into a uvd which is replaced by a timing on the dna the main limitation of that technique is the chemical modification of the city will occur in a narrow window located at 12 to 16 base pair from the ngg pan we have initially used base editing technology to develop a treatment for alzheimer's disease the alzheimer's disease is produced by an abnormal metabolism of the ammunition protein normally this protein is cut by the alpha secretaries followed by a cut by the gamma cell this proteins peptides and protein fragments that are degraded without causing any problems however this protein may also be cut by the beta sequence followed by a cut by the gamma sequences and this produced short 40 42 amino acid long beta amigo infections that aggregates to one another forming amino acids that interfere with synaptic transmission leading to neuron death and the memory problems this scheme illustrates the amino acid sequence of the transmembrane part of the anaerobic repressor protein we can see the position of the beta alpha and gamma separates calcite all the amino acids in the star above their main are amino acids which are modified leading to family or form of alzheimer's disease please note the position with the red arrow this is the alanine in version 673 when this adenine is changed by valley this leads to severe early onset alzheimer's disease and you are a zemer at the age of 40.

However when this adenine is changed by a journey you're not alzheimer even when you're 95 105 years old as shown by johnson nepal in nature 2012. The presence of the a673t mutation also known as the icelandic reduce mutation secretion of a beta 40 in our data quality peptide for the wild-type appg and for appg containing the london mutation our experiments have shown that the presence of the h673t mutation reduce the secretion of a beta 40 and a beta 42 peptide by the epp genes not only for the wild-type gene but also for app genes containing several familial alzheimer's disease mutations as mentioned before the crispr cas9 base editing technology permit to modify the city dean into a timing we just have used the base editing technology to target the cytoplane in the antecedent strand of the alanine codon transforming that cytodine into a timing and just changing the alanine codon into a triangle the main problem with this approach is that although we want to modify the cytodine into the ents decent strand of the adenine codon there are other acidity nucleotides nearby that are also affected by the base editing approach we have constructed 14 different base editing enzymes to be able to quantify more specifically by modifying the city and the antisense codon of led we have been able to introduce mutation in up to 17 of the avpg however other acidity also located in the antecedent strand were also modified by the subject a new fantastic technology called privacy has recently been developed by pencil on top with permits in principle the prime editing technology use a cas9 new case fuse with a reverse transcriptase it also requires a prime editing guide rna known as a pig rna the pig rna is essentially a prolonged single guide rna thus has a single guide rna it contains a spacer sequence which react with 20 nucleotide in the target dna it also contains the constant scaffold of the single guide rna which is in red and then at its five prime end there is a prolongation with the primer bending side which is a sequence of 10 to 17 nucleotide reacting with the upper strand of dna this is followed by the reverse transcriptase template again which is 10 to 17 nucleotides in length and which will contain some modified nucleotide in red in this case indicating which nucleotide has to be modified by the reverse transcriptives a plasmid designed by anzalo natal is available at edgy to construct new peg rna this plasmid contained a red fluorescent protein gene which may be removed by bsa1 cuts which produce a backbone of the protein and then the other components are the spacer primer binding side the reverse transcriptase template and the pig rna scalp all of these sequence are single stranded polygon nucleotide that may be purchased from idt these four parts are then assembled together to produce a new pay guarantee to use the prime editing technology we first have to identify photo spacer adjacent positive tab which is njj for the class nine of streptococcus pyrogene this will permit to the cas9 to bind to the dna then the spacer sequence of the peg rna will bind to a 20 nucleotide sequence of dna in this case lower strand and the formation of the complex between the peg rna class 9 decades and the dna will induce the nick at exactly 3 nucleotides from the path in the upper strand of dna this will release the upper strand of dna to be able to interact with the primer binding side of the pile the peg rna and then the reverse transcriptase template which contained a few nucleotides to be mutated will be available for the reverse transcriptase to synthesize a new dna upper strength meaning duchenne muscular dystrophy patient have a stop called on the dmdg since we did not have access to cells of patient containing such pointation we decided to introduce these stop codons using the prime editing technology so in each case we had to identify an ngg pam identify the peg rna photo spacer sequence and then modificar o modelo de transcriptase reversa para entrar para modificar o códon para um aminoácido em um códon de parada neste caso nós mudamos acetaminofeno para introduzir o material tga usamos com sucesso essa abordagem para apresentar os acionistas no exame 9 20 35 42 55 e 61.

Acabamos de projetar vários peg rna direcionados ao dmd exon 35, como você pode ver, variou o molde da transcriptase reversa em azul de 10 a 16 nucleotídeos e também variamos o sítio de ligação primária em verde de 10 a 15 nucleotídeos e no local de mutação desejado que introduzimos em introduzir essa mutação nossos experimentos iniciais foram feitos em células hek293t we tried to reproduce the mutation of mx1g and to target dmd exon 35.

So essentially these cells were transfected with plasmid coding for the cas9 djs fuse with the reverse transmitted days and a paid rna targeting either exon 35 of the dmd gene or the e mx1 g the dna was extracted three days later and the targeted sequence was pcr amplified and sequenced using center method the sequence were analyzed using the edit r online program essentially we observe a 32 percent correction mutation of the air x1 gene has done by heads along at all however for the exon 35 mutation we had a two percent background in the sequence of the control negative control and with the different peg rna we have mutation ranging from four to eight percent so this was not as great as for the e m x one g we have just tried different method to try to increase the percentage of genome editing of exon 35.

The first method that we have tried is to repeat the treatment three times essentially the cells were transfected with plasmid at the 0 6 12 and dna was extracted three days and six days after each treatment we have amplified the pcr exon 35 at each of the extraction date and sequence it by center and analyzer sequence as you can see the percentage of genome editing increased from day 3 to day 18 with the treatment and this was the case for all three peg rna network targeting exon 35.

