BACKGROUND
gene therapy is currently being evaluated for a wide range of acute and chronic lung diseases including those caused by single gene defects such as cystic fibrosis, alpha- <dig> antitrypsin deficiency and surfactant protein b deficiency  <cit> .
while the lung provides a natural route of entry into the host and can be accessed in a comparatively easy and noninvasive manner, methods for consistent and efficient delivery of viral vectors to the respiratory tract are required.
there have been a number of studies evaluating variables such as volume of inoculum, effect of diluent, nature of anesthesia , and position of mouse  <cit>  and how these affect intranasal delivery of therapeutic substances in mouse models, but a comparison of different delivery methods using viral vectors has not been done.
targeted delivery of viral vectors to the mouse respiratory tract can be performed by a variety of different methods, some of which require extensive skill and training.
in this report, four different methods of delivering viral vectors to the mouse respiratory tract were evaluated for ease and consistency, vector distribution, and invasiveness of the procedure with the aim of identifying the most effective and consistent method of delivery requiring minimal skill development.
for this study, a lung-tropic aav serotype  <dig> vector  <cit>  expressing the human placental alkaline phosphatase  reporter gene was administered to the respiratory tract of c57bl/ <dig> mice by intranasal, modified intranasal, endotracheal intubation, or intratracheal injection  and subsequently evaluated for transduction efficiency.
a total of 1x <dig> vector genomes  were delivered in a fixed volume of 80 μl  using isoflurane as the anesthetic.fig.
<dig> overview of the four different delivery methods used to administer viral vector to the mouse respiratory tract.
a intranasal technique.
mice were anesthetized with isoflurane and the viral vector administered drop wise over the nares allowing for passive inhalation of the substance.
b modified-intranasal technique.
mice were anesthetized with isoflurane and the viral vector administered drop wise over the nares.
the mouth is covered during administration to promote inhalation through the nose and subsequent distribution of the vector throughout the lower respiratory tract as well as to prevent swallowing of the administered substance.
c intubation technique.
mice were anesthetized with isoflurane in oxygen and a 22-gauge catheter placed into the trachea.
the viral vector was administered to the airways through the catheter.
d intratracheal injection technique.
mice were anesthetized with isoflurane and the viral vector injected directly into the trachea following surgical visualization
here we show that the modified-intranasal technique resulted in a similar and consistent distribution of vector to the upper and lower respiratory tract, including all lung lobes, in all mice examined.
while the intratracheal and intubation methods resulted in somewhat inconsistent distribution of vector, particularly to one lung lobe, the efficiency of lung gene delivery was similar to that of the modified intranasal technique.
however, only the intubation method resulted in consistent vector transduction of the distal most aspect of the lung.
intranasal delivery efficiently targeted vector to the upper respiratory tract and as expected, led to robust transduction of the nasal epithelium with significantly less vector transduction in the lung relative to the other three methods of vector administration.
in conclusion, the modified-intranasal technique proved to be the most efficient and consistent method of vector delivery requiring the least amount of skill.
use of the modified-intranasal technique for delivery of viral vectors to the mouse respiratory tract may improve the accuracy and precision of lung delivery, which may reduce variability between studies from different laboratories and can have animal welfare implications by reducing the number of animals required for detecting statistically significant differences.
RESULTS
gross analysis of transgene expression
grossly, hplap transgene expression was visible in all nasal cavities, trachea, and the proximal to middle aspect of all lung lobes  for all four methods of vector administration.
the intranasal technique resulted in staining of the proximal aspect of the lower respiratory tract with little to no staining of mid to distal aspects of lung lobes, respectively .
the modified-intranasal technique resulted in hplap staining in the proximal to mid-distal aspect of the lower respiratory tract, with no staining of the most distal aspects of lung lobes .
the intubation  and intratracheal injection  techniques resulted in hplap staining of the lower respiratory tract, up to and including distal aspects of lung lobes.
for the modified-intranasal technique, hplap expression was consistent between locations and between animals, whereas the intubation method resulted in markedly less hplap expression observed grossly in the middle lung lobe of half of the animals .
in one of four animals subjected to intratracheal administration of vector, hplap staining was restricted to the proximal aspect of the lower respiratory tract .
while more robust hplap expression was observed grossly in the nasal cavity following intranasal  and modified intranasal delivery of vector , both the intubation and intratracheal delivery methods resulted in varying degrees of hplap expression in the nasal cavity .fig.
