





































Berkeley
Pharma Tech
Journal of Medicine

Correspondence:
kellyyan@berkeley.edu

Keywords:
Autism, Gene Therapy, 
Neurodevelopment, Genetic Factors, 
Epigenetics

Submitted: December 11, 2022 
Accepted: January 27, 2023 
Published: June 30, 2023

Full Open Access

Creative Commons Attribution 
License 4.0

Abstract
Autism spectrum disorder (ASD) is a range of developmental disorders 
characterized by impaired traits associated with three distinct domains: 
communication, social interaction, and stereotypic repetitive behavior. 
Although the etiology of ASD depends on various components, current 
research mainly focuses on the genetic factors that contribute to the 
development of ASD and its effects. A big treatment consideration is gene 
therapy, which has disease-modifying potential. This article provides 
insight into the foundation of ASD and the leading gene therapies that aim 
to  address impaired neurological behavior. We will discuss how certain 
genetic factors can have large contributions to ASD development and how 
scientists can go about targeting these factors for potential remedies.

Gene Therapy as a Promising Approach 
for the Underlying Causes of Autism 
Spectrum Disorder
By: Kelly An, Fadel Batal, Sonya Svyatskya, Vanloan Nguyen

AutismSpectrumDisorder



 Introduction 

 Autism  spectrum  disorder  (ASD)  is  a  neurodevelopmental  condition 
 that  a�ects  communication  and  social  interaction.  It  is  a  complex 
 condition  with  a  range  of  symptoms  and  severity  levels,  and  the  exact 
 causes  are  still  not  fully  understood.  1  However,  genetic  factors  are 
 believed to play a signi�cant role in the development of ASD.  2

 Symptoms  of  ASD,  which  typically  appear  in  early  childhood,  include 
 impaired  social  interaction  and  communication,  repetitive  behaviors  and 
 interests,  and  sensory  processing  issues.  These  symptoms  range  from  mild 
 to severe and may a�ect an individual's ability to function in daily life.  1, 3

 At  a  brain  developmental  level,  ASD  is  thought  to  result  from  abnormal 
 development,  particularly  in  regions  of  the  brain  involved  in  social 
 interaction  and  communication.  Some  studies  have  suggested  that 
 individuals  with  ASD  may  have  di�erences  in  brain  structure  and  function, 
 such as altered connectivity between di�erent regions.  4

 At  a  genetic  level,  ASD  is  believed  to  be  caused  by  a  combination  of  genetic 
 and  environmental  factors.  While  the  exact  causes  of  ASD  are  still  not  fully 
 understood,  research  has  identi�ed  a  number  of  genetic  variations  that  are 
 associated  with  an  increased  risk  of  developing  the  condition.  These  genetic 
 factors  may  interact  with  environmental  factors  to  in�uence  brain 
 development, ultimately resulting in ASD.  1

 Autism  is  one  of  the  most  heritable  neurodevelopmental  disorders, 
 a�ecting  78  million  people  or  1.5  %  of  the  world’s  population.  5  Despite  its 
 ubiquitous  existence,  there  are  aspects  of  the  disorder  that  science  has  yet 
 to  unveil.  With  the  etiology  of  autism  still  in  question,  scientists  have  been 
 able  to  conclude  that  at  least  40%  of  neurobehavioral  disorders  within  the 
 autism spectrum are the result of genetic abnormalities.  6

 The  current  treatment  for  ASD  falls  into  two  categories:  those  that  target 
 core  symptoms  which  include  impaired  communication,  social  interaction, 
 and  repetitive  behaviors;  and  those  that  target  secondary  or  consequential 
 symptoms  such  as  ADHD  and  irritability.  Gene  therapy,  a  treatment  that 
 targets  core  symptoms,  focuses  on  mending  genetic  building  blocks.  It  aims 

 Berkeley Pharma Tech Journal of Medicine |  11 



 to  alter  abnormalities  in  hopes  of  preventing  domino-e�ects  in  the  body 
 that  result  in  the  neurobehavioral  de�cits  which  characterize  ASD.  Using 
 viral  vectors  such  as  recombinant  adeno-associated  viruses,  gene  therapy  is 
 able  to  introduce  new  genetic  material  to  counteract  the  establishment  of 
 ASD  symptoms.  7  This  paper  will  be  discussing  studies  and  their  adaptation 
 of  gene  therapy  to  varying  pathophysiologies  of  ASD  in  order  to  o�er 
 e�ective  treatment  methods.  This  paper  will  cover  the  underdevelopment  of 
 neurons,  impaired  neural  migration,  impaired  synaptogenesis,  and  dendritic 
 morphogenesis.  8 