We have then tested a second method to try to increase the percentage of mutation in exon 35. It is to induce a second nick in the target g this is the pe3 method essentially we have identified two pam sequence which permitted to kite rna to induce a second nick at either 57 nucleotide from the original big irony neighbor 24 nuclear time or at 57 nucleotides induced by the pig there was a significant increase in the addition of the target gene propaganda 35 four and figure eight by six but not for bigger only 20.5 you still have to understand why i method to improve the percentage of mutation in the target gene is to mutate the pam used by the peg harmony we just designed pig rna that were not only able to introduce a stop codon mutation but we're also able to mutate simultaneously the pan and the use mutation of the pan used by the peg rna improve the percentage of mutation in the stop codon for two of the three peg rna that we have tested combining the two method that is inducing a second nick in the target g and mutating the pan used by the peg rna further increase the mutation of dmd exon 35 to 39 with all three pegs that we have tested we are currently starting new project to correct mutation responsible for other heading steady disease we are working on cystic fibrosis due to mutation in the cftr chloride channel on congenital muscular dystrophy due to mutation in the rheanodine receptor and on ataxia 8 due to mutation in the nkx6 type 2 gene for each hearing steady disease due to a point mutation for example here with attacks at type 8 it is possible to correct in principle the mutated gene using the prime editing technology in this case here we can identify that the mutation is an adenosine change for timing and thus we can identify a pan ng for the sp cas9 which is close to the mutated nucleotide we can then design a plane rna that will introduce the desired mutation in this case we are introducing two mutations one to correct the mutation to reverse the timodine into an adenosine and the second midpoint mutation is to modify the path so that following the correction the cation enzyme can no longer bind to the dna crisper casino derived technologies may not be used to treat many different city diseases the main problem remained the inhibit delivery of the editing agents thank you for your attention okay thank you professor jax i'll take the turn now to make a general comment for those who the who doesn't speak uh those who don't speak english okay thank you for your presentation thank you of i foreign a he summarizes the your speech okay not the song yes sir thank you very much for the translation of my summary in portuguese okay um foreign is gabriel thank you for having me uh let's see if we can get it started yeah uh can you see it hi my name is and today i'll be discussing the detection of stars using crystal technology named the test that we've been developing and dr tremblay's Hospital studies have confirmed that many individuals infected with earth ob2 virus or asymmetric carriers you can't hear yet no yes can you hear it no just maybe you should talk okay uh i can actually try streaming it one other way um if that's the case so okay hi my name is gabriel lamot and today i'll be discussing the detection of cyrus kovi yeah that's much better is the sound better now e2 using crispr technology oh perfect namely the test that we've been developing in dr trombley's laboratory studies have confirmed that many individuals infected with osiris-cov2 virus are asymptomatic carriers of the virus this is problematic in that the pandemic might be growing without adequate supervision simply focusing the testing on individuals with symptoms as opposed to entire populations is not enough to better contain the virus until a large-scale distribution of an effective vaccine is underway large-scale and continuous testing is mandatory the current diagnostic pipeline for detecting sars cov2 is a three-step process the first step is to obtain nasopharyngeal swabs from patients the next step is to isolate the total rna from said samples and provide the rna required for this third step which is to use real-time quantitative vcr machines the rna is first reverse transcribed into dna and then amplified a dye is added to the amplification reaction to signal the presence of dna strands after a certain number of amplification cycles a positive result will give a strong enough signal that the machine flags it as containing the original virus now this process is problematic for a few reasons the first is that real-time pcr machines are extremely expensive most can range from fifteen thousand us dollars to well over ninety thousand us dollars given that it's a fairly specialized piece of equipment and that not all molecular biology laboratories need one some get along just fine with a regular pcr machine which costs a much more reasonable 5000 us dollars as such widespread testing for the coronifiers using these machines isn't ideal acquiring the large number of machines required for widespread testing is a feat in and of itself and is likely one of the contributing factors for the sometimes long wait times for results many reports have stated that it can take 3 to 24 hours or more to obtain results in a clinical diagnostic laboratory over here in canada ontario reports wait times of up to four days to get results high density metropolitan areas like new york are also subject to long lines for testing and then a time to results of about seven days for some clinics in that city the creation of new types of detection tests is therefore an important step in curbing the spread of this virus an ideal test needs to follow the world health organization's criteria for assured that is the test needs to be affordable for as many people at risk of infection as possible tests that require extremely expensive reagents or machinery are not ideal the test must also be specific to avoid false negatives that way anyone who could potentially spread the virus can be identified and put into quarantine the test must be sensitive so that few people who don't have the virus are mislabeled as having it and treated improperly the test must also be user friendly that is it must be simple to perform and it must be rapid and robust that is it must give a time to results that is quick enough to make the most use from the results situations where it can take up to seven days to obtain results are clearly far from ideal as by the time the patient receives the information they might have infected many others the tests must be equipment free as much as possible tests that require too many rare and expensive machines aren't ideal for many countries finally the test must be deliverable to those who need it the discovery of cast 13 or c2 c2 was revolutionary in the field of molecular detection this class 2 type 6 crispr effector is similar to the protein cast 9 off of which base ending and prime ending were designed this protein is also a member of the crispr family unlike cas9 however cast 13 can be programmed to target rnas as opposed to dnas by binding to its crispr rna cast 13 is primed to target extremely specific rna sequences after locating a target sequence the cast 13 protein is activated this causes the protein to begin to cleave everything its surroundings in an event called collateral cleavage it cleaves its original target and then moves on to start cleaving all surrounding rnas researchers realized that this collateral cleavage could be used to turn the protein into a switch for a detection test once the target molecule is present the switch is flipped the cas13 activated and all surrounding rnas are degraded by tagging certain rnas in the solution with a fluorescent molecule and a quencher it would therefore become possible to resolve a signal researchers therefore started to develop a detection test based on this protein for viruses bacteria and human diseases such as cancer however they soon noted that there were certain limitations to the protein and that his limit of detection was far from sensitive enough for their needs recently certain tests have been designed using only cast 13 however these tests are less sensitive and tend to use a different cast 13 protein researchers at the broad institute decided to supplement the protein with a few others that would amplify the target rna to this end dr zhang and his team created a new test called specific high sensitivity enzymatic reporter unlocking or sherlock for short this test is based on the reverse transcription of target rna into dna afterwards the dna is amplified at a constant temperature by using a process called recombinase polymerase amplification this technique amplifies a small amount of dna at a constant temperature unlike the more commonly used pcr the incorporation of this step also means that the ensuing detection test can target dna since the rpa reaction simply amplifies a determined section of dna using two primers the initial genetic sequence can either be rna or dna so long as reverse transcriptase enzymes are present for rna the amplified sequences are then transcribed using a t7 rna polymerase to create vast quantities of the target rna this then allows the cas13 to have a far larger pool of target molecules which can activate the enzyme once the enzyme is active it does what we discussed before and starts to cleave all surrounding rnas including reporter rnas which give off the easily detectable signal which makes the test work in their initial version of the sherlock test two different reporter molecules were used the first used a fluorescent molecule as well as a quencher and it was able to produce a lot of fluorescence after cleavage of the rna the other was designed to interact with a few antibodies and when it was cleaved it appeared as a distinct second band on a strip that was quite similar to a pregnancy test while this test was good in many ways it had certain drawbacks at the beginning of the pandemic we attempted to replicate it and immediately had a few issues the biggest issue by far was that some of the materials required for the test to work were only available in extremely limited supply the kits required to perform rpa reactions for example took approximately two months to arrive and even then we only received enough materials to perform 200 tests this clearly wasn't going to be enough to produce sufficient tests in the future additionally the reporter that used a pregnancy style strip was remarkable and seemed to be very useful however since it was too difficult to acquire insufficient quantities we decided not to use it and to instead focus our efforts on using the fluorescent style reporter rna since the original version do teste não pôde ser usado