<dig> gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following intranasal administration of an aav vector expressing hplap.
aav vector  was administered by the intranasal route to 7-week-old c57bl/ <dig> mice and tissues evaluated for hplap expression after three weeks.
the intranasal technique resulted in intense hplap staining of the upper respiratory tract, including the nasal cavity and trachea, and only the most proximal aspect of the lower respiratory tract, with similar transduction efficiencies observed for all mice in the group.
letters a-d represent individual mice and numbers one through five indicate the accessory lobe , the left lobe , the right cranial lobe , the right caudal lobe  and the right middle lobe
fig.
<dig> gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following administration of an aav vector expressing hplap using the modified intranasal technique.
aav vector  was administered by the modified intranasal method to 7-week-old c57bl/ <dig> mice and tissues evaluated for hplap expression after three weeks.
the modified intranasal technique resulted in intense staining of the upper respiratory tract, including nasal epithelium and trachea, as well as the proximal and mid-distal aspect of the lower respiratory tract in all mice examined.
letters a-d represent individual mice and numbers one through five indicate the accessory lobe , the left lobe , the right cranial lobe , the right caudal lobe  and the right middle lobe
fig.
<dig> gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following administration of an aav vector expressing hplap using the intubation technique.
aav vector  was administered by endotracheal intubation to 7-week-old c57bl/ <dig> mice and tissues evaluated for hplap expression after three weeks.
injection via intubation resulted in somewhat variable hplap staining of the upper and lower respiratory tract, including the distal aspect of the lung.
staining in the nasal epithelium and trachea was not as intense as with the intranasal methods.
note that some lung lobes were highly transduced with vector distribution throughout the entire lung lobe .
letters a-d represent individual mice and numbers one through five indicate the accessory lobe , the left lobe , the right cranial lobe , the right caudal lobe  and the right middle lobe
fig.
<dig> gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following intratracheal administration of an aav vector expressing hplap.
aav vector  was administered by intratracheal injection to 7-week-old c57bl/ <dig> mice and tissues evaluated for hplap expression after three weeks.
intratracheal injection resulted in intense hplap staining in the trachea with some staining in the nasal epithelium.
this delivery method resulted in variable hplap expression in the lower respiratory tract, such that the proximal and mid-distal aspect of the lower respiratory tract were well transduced in three  of four mice and in two of the four mice, hplap staining was observed in the distal aspect of some of the lung lobes, mainly the largest lung lobe .
letters a-d represent individual mice and numbers one through five indicate the accessory lobe , the left lobe , the right cranial lobe , the right caudal lobe  and the right middle lobe
histological analysis of aav vector distribution in the mouse lung
efficiency of vector distribution in the lung at 21 days post-delivery of 1x <dig> vg of an aav <dig> vector expressing hplap by the intranasal, modified intranasal, intratracheal and intubation methods was evaluated histologically .
representative sections of paraffin-embedded lung tissue subjected to staining for hplap expression are shown in figs.
6a-h. histologically, ap staining was observed in bronchiole, bronchiolar, and alveolar epithelial cells in all mice for all techniques .
consistent with gross observations, a considerable amount of ap staining was observed in distal alveolar epithelial cells with the intubation  and intratracheal injection  techniques.
in addition, ap expression was predominantly detected within the proximal airway following the modified intranasal technique.
for all methods, ap expression was consistent between locations and between animals.fig.
<dig> histological analysis of alkaline phosphatase staining in the lungs of mice 21 days after delivering 1x <dig> vg of an aav <dig> vector expressing hplap using four different methods of administration.
viral vectors were administered by the intranasal , modified-intranasal , intubation , or intratracheal injection  method.
tissue sections of one representative lung lobe stained for hplap expression and counterstained with nuclear fast red are shown at 4x  and 10x  magnification
quantification of vector genome copy number in lungs
we further evaluated the efficiency of aav <dig> vector transduction via the four different delivery methods by quantifying the number of vector genomes in the lungs of each of the experimentally infected mice at 21 days post-transduction.
vector copy number, which corresponds to viral genomes per ng of genomic dna, was quantified using a well-established taqman qpcr assay  <cit> .
as shown in fig.