 Etiology of Autism Spectrum Disorders 

 Although  the  details  of  the  origin  of  ASD  have  not  yet  been  discovered, 
 scientists  have  come  up  with  multiple  theories  answering  the  question  of 
 “how”  ASD  comes  to  be  in  individuals.  One  such  theory  on  the 
 pathophysiology  of  ASD  is  linked  to  neural  connectivity.  In  a  typical 
 human  body,  an  individual  develops  a  surplus  of  neurons  where  over  time, 
 non-functional  and  unnecessary  neurons  are  removed  through  various 
 mechanisms.  In  patients  who  have  ASD,  the  mechanism  that  targets  the 
 elimination  of  underdeveloped  neurons  is  damaged.  As  a  result,  the  excess 
 neurons impair the shaping and �ne-tuning of neural circuits.  9 

 Another  theory  focuses  on  neural  migration.  Similar  to  neural  connectivity, 
 neural  migration  also  contributes  to  well-formed  neural  circuits  which  aid 
 in  proper  communication  and  other  neural  behaviors.  In  ASD  patients,  the 
 misplacement  of  neurons  as  a  result  of  faulty  migration  during 
 development  increases  the  thickness  of  the  cortex  and  “smudges”  the 
 boundaries  of  white  matter.  This  mostly  a�ects  the  frontal  and  temporal 
 lobes:  key  elements  in  processing  language  and  emotion—functions 
 commonly  found to be impaired in ASD patients.  9 

 An  additional  theory  that  de�nes  the  mechanism  by  which  ASD  is 
 established  focuses  on  impaired  synaptogenesis  and  dendritic 
 morphogenesis.  The  normal  development  of  synapses  and  dendrites  entails 
 excess  formation  followed  by  the  purging  of  faulty  expression.  In  patients 
 with  ASD,  the  suppression  mechanism  responsible  for  the  expulsion  of 

 Berkeley Pharma Tech Journal of Medicine |  12 



 faulty  signaling  factors  is  impaired.  The  overwhelming  number  of  defective 
 synapses  and  dendrites  damages  the  pathway  of  signals  within  the  body, 
 resulting in the core symptoms associated with ASD.  9

 Key Genetic Factors 

 The  build-up  of  faulty  factors  as  a  result  of  impaired  mechanisms  stems 
 from  mutations  present  in  the  genetic  factors  that  regulate  such  processes 
 within  the  human  body.  The  existence  of  these  mutations  is  what  has  led 
 scientists  to  link  ASD  to  genetic  abnormalities.  In  typical  human 
 physiology,  genetic  factors  work  in  a  factory-line  fashion  to  facilitate  proper 
 operations  in  the  formation  of  neurons,  synapses,  and  dendrites  as  well  as 
 key  neural  factors.  The  malfunction  of  any  genetic  factor  in  the  factory  line 
 creates  a  “domino  e�ect”  that  leads  to  forms  of  neurobehavioral  impairment 
 associated  with  ASD.  Although  there  are  many  di�erent  factors  that 
 contribute  to  proper  neurobehavior,  this  paper  focuses  on  the  mutation  of 
 three in particular: TCF4, RELN and MECP2.  10, 11, 12,  13, 14

 Studies: Linking Etiology of ASD and Mutations of ‘Key 
 Genetic Factors’ 

 4.1  TCF4 Gene Study 

 Transcription  Factor  4  (TCF4)  is  responsible  for  encoding  a  helix-loop-helix 
 transcription  factor  expressed  most  often  during  brain  development.  An 
 alteration  in  TCF4  causes  Pitt-Hopkins  Syndrome  (PTHS):  a  disorder 
 characterized  by  profound  cognitive  and  motor  disabilities  and  de�ned 
 under  the  umbrella  de�nition  of  autism  disorders.  Patients  with  PTHS  have 
 mutations  that  either  knock  out  the  functional  TCF4  gene,  eliminate  its 
 essential  DNA-binding  domain,  or  impact  one  of  its  transcriptional 
 activation domains.  10

 The  University  of  California  San  Diego  Medical  School  sought  to 
 understand  how  a  mutated  TCF4  gene  results  in  this  form  of  autism.  11,  12  To 