por nós da maneira que se pretendia, decidimos redesenhá-lo ao redesenhar o teste tínhamos várias opções e caminhos que podíamos seguir sabíamos que queríamos continuar usando cast 13, porque tínhamos um grande suprimento dessa enzima devido a uma colaboração contínua com o dr alain Yani também sabíamos que queríamos a todo custo manter a natureza isotérmica do neste teste uma das maiores vantagens do sherlock foi que ele não exigia grandes quantidades de equipamentos caros simplesmente usando um bloco de aquecimento ou algo do tipo era possível obter uma leitura visual decidimos, portanto, empregar amplificação isotérmica mediada por loop ou lâmpada para encurtar essa amplificação estratégia foi bastante bem documentada e fácil de executar com base no uso de seis primers esta técnica pode amplificar rapidamente grandes quantidades de dna alvo, ao contrário de rpa e pcr, no entanto, a lâmpada não cria amplicons idênticos de tamanho discreto, em vez disso, os seis primers podem interagir para criar moléculas muito maiores de uma sequência de repetição inicialmente era um pouco difícil incorpore essa estratégia de amplificação isotérmica em nosso teste, pois você pode se lembrar da etapa seguinte rpa no teste sherlock original foi aquele que produziu muitos e muitos rna alvo essa produção de rna alvo só foi possível, porque a etapa rpa adicionou uma tag que deixou o rna t7 polymerase bind to the dna and begin transcribing it however no one had seemingly ever done that with lamp and since this new technique used six primers two of which looped back on themselves to create weird dumbbell-like structure structures it was difficult to figure out exactly where to incorporate this tag after a while we decided to include the tag right in the middle of one of our looping primers this has consistently given us very strong results we were therefore able to retrofit the original sherlock test with a new amplification strategy all the while still benefiting from cast 13's specificity and signaling the new test is therefore using rt lamp as opposed to rt rpa of course should we become interested in targeting dna in the future for either certain types of viruses or certain bacteria this test would be readily applicable to that as well we also decided to use a fluorescent reporter rna instead of the pregnancy style one that we spoke about previously because it seems to be the most readily available material since it doesn't take any particularly advanced machinery to make it work we were happy to choose this one now that you know how the test works from a theoretical standpoint let's discuss it from a more practical point of view for now the test requires that we start with rna that was extracted from a virus using a commercially available kit one microliter of this solution is transferred over to the tube containing the rt lamp reaction ideally in the future we would like to modify the test so that it becomes possible to directly add a patient's saliva to it and continue on from there that would noticeably cut down on the labor required to obtain each reading cut down on the overall cost and make it much faster for the results to come in after transferring one microliter of the rna extract to the first tube the tube must then be incubated at 65 degrees celsius for 30 minutes after that one microliter is transferred from the first tube into the second which contains the t7 and cast 13 solution after tapping the tube a few times to mix it and microcentrifuge it down for a few seconds all you need to do is incubate that tube at 37 degrees celsius for 30 minutes at that point all of the reactions are done and that the results are ready to be read by exposing the tubes to a wavelength of approximately 490 nanometers it's possible to easily detect which samples contain cyrus kov2 those ones will fluoresce a bright green those that didn't contain any of the viral specimens of interest will be completely colorless and indistinguishable from a tube filled with a little water i'll show you that later now it's worth noting that the dna and rna sequences that we have used in this first version of our test were all previously published by different groups as such we do not believe that they will result in a positive signal when in the presence of a relevant rna this is particularly important because your saliva is full of rna the extraction process for the virus does indeed result in viral rnas being captured but there tends to be a whole lot more human and bacterial rnas too in addition other coronaviruses that are much less dangerous are fairly common in the human population if our test gave positive signals every time someone came in with a common cold then it wouldn't be very effective as such we have based ourselves on previously described templates to try and avoid this as much as possible ao projetar este teste, tentamos garantir que ele não dependesse excessivamente de máquinas complicadas queríamos que todo o teste fosse fácil de realizar usando apenas alguns aparelhos comuns de laboratório como resultado, com esta iteração atual do teste, tudo o que precisamos é de uma máquina que possa aquecer tubos estilo pcr a 65 graus celsius e um que pode aquecer os tubos pcr a 37 graus celsius também precisamos de dois p2 ou p10 pipetas por motivos que abordaremos mais tarde, juntamente com suas pontas de pipeta associadas uma pequena microcentrífuga capaz de receber tubos de PCr também é necessária apenas para garantir que a reação misturas não tocam no topo do tubo quando você os abre e potencialmente contaminam todos os tubos circundantes, finalmente, é necessária uma máquina capaz de emitir comprimentos de onda ultravioleta nesta imagem você verá a máquina que tenho usado é mais comumente usada para visualizar géis em uma biologia molecular lab, mas faz um trabalho muito bom ao visualizar nossas amostras positivas em nosso teste para as duas incubações períodos várias opções diferentes estão disponíveis eu tenho usado um termociclador antigo hoje como considera-se clinicamente que ele faz um bom trabalho de aquecimento uniforme dos meus tubos, vale a pena notar aqui que isso significa that even old and run-down thermocyclers that no longer do a very good job of cycling between temperatures can be readily used in this test unlike the current rtq pcr gold standard that requires specialized real-time thermocyclers here any old machine can be used it's also possible to use other common laboratory apparatuses such as block heaters that have an adapter for pcr tubes please note that in this picture the block heater has an adapter for 1.5 millimeter microcentrifuge tubes so the true adapter will look a little different i've also used incubators to great success even material incubators that operate at a steady 37 degrees celsius can perfectly suit the needs of this test so long as the tubes are readily exposed to the ambient temperature then positive signals should have no difficulty in resolving please note however that when using incubators it's better to have the tubes exposed to the air that way they don't waste time as their rack slowly begins to heat up water baths are one final option that you can choose personally i tend to avoid this option because i find the risk for cross-contamination between samples to be much greater and therefore unideal however i have tried to perform rt lamp reactions with this system and so long as you're careful it's very possible to do it once the reactions have been completed it's possible to use various different machines to visualize the tubes depending on the materials your laboratory has personally my machine of choice is a gel uv emitter these machines which are more commonly used to visualize agarose gels do a very good job of safely showing which tubes are fluorescent and which aren't black lights can also be used for this test however i have found them to be slightly less desirable since the reporter rna best absorbs wavelengths of 490 nanometers i have found that black lights which produce wavelengths of 390 nanometers to be less favorable for this test for them to work it's better to visualize the tubes in a dark room in both cases the machine should really be placed in a darker room away from excessive light a little background light seems to have less of an effect in the case of the gel uv light however this picture seen on the left was taken using a generic iphone when the tubes were placed on the gel uv light the lights in the room were only partially turned off resulting in lots of ambient lighting even if there were no lights directly pointed at the machine given the strong and even radiation that the tubes received on this machine it's easy to determine which samples were positive and which were negative in the case of the black light on the right it's clear which samples are positive and which are negative of course however the fluorescence is not even and it's a little bit harder to detect by the naked eye in this case the camera on the iphone took a very good picture and made it easier to visualize the tubes overall now there are several important considerations to be had when using this test for it to work properly and not give either false positives or false negatives it's important to first designate proper working stations for each section of the test it's also necessary to follow the predetermined reaction times without varying otherwise the results might start to differ slightly finally those handling the test must be careful not to contaminate any of the tubes with rnases the first consideration to separate working areas is extremely important because lamb is excessively sensitive this technique is known to produce lots and lots of concentrated target dna we as well as others have noted that when we opened tubes that completed the lamp amplification in the same area that we originally used to set up the reaction it's possible to contaminate the working area with aerosols containing the product if you continue to set up the lamp reactions in those areas afterwards then you run the risk of contaminating your new tubes with amplicons from the previous reactions by touching your desk or various other contaminated surfaces with your gloves you run the risk of artificially seeding your reaction mixtures that contain patient rna with no sars cov2 and therefore producing positive results when there really shouldn't have been any by separating the areas in which you set up the lamp reactions and those in which you use the amplified sequences you run less of a risk for this to happen it's also necessary to have designated lab coats pipettes and other such materials for each working station if those in workstation 2 become contaminated you don't want that coming back into workstation 1.