<dig>  the mean copy number of aav vectors in the lung after intranasal delivery was approximately four-fold lower in comparison to the other three methods of vector delivery.
the other three methods of vector delivery did not yield statistical differences in aav copy number in the lung.fig.
<dig> vector genome copy numbers in lung tissue.
aav genome copy numbers/ug of genomic dna were determined 21 days after administration of 1x <dig> vg of an aav <dig> vector expressing hplap.
genomic dna was isolated from lung tissue slices tissues and 100 ng of each was used in triplicate to determine vector genome copies using taqman qpcr.
levels of significance were determined using one-way analysis of variance followed by tukey’s post-hoc test.
the data are shown as mean values ± sem.
*
p <  <dig>
discussion
it has been shown that relative distribution of a substance between the upper, lower respiratory, and gastrointestinal tract in mice following intranasal administration is largely dependent on delivery volume and level of anesthesia  <cit> .
studies have shown that small volumes, specifically those less than 10 μl, resulted in detection of the administered substance predominantly in the upper respiratory tract, whereas efficient detection in the lower respiratory tract required administration of volumes greater than 50 μl  <cit> .
in addition, intranasal installation was more efficient in anesthetized animals versus awake animals  <cit> , and in animals anesthetized with inhalant anesthetic versus parenteral  <cit> .
based on these findings, the current study evaluated transduction of the respiratory tract of mice following delivery of 1x <dig> vg of an aav serotype  <dig> vector expressing the human placental alkaline phosphatase  reporter gene in a fixed volume of 80 μl to animals that were anesthetized with isoflurane at deep plane in order to contrast and compare four different methods of administration.
the modified-intranasal technique resulted in a similar and consistent distribution of vector to the upper and lower respiratory tract, including all lung lobes, with vector seldom reaching the most distal aspect of the lung.
intratracheal and intubation methods resulted in less consistent vector transduction with a lack of localization to the right middle lung lobe; however, both methods resulted in vector transduction of the distal most part of the lung.
the intranasal technique resulted in vector localization primarily within the upper respiratory tract and significantly less vector transduction in the lung compared to the other three methods.
these results indicate that the method of vector administration should be chosen based on preferred target site and expertise of the researcher, and that appropriate technique selection may influence experimental outcomes.
the skills and/or amount of training required to perform each technique varied considerably.
the intratracheal injection method required basic surgical skills for closure of the skin incision.
the intubation method was inconsistent, likely due to the challenging nature of the technique.
both of these techniques required considerable practice in order to ensure the well-being of the animal following recovery, and efficient/consistent administration of the viral vector to the respiratory tract.
in contrast, both intranasal delivery methods required minimal training and efficient and consistent transduction was observed in the respiratory tract following a relatively short time commitment to practicing the modified-intranasal technique.
see table  <dig> for a summary of the advantages and disadvantages of the four different delivery methods evaluated.table  <dig> summary of the advantages and disadvantages of the four delivery methods evaluated
- tracheal epithelium
- little skill/training required
- can be performed by one person
- inexpensive
- best option if wanting to target the upper respiratory tract
- inefficient delivery to the
- tracheal epithelium
- little skill/training required
- inexpensive
- least variable delivery method
- tracheal epithelium
consistently reaches the distal aspect of the lung lobes
- requires more equipment than the other methods
- variable
- time consuming
- possibility of losing
vector if esophagus is penetrated
- tracheal epithelium
- proximal to middle
aspect of lung lobes
respiratory tract
- limited delivery to the nasal epithelium
- requires surgical skills
- variable
- invasive
- requires analgesic
previous studies have shown that depth of anesthesia can influence the amount of administered substance lost to the gastrointestinal tract  <cit> .
for both the intranasal and the modified-intranasal technique, the mouse was briefly removed from the source of isoflurane anesthesia prior to vector administration.
therefore, quick recovery and a light plane of anesthesia may have resulted in an increased loss of vector to the gastrointestinal tract.
according to our criteria, which were established based on previous experiments, swallowing was documented to have occurred if liquid was found pooling in the mouth of the mouse immediately following vector administration or if bubbles of liquid were released from the mouth during or immediately following vector administration.