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 explore  this  idea,  scientists  used  pluripotent  stem  cells  (iPSC),  adult  somatic 
 cells  that  have  been  reprogrammed  to  be  in  an  embryonic  state,  of  5  patients 
 with  PTHS.  12  To  guarantee  a  controlled  experiment,  mutated  TCF4  was  the 
 only  chromosomal  abnormality  present  in  the  experimental  PTHS  iPCS 
 lines.  These  iPSC  lines  were  used  to  generate  neuron  progenitor  cells 
 (NPCs),  the  precursors  of  most  of  the  neurons  that  make  up  the  central 
 nervous  system.  The  PTHS  NPCs,  produced  by  the  PTHS  iPSc  lines, 
 presented  a  reduction  in  TCF4  expression.  This  resulted  in  neurons  with 
 altered  expressions  of  CNTNAP2  and  KCNQ1,  which  are  usually 
 regulated  by  the  TCF4  gene.  10,11  For  further  analysis,  scientists  used  the 
 control  and  PTHS  iPSCs  line  to  create  brain  cortical  (CtO)  organoids,  an 
 arti�cially  manufactured  tissue  resembling  the  functionality  and  structure 
 of the human brain.  11

 In  comparison  to  the  control  organoid’s  normal  spheroid  form,  the  PTHS 
 CtO  organoid  presented  an  abnormal  structure  and  size.  Through  single 
 cell  RNA  sequencing,  it  was  concluded  that  the  PTHS  cortical  organoid’s 
 diminished  size  and  characteristic  factors  were  the  result  of  a  loss  of  cellular 
 diversity.  To  make  certain  that  this  loss  was  not  a  result  of  mispatterning, 
 the  UC  San  Diego  team  performed  single  cell  transcriptomic  testing  on  the 
 organoids.  The  telencephalic  marker,  FOXG1,  expression  analysis  in 
 combination  with  the  single  cell  RNA  sequencing  concluded  that  the  loss 
 of  cell  diversity  in  the  PTHS  cortical  organoid  was  due  to  a  higher 
 percentage  of  underdeveloped  neurons,  known  as  neural  progenitor  cells 
 (NPC).  The  e�ects  of  the  mutated  genetic  factor  TCF4  on  the  brain 
 correlates  to  the  neural  connectivity  theory.  This  theory  posits  that  a  surplus 
 of  de�cient  development  of  neurons  due  to  impaired  progenitor 
 proliferation,  alters  the  function  and  structure  of  the  brain  and  its  neural 
 circuits  responsible  for  functions  such  as  communication  and  behavior. 
 This  excess  of  underdeveloped  neurons  prevents  the  idealized  function  of 
 neural circuits.  11

 With  an  understanding  of  the  �rst  domino  in  the  developmental  pathway 
 of  PTHS,  the  UC  San  Diego  team  focused  on  abolishing  its  molecular  and 
 cellular  characteristics  by  correcting  the  expression  of  the  TCF4  gene.  The 
 �rst  method  used  two  viral  vectors  and  a  CRISPR-  based  transepigenetic 

 Berkeley Pharma Tech Journal of Medicine |  14 



 strategy.  All  three  cassettes  worked  together  to  enhance  the  transcription  of 
 the TCF4 gene, ultimately correcting the downstream targets.  11

 Figure  1:  CRISPR-based  transepigenetic  strategy  for  correction  of  
abnormally  low  TCF4  expression;  a  complex  comprising  of  gRNA  targeting  
promoters  of  TCF4, a transcriptional activation complex (MPH) and dead Cas9 

 However,  the  use  of  two  viral  vectors  created  another  problem:  a  decrease  in 
 cellular  aggregation  in  the  brain  organoids.  The  scientists  attempted 
 another  method  called  virus-mediated  overexpression  in  which  a  wild-type 
 copy  of  TCF4  was  overexpressed  in  hopes  of  overriding  ectopic  expression 
 of  the  mutation.  PTSH  organoids  exposed  to  overexpression-type  gene 
 therapy  showed  improvements  in  two  key  regions  that  marked  the 
 downstream  corrected  function  of  the  TCF4  gene:  increased  �ring  rates  and 
 number  of  network  electrical  bursts.  The  combination  of  these  two 
 methods,  provided  that  the  impaired  proliferation  of  neurons  from  a 
 mutated  TCF4  can  be  genetically  corrected,  could  prove  to  lessen  the 
 symptoms of other genetic autism disorders.  11