Please note that this is much less stringent when bringing materials from estação de trabalho 1 para estação de trabalho 2. Já que é a reação da lâmpada rt que corre o maior risco de contaminação trazer material por exemplo luvas dessa estação para a próxima não deve ser um problema, desde que as luvas ainda estejam relativamente limpo, observe que esta situação de contaminação por aerossol é de muito menos importância durante a segunda etapa do teste, pois discutimos anteriormente o cas13 proteína que estamos usando requer uma quantidade relativamente grande de rna para ser funcional no momento, não estamos nem um pouco preocupados que uma contaminação por aerossol da lâmpada pode produzir falsos positivos de nossas reações do elenco 13, pois ainda temos que ver uma única situação onde isso aconteceu, a segunda consideração digna de nota é que o tempo de reação deve ser seguido uma pequena discrepância pode ser tolerada que seja um minuto a mais ou a menos não realmente parecem ter um grande impacto no teste, no entanto, mais do que isso pode começar a se tornar problemático no caso das equipes externas de reação à lâmpada rt, assim como nós vimos que dobrando a reação tempo para a lâmpada podem surgir falsos positivos como você pode ver aqui em ambas as fotos uma reação bem-sucedida da lâmpada também pode ser visualizada em um gel de agarose, as reações bem-sucedidas são caracterizada pela presença de várias bandas grandes de tamanhos variados, no entanto, quando a lâmpada reactions go on for too long it's possible to begin amplifying samples with nothing in them for example on the left you'll see a gel that was taken from an article in which at 30 minutes the wells containing reactions with 10 copies of the target are viral rna and no copies of the target viral rna had no bands in the case of the gel on the right a gel that i made three of the wells contained reactions that had no target rna to speak of only the n fragment well contained target rna at 30 minutes these negative controls did not demonstrate any amplification as we expected however at 60 minutes both the published gel and mine started to give amplified products in their case the 10 and 0 copies both started to be amplified and in my case one of my negative samples started to get amplified i've performed several experiments so far with 30 minute incubation times and so far have never gotten false positives that's not to say that it's entirely impossible simply that it's very unlikely as a side note this is a good time to mention that changing gloves frequently when performing the first reaction is a good way to avoid these kind of unlikely false positives after prolonged use gloves might become contaminated with small amounts of viral rna and that could potentially lead to false positives even if we're only incubating at 30 minutes a consideração final a ser observada é a presença de rnases para aqueles de vocês que podem não estar rnases familiares são enzimas que clivam todos os rnas que encontram ambas as etapas para este teste são sensíveis a rnases a lâmpada arty inicial começa sua amplificação do rna, de modo que a presença de rnases pode ser muito prejudicial e, portanto, levar a falsos negativos o segundo passo do teste o t7 e o cast 13 passo usa um rna repórter para sinalizar tubos positivos se enquanto você estiver usando os testes você começar a introduzir rnases então você corre o risco de obter falsos positivos rnases são extremamente difíceis de remover, uma vez que eles são introduzidos em um sistema, então o melhor meio de proteger seu teste é usando a prevenção, uma vez que as suas mãos produzem muitos rnases é obrigatório o uso de luvas sempre ao trabalhar com o teste ao fazer isso, você evitará que as rnases naturais do seu corpo degradem as estrelas iniciais cov2 rna e proteger o repórter fluorescente no final as luvas também devem ser trocadas sempre que eles entram em contato com o cabelo da pele ou outros objetos frequentemente manipulados como maçanetas e dispositivos pessoais o uso de rnas e dnase free pipetas filtradas também é importante para este teste usando esses tipos de pontas especializadas you can make sure that any possible contaminations you might have in your pipettes doesn't have a chance to contaminate your test in areas where the risk for rnas contamination is fairly high it might be worthwhile to treat all surfaces or tools with an rnas removal solution these kinds of solutions can degrade rnases that might otherwise stick to your gloves and contaminate your samples please note i haven't had to use the solution very often so long as you maintain a clean working schedule you should be fine the second component for the test the one containing t7 and cast 13 is also designed to be able to tolerate a certain level of contaminating rnases there is an rnas inhibitor added to the reaction mixture to prevent the degradation of the reporter in case the original sars kobe 2 rna wasn't completely clean or in case contaminations occurred while you were repairing the tests that said the presence of excessive rnases can still fully activate the test seen here on the right one microliter of very concentrated rnase a is capable of causing the test tube to become fluorescent it's therefore important to take the required cabriolet test that can be widely distributed while also meeting this limit of detection is fully functional for curbing the spread of this pandemic while rtq pcr is much more sensitive do que isso, sua detecção limitada aprimorada pode não ser necessariamente tão benéfica, uma vez que esta técnica pode recolher rnas libertados por células infectadas e mortas após a infecção ter terminado essa forma de triagem pode fazer com que indivíduos nominalmente sars kovi positivos, mas não são realmente infecciosos para serem isolados desnecessariamente como você pode ver aqui à nossa esquerda, nosso teste de detecção é capaz de detectar amostras que possuem 100 exemplares por microlitro como foi estipulado pelo modelo mais comparação recente entre os diferentes primers que usamos para a lâmpada demonstrou que uma versão do nosso teste pode detectar amostras contendo apenas 80 cópias por microlitro enquanto o outro só pode detectar amostras contendo 1.000.