unfortunately, because we did not harvest the esophagus and stomach from the animals used in this study, we cannot substantiate these observations.
nevertheless, pooling of liquid in the mouth and production of bubbles was commonly observed with the intranasal delivery method and may have contributed to significantly less vector particles being delivered to the lungs.
however, modifying this technique such that the mouth was covered and the animal maintained upright at a 45 °c angle during vector administration resulted in no apparent loss of vector into the gastrointestinal tract.
repeat-administration using the intratracheal injection technique may be challenging.
the intratracheal injection technique requires a skin incision, which will begin to heal immediately following closure.
if re-administration is required within a relatively short timeframe, the same incision site could be re-opened since healing would be minimal.
greater than seven days following closure will result in significant fibrosis of the incision site, which may make re-incision challenging.
the second incision may need to be made slightly lateral to the first; however, this could impede proper visualization of the trachea.
for this reason, if repeat administration is required, an alternate method of administration is recommended.
additional studies are required to evaluate the extent and character of inflammation resulting from each technique.
control mice received saline in order to evaluate the effects of each technique at the tissue level.
no changes consistent with trauma, inflammation or repair were observed .
however, histopathology at several time points immediately following each technique would need to be evaluated in order to properly characterize whether tissue damage or local inflammation resulted.
the intratracheal injection technique likely resulted in the most inflammation locally, at the site of incision; however, evaluation of an acute immune response may be confounded by a local immune response at the site of the incision.
when performing the intubation technique, considerable swelling of the tongue and throat may occur if repeated attempts at insertion of the catheter are required.
this is more likely to occur if the individual performing the procedure does not practice on a regular basis.
if extensive swelling does occur, the procedure must be halted until the swelling has subsided, usually within  <dig> to 48 hrs.
the local and systemic effects of tissue inflammation should be taken into consideration when selection of method of administration is made.
lastly, analgesia is required for the intratracheal injection technique.
the use of certain analgesics may be contraindicated in a particular study.
examples include opioids in behavioural research  <cit>  and nsaids in cancer research  <cit> .
however, this should not supersede the use of analgesics  <cit> .
if this is the case, it is recommended that an alternate mode of administration be chosen or a different class of analgesics be utilized.
CONCLUSIONS
in summary, we evaluated three methods of aav vector administration to the upper and lower respiratory tract in mice: intranasal, intubation, and intratracheal injection.
in addition, we presented a modified-intranasal delivery technique .
all methods of viral vector administration resulted in transduction of the trachea and nasal epithelium, albeit to a lesser extent with the intubation and intratracheal methods of delivery.
intubation was the only method that resulted in transduction of alveoli in the distal most aspect of the lung.
the modified-intranasal technique resulted in the most efficient distribution of vector to the upper and lower respiratory tract with the least amount of inter-animal variability and requiring the least amount of training.
technique selection may greatly influence experimental outcomes and should be selected based on a variety of factors including time, skill, and desired target site.