 4.2  RELN Study 

 RELN  is  a  gene  that  encodes  for  a  RELN  glycoprotein  in  the  extracellular 
 matrix  of  GABAergic  (Gamma-Aminobutyric  Acid)  neurons.  These  cells 
 perform  important  functions  in  neural  migration  and  cortical  lamination.  13

 Neural  migration  is  an  important  process  that  occurs  in  mammalian 
 nervous  system  development.  14  Cortical  lamination  is  the  layering  of  cells  of 

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 the  outer  regions  of  the  brain’s  cerebrum,  helping  to  maintain  neuronal 
 cytoskeletal  stability.  Reduced  RELN  levels  have  been  shown  to  be 
 associated  with  di�erent  psychological  disorders  including  bipolar  disorder, 
 Alzheimer's  disease,  and  autism  spectrum  disorder.  Studies  have  shown 
 signi�cantly  lower  levels  of  RELN  in  ASD  patient’s  superior  frontal  cortex, 
 parietal  cortex,  cerebella  and  plasma.  This  could  potentially  be  because  of 
 RELN  promoter  hypermethylation  that  occurs  in  GABAergic  neurons,  as 
 seen in seizure disorder patients.  13 

 In  addition,  sex  hormones  can  also  play  a  role  in  methylation  of  the  RELN 
 promoter,  which  has  shown  to  increase  ASD-associated  behaviors.  After 
 postmortem  cerebellar  studies  of  ASD  patients,  it  was  found  that  lower 
 RELN  mRNA  levels  are  associated  with  higher  MECP2  (another  essential 
 protein  for  nerve  cells)  binding,  increasing  gene  regulator  5-hmC  at  the 
 RELN  promoter  and  in  turn  decreasing  transcription  and  protein  levels. 
 Such  RELN  mutations  that  cause  disruptions  in  signaling  pathways  are 
 connected  to  ASD  disorders.  One  example  is  the  loss  of  Purkinje  cells, 
 regulated  by  RELN,  which  increases  the  risk  of  cognitive  delay  and  epilepsy. 
 This is a phenomenon also seen in ASD patients.  15 

 Investigating  the  role  of  RELN  on  neuronal  signaling  and  ASD 
 development  can  produce  promising  leads  for  potential  treatments  of  ASD. 
 Increasing  levels  of  RELN  protein  can  alleviate  behavioral  symptoms  of 
 RELN  that  ASD  patients  also  experience.  However,  although  there  is  much 
 evidence  for  RELN’s  in�uence  on  ASD,  it  is  not  the  sole  factor  of  ASD 
 development.  For  the  diagnosis  of  ASD,  there  are  usually  secondary  genetic 
 or environmental factors that contribute to the disorder’s development.  16 

 4.3  MeCP2 Gene Study 

 Many  studies  have  shown  MeCP2  protein’s  role  in  brain  development  and 
 regulation,  such  as  expression  of  the  brain-derived  neurotrophic  factor 
 (BDNF)  gene  and  regulation  of  synaptic  homeostatic  plasticity.  MeCP2  is  a 
 part  of  the  methyl-CpG-binding  protein  family  which  regulates  gene 
 expression  by  modifying  chromatin,  a  protein  complex  of  DNA  and 
 histone  proteins  that  package  DNA  in  chromosomes.  MeCP2  performs 
 regulation of DNA methylation via recruitment of histone deacetylases.  17, 18 

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 Figure 2:  MeCP2 protein structure 

 Figure 3:  The MECP2 gene

 MeCP2  is  found  in  high  concentrations  in  neurons.  The  gene  locus  is 
 located  on  the  long  (q)  arm  of  the  X  chromosome  in  band  28  (“Xq28”). 
 MeCP2's  main  role  is  "repressing"  or  "silencing"  other  genes,  preventing 
 transcription  and  translation  when  they  are  not  needed.  Recent  studies  have 
 suggested  that  MeCP2  can  act  as  an  activator,  but  this  is  still  a  new  and 
 controversial  theory.  MeCP2  represses  gene  expression  by  recognizing  and 
 binding  to  methylated  cytosine  residues  in  DNA  called  5MeCyt;  regions 
 enriched  with  A/T  neighbor  bases.  MeCP2  is  also  able  to  bind  to 
 hydroxymethylated DNA called 5-hydroxy methylated cytosine.  18 