No futuro, provavelmente focaremos nossa esforços no primeiro conjunto de primers para garantir que atingimos o limite de detecção que foi estipulado pelo modelo como nota final, temos o prazer de informar que nosso teste funciona não apenas em fragmentos do genoma cyrus cov2, mas também quando exposto ao perfil de rna completo do vírus inicialmente começamos o desenvolvimento de nosso teste usando fragmentos dos genes de vírus que nós mesmos havíamos transcrito no laboratório recentemente, usamos o teste em vírus sars cov2 reais e obtiveram os resultados esperados como você pode ver à esquerda, a primeira seção da reação, a reação da lâmpada rt funciona da mesma forma expected on the viral rna the top left wells that were run on the agarose gel clearly demonstrate a strong amplification below them three reactions were performed with cells that had not been cultured with the virus the viral rnas were created by first isolating the viruses from a patient these viruses were then cultured in vero e6 cells and after a few days the culture medium was treated to extract any and all rnas that were present the viruses were therefore destroyed and their rna was obtained when the lab amplicons that had amplified the real virus were transferred over to the second stage of the test they gave the expected strong fluorescent signals at the moment what remains to be done is to use our tests on samples that have been confirmed positive or negative by rtq pcr the current gold standard in sarsko v2 detection and see how our test compares this will give us a better indication of what our tests sensitivity and specificity are just to wrap up i would like to thank dr gary cobinger dr alain ghani and dr ghiblave for having helped to provide finances or materials for this project i'd also like to thank dr tremblay and everyone in his team for having helped to provide certain insights as i was redesigning this test finally i'd like to thank Dr.