 gene therapy is currently being evaluated for a wide range of acute and chronic lung diseases including those caused by single gene defects such as cystic fibrosis, alpha- antitrypsin deficiency and surfactant protein b deficiency. while the lung provides a natural route of entry into the host and can be accessed in a comparatively easy and noninvasive manner, methods for consistent and efficient delivery of viral vectors to the respiratory tract are required. there have been a number of studies evaluating variables such as volume of inoculum, effect of diluent, nature of anesthesia, and position of mouse and how these affect intranasal delivery of therapeutic substances in mouse models, but a comparison of different delivery methods using viral vectors has not been done. targeted delivery of viral vectors to the mouse respiratory tract can be performed by a variety of different methods, some of which require extensive skill and training. in this report, four different methods of delivering viral vectors to the mouse respiratory tract were evaluated for ease and consistency, vector distribution, and invasiveness of the procedure with the aim of identifying the most effective and consistent method of delivery requiring minimal skill development. for this study, a lung-tropic aav serotype vector expressing the human placental alkaline phosphatase reporter gene was administered to the respiratory tract of c57bl/ mice by intranasal, modified intranasal, endotracheal intubation, or intratracheal injection and subsequently evaluated for transduction efficiency. a total of 1x vector genomes were delivered in a fixed volume of 80 μl using isoflurane as the anesthetic.fig. overview of the four different delivery methods used to administer viral vector to the mouse respiratory tract. a intranasal technique. mice were anesthetized with isoflurane and the viral vector administered drop wise over the nares allowing for passive inhalation of the substance. b modified-intranasal technique. mice were anesthetized with isoflurane and the viral vector administered drop wise over the nares. the mouth is covered during administration to promote inhalation through the nose and subsequent distribution of the vector throughout the lower respiratory tract as well as to prevent swallowing of the administered substance. c intubation technique. mice were anesthetized with isoflurane in oxygen and a 22-gauge catheter placed into the trachea. the viral vector was administered to the airways through the catheter. d intratracheal injection technique. mice were anesthetized with isoflurane and the viral vector injected directly into the trachea following surgical visualization here we show that the modified-intranasal technique resulted in a similar and consistent distribution of vector to the upper and lower respiratory tract, including all lung lobes, in all mice examined. while the intratracheal and intubation methods resulted in somewhat inconsistent distribution of vector, particularly to one lung lobe, the efficiency of lung gene delivery was similar to that of the modified intranasal technique. however, only the intubation method resulted in consistent vector transduction of the distal most aspect of the lung. intranasal delivery efficiently targeted vector to the upper respiratory tract and as expected, led to robust transduction of the nasal epithelium with significantly less vector transduction in the lung relative to the other three methods of vector administration. in conclusion, the modified-intranasal technique proved to be the most efficient and consistent method of vector delivery requiring the least amount of skill. use of the modified-intranasal technique for delivery of viral vectors to the mouse respiratory tract may improve the accuracy and precision of lung delivery, which may reduce variability between studies from different laboratories and can have animal welfare implications by reducing the number of animals required for detecting statistically significant differences.  gross analysis of transgene expression grossly, hplap transgene expression was visible in all nasal cavities, trachea, and the proximal to middle aspect of all lung lobes for all four methods of vector administration. the intranasal technique resulted in staining of the proximal aspect of the lower respiratory tract with little to no staining of mid to distal aspects of lung lobes, respectively. the modified-intranasal technique resulted in hplap staining in the proximal to mid-distal aspect of the lower respiratory tract, with no staining of the most distal aspects of lung lobes. the intubation and intratracheal injection techniques resulted in hplap staining of the lower respiratory tract, up to and including distal aspects of lung lobes. for the modified-intranasal technique, hplap expression was consistent between locations and between animals, whereas the intubation method resulted in markedly less hplap expression observed grossly in the middle lung lobe of half of the animals. in one of four animals subjected to intratracheal administration of vector, hplap staining was restricted to the proximal aspect of the lower respiratory tract. while more robust hplap expression was observed grossly in the nasal cavity following intranasal and modified intranasal delivery of vector, both the intubation and intratracheal delivery methods resulted in varying degrees of hplap expression in the nasal cavity.fig. gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following intranasal administration of an aav vector expressing hplap. aav vector was administered by the intranasal route to 7-week-old c57bl/ mice and tissues evaluated for hplap expression after three weeks. the intranasal technique resulted in intense hplap staining of the upper respiratory tract, including the nasal cavity and trachea, and only the most proximal aspect of the lower respiratory tract, with similar transduction efficiencies observed for all mice in the group. letters a-d represent individual mice and numbers one through five indicate the accessory lobe, the left lobe, the right cranial lobe, the right caudal lobe and the right middle lobe