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 Figure 4:  The varied molecular functions of MECP2 protein 

 The Relationship Between Autism and MECP2 

 Mutations  that  alter  typical  MeCP2  function  will  lead  to  severe 
 neurodevelopmental  disorders  such  as  Rett  syndrome  (RTT)  and  ASD. 
 More  than  95%  RTT  patients  carry  Mutant  MeCP2,  and  some  MeCP2 
 mutations  have  been  reported  in  ASD  patients.  ASD  and  RTT  overlap  in 
 certain  phenotypes,  including  stereotypical  body  movements,  social 
 avoidance, and anxiety.  17, 18

 In  a  study  conducted  by  Zhu  Wen  at  School  of  Life  Sciences  in  Peking 
 University,  Whole-Exome  Sequencing  (WES)  was  performed  on  120  ASD 
 cases.  This  experiment  was  able  to  identify  three  mutations  in  the  coding 
 regions  of  the  MeCP2  gene.  They  found  that  the  MECP2  gene  was  linked 
 with a host of neuropsychiatric disorders and neurological phenotypes.  18 

 5.1  Methods: 

 120  Han  Chinese  families  were  selected  with  probands  diagnosed  with  ASD 
 from  2013-2015  in  the  Department  of  Child  and  Adolescent  Psychiatry  in 
 Shanghai  Mental  Health  Center.  The  range  of  age  of  the  patients  was 
 between  2  to  18  years  old  and  included  18  females  and  102  males.  The  study 
 excluded patients with severe somatic disorders RRT.  18

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 In  terms  of  clinical  assessment,  comprehensive  pro�les  of  each  patient  were 
 collected  in  the  case  report  form.  Researchers  classi�ed  the  symptoms  into  4 
 categories:  social  interaction,  language,  repetitive  behaviors,  and  functional 
 impairment.  2–3  μg  of  genomic  DNA  was  extracted  from  each  patient  and 
 libraries  in  order  to  prepare  cluster  generation  and  sequencing.  Based  on  the 
 patients’  WES  experiment,  families  within  probands  carrying  MECP2 
 variations  were  selected  for  Sanger  sequencing  to  determine  if  the  variations 
 were  de  novo  or  inherited.  The  WT-  MeCP2  gene  (the  E2  isoform  of  rat 
 Mecp2  cDNA)  and  other  mutated  plasmids  such  as  MeCP2-P152L, 
 MeCP2-R294X, and MeCP2-P376S were used to detect ASD probands.  18

 On  days  15–16  of  mouse  embryo  development,  cortical  neurons  and 
 HEK-293  cells  were  individually  cultured  and  electrophoretically 
 transfected  into  each  group  at  0  days  in  vitro  individual.  HEK-293  cells  and 
 mouse  cortical  neurons  were  collected  after  3  days  in  vitro  for  Western  blot 
 and immuno�uorescence analysis, respectively.  18 

 5.2  Results: 

 Three  mutations  of  MeCP2  were  detected  among  120  ASD  patients  via 
 WES.  p.P152L  (c.455C>T)  and  p.P376S  (c.1162C>T)  were  missense 
 mutations  and  p.R294X  (c.880C>T)  was  a  truncating  mutation.  Sanger 
 sequencing  showed  that  p.P152L  and  p.R294X  were  de  novo  mutations, 
 but  p.P376S  was  inherited  maternally.  They  did  not  �nd  any  of  these 
 mutations in GnomAD, indicating that they were rare mutations.  18 

 In  terms  of  clinical  features,  poor  social  interaction  and  functional 
 impairment  were  major  symptoms  for  all  three  mutations,  but  language  use 
 and  repetitive  behavior  di�ered  greatly  between  them.  Also  noted  were 
 abnormalities  of  dendritic  and  axonal  growth  found  after  autism-related 
 MeCP2  mutants  were  expressed  in  mouse  cortical  neurons,  suggesting  that 
 autism-related MECP2 mutations impair proper development of neurons.  18 

 The  results  strongly  suggest  that  MeCP2-P152L,  MeCP2-R294X,  and 
 MeCP2-P376S  a�ect  the  proper  physiological  function  of  the  MeCP2 
 protein and may contribute to the pathogenesis of autism.  18 