Marcello Bossoisfor tendo ajudado a organizar todo este encontro e o instituto canadense de pesquisa em saúde e os frqs por terem me ajudado a me financiar enquanto eu seguia este projeto ok então essa foi minha apresentação uh sim obrigado gabriel uh eu vou tomar o tempo apenas para fazer um para resumir o melhor que posso aqui para os que não falam inglês muito obrigado pela apresentação obrigado ok então é é esqueceram positivo obrigado matilda e obrigado dr graviol professor jackson do you like to talk something at this point uh i have nothing to add except if there is some question that i can answer otherwise i thank you for organizing this session thank you question is uh writing english can you read the commentary first one can you read the comments yeah but i think that most of them are in portuguese no i asked you to write in english i have in the comments oh okay i was looking at private chat i'm going up the list oh boy lots of comments are you in commencement chat yeah i'm just looking at the comments very quickly uh grow up anachrestia is the probability that the crispr test will give false negative results that's a question mostly for gabrielle gabriel can you answer that uh so at the moment we don't entirely know with my test generally um tests similar to this one have a sensitivity of about 90 percent so the probability of giving a negative result is very low because it also tends to have a sensibility of about 80 to 90 as well so overall it's better than some of the alternatives that exist that aren't rtq bcr another question for you it's also from anna christina does the crisper test apply to other disease like ebola zika or dank so we actually intend to adapt the test to ebola uh as soon as we can um we're toying com a ideia de fazer zika leps e alguns outros, porque tudo o que precisamos fazer é mudar alguns dos sequências que estamos usando para que os primers da lâmpada e o guia para a proteína 13 mais nítida e nesse ponto, seremos capazes de começar a detectar esses vírus imediatamente, então agora que o teste existe, é apenas um questão de otimizá-lo para cada vírus individual ok, há outro pergunta da frança antártica nouvelle uh quais são as perspectivas de usar o crispr tecnologia castline para tratar o hiv devo admitir que não sou um especialista em hiv eu sei que alguns grupos estão trabalhando nisso eu não sei quais são as chances disso se isso funcionará ou não, mas acho que isso é muito zonal, você conhece as bactérias usam a tecnologia crispr cat9 há milhões de anos para cortar o vírus e, portanto, a ideia aqui é usar a tecnologia em cascata para esmagar para cortar o vírus hiv considera-se clinicamente que isso é bastante notável de fato no início da pandemia do cobia 19 eu vi um grupo nos unidos Estados que se propunham a usar a tecnologia crispr cast 9 de fato, para cortar o vírus da cópia 19, então é a mesma abordagem para o hiv i acho que esta é uma abordagem razoável, meu laboratório não está trabalhando nessa abordagem também desde o início, uh, essa tecnologia pode ser antibiótica em um futuro próximo para example to treat multi-resistant tuberculosis again i think it's a good suggestion and yes indeed the main problem with the with these crispr cas9 technology is to find an adequate technique to deliver either the cast 9 protein of the castline gene and so far in gene therapy what is being used is the adeno associated virus unfortunately i didn't know associated virus custom fortune to produce in good manufacturing practice so we have to find another delivery method to deliver into the cas9 protein or the cas9 gene and then it would ease using it because right now it would be just too expensive to be used instead of an antibiotic also from french and tactic crispr cast 9 may be an option in the near future to treat koben ebola zika malaria well all of that i think is feasible if i would have more money in my lab we could start working on that also i i think these are a reasonable project many of the comments are in portuguese unfortunately there is no question for you yes only comments no questions let me try to help is there any questions only command yeah i see that i vary between two and five stars well one question from vitor is it like to know it is i'd like to know if crispr is affordable to public health system and if it can contribute to the cost redução da terapia genética que já existe a principal causa de terapia gênica que existe atualmente é a produção do adenocarcinoma vírus associado em condições de boas práticas de fabricação na última vez em que penso em fazer um ensaio clínico ou uma entrega sistêmica de alguma terapia genética você levou cerca de um milhão de dólares para produzir aav suficiente para tratar um paciente para fazer um ensaio clínico em 10 pacientes que você precisava apenas para iniciar um orçamento de 10 milhões e claro que até agora não consegui obter um orçamento tão grande uh o único pessoas que têm um orçamento tão grande são empresas americanas que vão gastar uma fortuna fazendo isso ensaios clínicos e eles têm milhões de dólares disponíveis, o problema é que após o tratamento ter se mostrado eficaz, o custo do tratamento é de 2,3 milhões dólar por pessoa, então considera-se clinicamente que é o futuro que precisamos para desenvolver genes terapia para muitas doenças de estudo, mas também precisamos desenvolver uma terapia genética barata que você conhece ii acho que idealmente uma terapia genética deveria custar cerca de 100.000 nem um milhão de dólares, porque a maior parte do sistema de saúde pública não pode se dar ao luxo de fazer isso considera-se clinicamente que é apenas nos Estados Unidos que as pessoas ricas podem pagar por aqueles e os pobres não terão acesso ao tratamento ainda procurando a lista para ver se há alguma outra pergunta desculpe-me professor jax só para fazer um pequeno comentário aqui para aqueles que não falam inglês vou tentar pegar a ideia geral das respostas que você acabou de dar e pegar gabriel também uh só um segundo princípio estrangeiro esqueceram tecnologia obrigado pelo seu tempo e se houver alguma dúvida, você pode continuar lendo e respondendo bem, se houver outras perguntas, as pessoas sempre podem me enviar algumas e-mails ok uh obrigado professor jack england por este encontro maravilhoso obrigado gabrielle obrigado barbara dr barbara obrigado matteo shots e espero que você tenha outro encontro como este obrigado muito obrigado adeus obrigado tenha um bom esqueceram.

Desdobramento em Medicina Baseada em Evidências: Correlação Fisiopatológica e Conduta Clínica

Sob os princípios da Medicina Baseada em Evidências (MBE) aplicados à Alergia e Imunologia Clínica, a interpretação de sinais e sintomas respiratórios, cutâneos, gastrointestinais ou sistêmicos exige a correlação rigorosa entre a história clínica detalhada (fenótipo) e os mecanismos imunológicos subjacentes (endótipo — mediado por IgE, por linfócitos T, por imunocomplexos ou por vias da imunidade inata). A investigação diagnóstica direcionada — utilizando testes cutâneos padronizados, dosagens laboratoriais específicas e provas funcionais — permite estabelecer o diagnóstico diferencial preciso, evitar a automedicação e instituir um plano terapêutico individualizado voltado ao controle sustentado da inflamação e à preservação da qualidade de vida do paciente.

Saúde da Barreira Cutânea, Inflamação Imunológica e Controle do Prurido

Disfunção Epitelial, Microbioma da Pele e Sensibilização Tardia

Nas dermatites eczematosas e quadros de prurido crônico, diferenciar a atopia constitucional da dermatite alérgica de contato (uma reação de hipersensibilidade tardia do tipo IV mediada por linfócitos T sensibilizados a metais, conservantes cosméticos, fragrâncias, resinas ou borracha) é indispensável. A exposição repetida ao agente sensibilizante mantém a inflamação cutânea ativa mesmo sob uso de pomadas anti-inflamatórias, exigindo identificação exata do fator desencadeante.

Hidratação Terapêutica, Testes Cutâneos e Imunoterapia

O pilar diário de proteção consiste na hidratação intensiva imediata após banhos rápidos e mornos com higienizadores sindet (sem sabão alcalino), aliada a protocolos personalizados de vacina para dermatite. Na investigação causal, o teste de alergia (seja o prick test para aeroalérgenos ou o patch test de contato para substâncias químicas) orienta medidas precisas de evitação e viabiliza a indicação de vacina para alergia em pacientes sensibilizados a ácaros.

Mecanismos Vacinais, Excipientes e Avaliação da Competência Imunitária

A imunização preventiva representa uma das maiores conquistas da medicina para reduzir hospitalizações e complicações infecciosas graves, especialmente em portadores de doenças respiratórias crônicas (como asma e DPOC), cardiopatias, hipertensão arterial e diabetes. Pacientes alérgicos a aeroalérgenos, alimentos ou medicamentos comuns não possuem contraindicação geral às vacinas; a atenção alergológica concentra-se na avaliação específica de raros casos de anafilaxia prévia a componentes isolados da fórmula (como polietilenoglicol, polissorbato ou traços proteicos específicos), situações que hoje contam com protocolos seguros de aplicação assistida.

Prevenção Ambiental, Adesão Terapêutica e Monitoramento Contínuo

O sucesso a longo prazo no manejo das condições alérgicas e imunológicas depende diretamente da aliança entre tratamento médico direcionado e medidas educativas cotidianas. Manter ambientes bem ventilados e livres de acúmulo de poeira doméstica, preservar a hidratação fisiológica das mucosas e da barreira cutânea, respeitar rigorosamente os horários e a técnica de uso das medicações preventivas prescritas e realizar revisões periódicas permitem estabilizar a inflamação subclínica antes que ela evolua para exacerbações agudas.

Critérios de Segurança Clínica e Sinais de Alerta em Alergologia

Reconhecer precocemente os sinais de alerta que exigem reavaliação médica prioritária faz parte da educação em saúde de todo paciente e familiar. Sintomas como dispneia progressiva (dificuldade para respirar ou falar frases completas), chiado torácico refratário, edema súbito de lábios, língua ou glote, tontura por queda pressórica, febre persistente associada a lesões de pele ou acometimento de mucosas demandam atendimento imediato de urgência, seguido de investigação alergológica estruturada para prevenir recorrências futuras.

Impacto na Qualidade de Vida, Sono e Bem-Estar Sistêmico

Além das manifestações físicas diretas, quadros não controlados ligados a Seminário Internacional de Ciências interferem profundamente na arquitetura do sono reparador, na capacidade de concentração escolar e profissional e no equilíbrio emocional. O estresse crônico, por meio de mediadores neuroendócrinos e alarminas epiteliais, retroalimenta a resposta inflamatória imunitária. Dessa forma, instituir uma abordagem integral de reabilitação clínica e ambiental restaura a autonomia diária e o bem-estar global do indivíduo.

Importância do Diagnóstico Diferencial e da Anamnese Detalhada

Na especialidade de Alergia e Imunologia, dois pacientes com sintomas aparentemente semelhantes envolvendo Seminário Internacional de Ciências podem apresentar mecanismos fisiopatológicos completamente distintos. Por essa razão, a história clínica minuciosa — investigando cronologia das crises, ambiente domiciliar e profissional, histórico familiar de atopia, uso de fármacos concomitantes e hábitos alimentares — funciona como bússola indispensável para interpretar corretamente exames laboratoriais (como dosagem de IgE total e específica) e provas funcionais. Um resultado laboratorial isolado jamais substitui o julgamento clínico presencial ou por telemedicina especializada, prevenindo diagnósticos equivocados e tratamentos desnecessários.

Perguntas Complementares Frequentes na Prática Clínica

Reações Imunológicas a Picadas de Insetos e Prevenção de Crises: Na prática alergológica, distinguir um edema inflamatório local de uma reação alérgica sistêmica é decisivo para proteger a vida do paciente. Na hipersensibilidade a mosquitos, ocorre resposta imune mista (imediata mediada por IgE e tardia celular por linfócitos T), fazendo com que uma única picada reative lesões antigas pelo corpo. Já nas reações a abelhas e marimbondos, sintomas à distância do local da ferroada — como placas generalizadas, rouquidão, broncoespasmo ou queda de pressão arterial — caracterizam anafilaxia e demandam conduta imediata.

Mecanismos da Alergia Alimentar, Diagnóstico Preciso e Manejo Nutricional: Na alergologia contemporânea, é essencial diferenciar a reação imunológica contra proteínas alimentares — como caseína, alfa-lactoalbumina e beta-lactoglobulina no leite de vaca, ovomucoide no ovo ou tropomiosina nos crustáceos — das intolerâncias digestivas não imunes, a exemplo da deficiência da enzima lactase. Enquanto a intolerância provoca distensão abdominal, flatulência e diarreia osmótica restritas ao tubo digestivo, a alergia alimentar envolve linfócitos Th2 e anticorpos IgE ou inflamação celular eosinofílica, podendo causar urticária aguda, angioedema, broncoespasmo, proctocolite ou anafilaxia em minutos após a ingestão de traços.