 Berkeley Pharma Tech Journal of Medicine |  19 



 MECP2 Gene Therapy Methods 

 In  addition  to  these  studies,  there  have  been  a  number  of  experiments 
 utilizing  gene  therapy  for  the  treatment  of  ASD.  The  majority  of  these  trials 
 have  utilized  a  viral  vector  to  deliver  a  modi�ed  version  of  the  MECP2  gene 
 to  patients.  The  results  of  these  trials  have  been  promising,  with  patients 
 exhibiting  improved  social  interaction,  better  communication  skills,  and  an 
 improved quality of life.  6, 19

 Recombinant  adeno-associated  virus  (rAAV)-delivered  gene  substitution 
 has  been  shown  to  improve  behavior  in  several  investigations  employing 
 monogenic  animal  models.  For  example,  a  recent  study  has  shown  that 
 systematic  administration  of  a  rAAV9-MECP2  vector  su�cient  for  10% 
 CNS  transduction  (of  primarily  neuronal  cells)  in  an  RS  animal  model 
 resulted  in  modest  behavioral  improvements.  6  Comparatively,  25%  CNS 
 transduction  at  a  6-fold  higher  vector  dose  led  to  noticeable  behavioral  and 
 phenotypic bene�ts.  19 

 A  promising  MECP2  gene  therapy  strategy  is  using  antisense 
 oligonucleotides.  Antisense  Oligonucleotides  are  small,  modi�ed  nucleic 
 acids  that  can  selectively  hybridize  with  m-RNA  from  a  target  gene  and 
 silence  it.  MECP2  duplication  syndrome  has  been  associated  with  autism, 
 intellectual  disability,  motor  dysfunction,  and  anxiety,  and  is  one  of  the 
 most  common  genomic  rearrangements  seen  in  male  patients.  The  cause  of 
 these  de�cits  is  the  over-expression  of  methyl-CpG-binding  protein  2 
 (MeCP2).  This  syndrome  poses  a  challenge  to  traditional  therapeutic 
 approaches.  19 

 Some  symptomatic  mice  models  of  monogenic  loss  of  function  neurological 
 disorders,  including  the  loss  of  MECP2  in  Rett  syndrome,  have 
 demonstrated  reversal  of  phenotypes,  indicating  that  the  molecular 
 correction  of  the  underlying  dysfunction  could  potentially  restore  typical 
 physiology.  The  study  focused  on  the  restoration  of  normal  MeCP2  levels  in 
 MECP2 duplication syndrome using mice models.  17, 19 

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 Figure  5:  methods  used  in  Sztainberg  et  al.  trials  testing  use  of  human-
speci�c  antisense oligonucleotides (ASOs) to normalize NeCP2 levels in mouse 
models 

 The  contributors  generated  a  conditional  MeCP2  -  overexpression  mouse 
 model  and  showed  that  the  correction  of  MECP2  levels  e�ectively  reversed 
 the  molecular,  electrophysiological,  and  phenotypic  de�cits  using  antisense 
 oligonucleotides  (ASO).  It  was  found  that  antisense  oligonucleotide 
 treatment  resulted  in  broad  phenotypic  rescue  in  symptomatic  transgenic 
 MECP2  duplication  mice  and  corrected  MeCP2  levels  in  lymphoblastoid 
 cells in a dose dependent manner.  19 

 Experimental  results  suggest  that  delivery  of  ASOs  to  CNS  could  prove  to 
 be  promising  for  treating  MeCP2  duplication  syndrome,  with  potential 
 applications  in  ASD  and  Rett  Syndrome.  Future  directions  should  focus  on 
 testing  di�erent  ASo  dosages,  determining  safety  margins  of  MeCP2  levels, 
 and screening MeCP2 ASOs for o�-target e�ects.  19 

 Antisense  oligonucleotides  (ASOs)  and  short  interfering  RNAs  (siRNAs) 
 are  two  methods  for  sequence-speci�c  suppression  of  mRNA  transcripts 
 which  can  then  be  used  to  mute  gene  expression.  In  a  conditional 
 MECP2-overexpressing  mouse  model  of  MECP2  duplication  syndrome,  it 
 was  demonstrated  that  halving  MECP2  expression  restored  cellular 
 function  and  phenotype  postnatally.  The  same  study  found  that 
 intraventricular  delivery  of  ASOs  speci�cally  directed  against  MECP2 
 caused  widespread  ASO  dispersion  throughout  the  CNS.  This  resulted  in 
 e�cient  knockdown  of  MECP2  to  levels  close  to  wildtype  and  a 
 long-lasting  phenotypic  reversal.  Both  studies  suggest  gene  replacement  and 
 RNA  knockdown  of  MeCP2  gene  may  be  a  method  worth  exploring  as  a 
 possible remedy for ASD.  19 