Imunologia da Mucosa Digestiva: Eixo Intestino-Vias Aéreas e Diagnóstico Diferencial: O equilíbrio entre a microbiota comensal e a mucosa digestiva regula diretamente a tolerância imunológica sistêmica. Proteínas como a gliadina podem modular a liberação de zonulina em indivíduos geneticamente suscetíveis (portadores de alelos HLA-DQ2 ou HLA-DQ8 na doença celíaca), ampliando a permeabilidade intestinal e perpetuando cascatas inflamatórias locais e extraintestinais. A distinção criteriosa entre autoimunidade celíaca, alergia ao trigo IgE-mediada e fermentação de carboidratos Fodmaps evita condutas inadequadas.

Imunologia das Doenças Autoimunes Cutâneas e Cuidado Integrado: Na interface entre a Imunologia Clínica, a Dermatologia e a Reumatologia, avaliar o paciente de forma sistêmica permite reconhecer precocemente comorbidades metabólicas, articulares e eventuais hipersensibilidades coexistentes. Exames como o Fator Antinuclear (FAN) e provas inflamatórias atuam como ferramentas de triagem que só adquirem significado diagnóstico real quando interpretados à luz do exame físico detalhado da pele, unhas, couro cabeludo e articulações.

Fisiopatologia das Urticárias e do Angioedema: Diagnóstico e Controle Seguro: As urticárias caracterizam-se pelo surgimento súbito de urticas (pápulas eritematosas e edematosas intensamente pruriginosas e fugazes, que desaparecem em menos de 24 horas no mesmo ponto sem deixar cicatriz), podendo acompanhar-se de angioedema nas camadas profundas da derme e submucosa. Nos quadros agudos (duração inferior a seis semanas), infecções virais, alimentos e medicamentos figuram entre os gatilhos clássicos; já na urticária crônica espontânea ou nas urticárias induzidas (dermográfica, colinérgica pelo suor/aquecimento, ao frio ou à pressão tardia), mecanismos autoimunes ou de hiper-reatividade mastocitária assumem papel central.

Controle da Inflamação Brônquica, Função Pulmonar e Prevenção do Remodelamento: O monitoramento objetivo por meio da espirometria (prova de função pulmonar com teste broncodilatador) é indispensável mesmo nos períodos em que o paciente acredita estar assintomático, pois detecta obstruções silenciosas em pequenas vias aéreas. Os corticoides inalatórios modernos atuam diretamente na mucosa respiratória em doses medidas em microgramas, oferecendo elevada segurança sistêmica, reduzindo exacerbações graves e protegendo a capacidade respiratória a longo prazo.

Via Aérea Unificada: Manejo da Rinite, Rinossinusite e Hipersensibilidade Nasal: O nariz exerce funções vitais de filtragem, aquecimento e umidificação do ar inspirado antes que ele alcance os pulmões. Na rinite alérgica e na rinossinusite crônica, o contato de aeroalérgenos — sobretudo ácaros da poeira doméstica (Dermatophagoides pteronyssinus e Blomia tropicalis), fungos anemófilos e epitélios — com anticorpos IgE fixados nos mastócitos da mucosa nasal dispara liberação imediata de histamina, leucotrienos e prostaglandinas. Esse processo gera obstrução nasal, prurido, espirros em salva, gotejamento pós-nasal (causa frequente de tosse crônica noturna) e fragilidade vascular que pode favorecer pequenos sangramentos (epistaxe) pelo trauma ao assoar ou pelo jato mal direcionado de sprays.

Segurança Farmacológica, Reações Adversas a Medicamentos e Uso Racional de Corticoides: Outro ponto crítico de farmacovigilância diz respeito ao uso indiscriminado de corticoides sistêmicos orais ou injetáveis de depósito de longa ação (como associações de betametasona intramuscular). Embora promovam alívio rápido aparente, aplicações repetidas suprimem o eixo hipotálamo-hipófise-suprarrenal, elevam a pressão arterial e a glicemia, desmineralizam a massa óssea, fragilizam a imunidade contra infecções e podem provocar dor neuropática local quando aplicadas inadequadamente na região glútea.

Imunização Segura, Resposta Imunológica e Proteção do Paciente Alérgico: A imunização preventiva representa uma das maiores conquistas da medicina para reduzir hospitalizações e complicações infecciosas graves, especialmente em portadores de doenças respiratórias crônicas (como asma e DPOC), cardiopatias, hipertensão arterial e diabetes. Pacientes alérgicos a aeroalérgenos, alimentos ou medicamentos comuns não possuem contraindicação geral às vacinas; a atenção alergológica concentra-se na avaliação específica de raros casos de anafilaxia prévia a componentes isolados da fórmula (como polietilenoglicol, polissorbato ou traços proteicos específicos), situações que hoje contam com protocolos seguros de aplicação assistida.

Medicina de Precisão: Como Funcionam os Anticorpos Monoclonais e Imunobiológicos: A escolha do imunobiológico ideal exige fenotipagem e endotipagem rigorosas pelo médico especialista, avaliando biomarcadores sanguíneos (contagem de eosinófilos periféricos, níveis séricos de IgE total e fração exalada de óxido nítrico) e comorbidades coexistentes, como asma grave, polipose nasal, urticária crônica espontânea refratária e dermatite atópica moderada a grave. As aplicações subcutâneas seguem cronograma regular sob supervisão assistencial segura.

Conclusão e Síntese das Orientações

Conclui-se que o manejo eficiente das questões relacionadas a Seminário Internacional de Ciências apoia-se na união entre informação científica de qualidade, prevenção ambiental e terapêutica direcionada ao perfil de cada paciente. Diante de sintomas persistentes ou recorrentes, buscar avaliação com médico Alergista e imunologista — integrando investigação diagnóstica criteriosa e opções consolidadas de cuidado especializado em alergia e imunologia sempre que indicadas — garante segurança clínica e proteção duradoura da saúde.

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