 Berkeley Pharma Tech Journal of Medicine |  21 



 Future Directions 

 In  addition  to  the  aforementioned  genetic  in�uences  on  ASD  cases,  there 
 are  other  factors  that  can  impact  the  pathophysiology  of  ASD.  For  example, 
 many  autism  patients  have  reported  gastrointestinal  disorders  (GI).  This  has 
 prompted  interest  in  possible  relationships  between  the  gut  microbiome 
 and autism.  7

 Up  to  70%  of  autism  patients  have  reported  dealing  with  GI  disorders. 
 These  GI  disorders  can  be  a  result  of  dysbiosis,  an  imbalanced  gut 
 microbiome,  characterized  by  reduced  microbial  diversity  and  increased 
 micro�ora  in  gut  microbiota.  The  gut  microbiome  generates  a  large  portion 
 of  metabolites,  and  is  responsible  for  energy  conversion,  signaling,  and, 
 most  importantly,  epigenetic  signaling.  An  imbalanced  gut  microbiome  can 
 create  an  excess  formation  of  S-adenosyl-methionine,  the  main  methyl 
 group  in  a  microbiome,  which  results  in  increased  methylation.  The 
 activation  and  deactivation  of  DNA  strands  through  DNA  methylation 
 could  potentially  lead  to  the  gene  impairment/variation  that  results  in  ASD. 
 Further  studies  into  this  phenomenon  may  have  implications  for  treatment 
 development  if  methylation  events  in�uencing  ASD  development  can  be 
 targeted  and  altered.  Future  research  studying  the  gut  microbiome’s  ability 
 to  interfere  with  methylation  patterns  can  be  used  to  inform  development 
 of gene editing in treating ASD pathogenesis.  1 

 Conclusion 

 While  there  is  currently  no  cure  for  ASD,  there  are  various  approaches  to 
 managing  its  symptoms  and  improving  quality  of  life.  These  include 
 behavioral  therapy,  medication,  and  support  services.  Research  into  the 
 causes  and  potential  treatments  of  ASD  is  ongoing,  and  new  developments 
 are constantly emerging.  2 

 Gene  therapy  is  one  promising  approach  that  holds  signi�cant  potential  for 
 treating  the  underlying  causes  of  ASD,  rather  than  just  managing  its 
 symptoms.  However,  more  research  and  clinical  trials  are  needed  to  fully 
 understand  the  safety  and  e�ectiveness  of  gene  therapy  for  ASD.  In  the  last 

 Berkeley Pharma Tech Journal of Medicine |  22 



 �ve  years,  clinical  research  has  shown  that  TCF4,  RELN,  and  MeCP2  are 
 linked  to  ASD,  providing  scientists  with  a  basis  to  explore  the  possibilities  of 
 gene  therapy.  The  CRISPR  strategy  and  viral  overexpression  in  relation  to 
 the  TCF4  gene,  MECP2  levels  in  relation  to  the  RELN  gene,  and 
 rAAV-delivered  transgenes  to  alter  gene  expression  all  show  promise  in 
 altering  the  behavioral  phenotypes  associated  with  autism  spectrum 
 disorder.  20

 However,  there  are  also  challenges  and  limitations  to  the  use  of  gene  therapy 
 for  ASD.  Gene  therapy  is  a  complex  and  expensive  approach  that  may  not 
 be  accessible  to  all  individuals  with  ASD.  In  addition,  there  are  ethical 
 considerations  surrounding  the  use  of  gene  therapy,  such  as  the  potential  for 
 unintended e�ects on other genes.  2 

 Overall,  gene  therapy  holds  signi�cant  potential  for  the  treatment  of  ASD, 
 but  further  research  and  clinical  trials  are  needed  to  fully  understand  its 
 safety  and  e�ectiveness.  If  successful,  gene  therapy  could  o�er  a  promising 
 new  approach  for  treating  the  underlying  causes  of  ASD  and  improving  the 
 lives of individuals with the condition. 

 Berkeley Pharma Tech Journal of Medicine |  23 



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