fig. gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following administration of an aav vector expressing hplap using the modified intranasal technique. aav vector was administered by the modified intranasal method to 7-week-old c57bl/ mice and tissues evaluated for hplap expression after three weeks. the modified intranasal technique resulted in intense staining of the upper respiratory tract, including nasal epithelium and trachea, as well as the proximal and mid-distal aspect of the lower respiratory tract in all mice examined. letters a-d represent individual mice and numbers one through five indicate the accessory lobe, the left lobe, the right cranial lobe, the right caudal lobe and the right middle lobe fig. gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following administration of an aav vector expressing hplap using the intubation technique. aav vector was administered by endotracheal intubation to 7-week-old c57bl/ mice and tissues evaluated for hplap expression after three weeks. injection via intubation resulted in somewhat variable hplap staining of the upper and lower respiratory tract, including the distal aspect of the lung. staining in the nasal epithelium and trachea was not as intense as with the intranasal methods. note that some lung lobes were highly transduced with vector distribution throughout the entire lung lobe. letters a-d represent individual mice and numbers one through five indicate the accessory lobe, the left lobe, the right cranial lobe, the right caudal lobe and the right middle lobe fig. gross images of alkaline phosphatase staining in the upper and lower respiratory tract of mice following intratracheal administration of an aav vector expressing hplap. aav vector was administered by intratracheal injection to 7-week-old c57bl/ mice and tissues evaluated for hplap expression after three weeks. intratracheal injection resulted in intense hplap staining in the trachea with some staining in the nasal epithelium. this delivery method resulted in variable hplap expression in the lower respiratory tract, such that the proximal and mid-distal aspect of the lower respiratory tract were well transduced in three of four mice and in two of the four mice, hplap staining was observed in the distal aspect of some of the lung lobes, mainly the largest lung lobe. letters a-d represent individual mice and numbers one through five indicate the accessory lobe, the left lobe, the right cranial lobe, the right caudal lobe and the right middle lobe histological analysis of aav vector distribution in the mouse lung efficiency of vector distribution in the lung at 21 days post-delivery of 1x vg of an aav vector expressing hplap by the intranasal, modified intranasal, intratracheal and intubation methods was evaluated histologically. representative sections of paraffin-embedded lung tissue subjected to staining for hplap expression are shown in figs. 6a-h. histologically, ap staining was observed in bronchiole, bronchiolar, and alveolar epithelial cells in all mice for all techniques. consistent with gross observations, a considerable amount of ap staining was observed in distal alveolar epithelial cells with the intubation and intratracheal injection techniques. in addition, ap expression was predominantly detected within the proximal airway following the modified intranasal technique. for all methods, ap expression was consistent between locations and between animals.fig. histological analysis of alkaline phosphatase staining in the lungs of mice 21 days after delivering 1x vg of an aav vector expressing hplap using four different methods of administration. viral vectors were administered by the intranasal, modified-intranasal, intubation, or intratracheal injection method. tissue sections of one representative lung lobe stained for hplap expression and counterstained with nuclear fast red are shown at 4x and 10x magnification quantification of vector genome copy number in lungs we further evaluated the efficiency of aav vector transduction via the four different delivery methods by quantifying the number of vector genomes in the lungs of each of the experimentally infected mice at 21 days post-transduction. vector copy number, which corresponds to viral genomes per ng of genomic dna, was quantified using a well-established taqman qpcr assay. as shown in fig.  the mean copy number of aav vectors in the lung after intranasal delivery was approximately four-fold lower in comparison to the other three methods of vector delivery. the other three methods of vector delivery did not yield statistical differences in aav copy number in the lung.fig. vector genome copy numbers in lung tissue. aav genome copy numbers/ug of genomic dna were determined 21 days after administration of 1x vg of an aav vector expressing hplap. genomic dna was isolated from lung tissue slices tissues and 100 ng of each was used in triplicate to determine vector genome copies using taqman qpcr. levels of significance were determined using one-way analysis of variance followed by tukey’s post-hoc test. the data are shown as mean values ± sem. * p <  discussion it has been shown that relative distribution of a substance between the upper, lower respiratory, and gastrointestinal tract in mice following intranasal administration is largely dependent on delivery volume and level of anesthesia. studies have shown that small volumes, specifically those less than 10 μl, resulted in detection of the administered substance predominantly in the upper respiratory tract, whereas efficient detection in the lower respiratory tract required administration of volumes greater than 50 μl. in addition, intranasal installation was more efficient in anesthetized animals versus awake animals, and in animals anesthetized with inhalant anesthetic versus parenteral. based on these findings, the current study evaluated transduction of the respiratory tract of mice following delivery of 1x vg of an aav serotype vector expressing the human placental alkaline phosphatase reporter gene in a fixed volume of 80 μl to animals that were anesthetized with isoflurane at deep plane in order to contrast and compare four different methods of administration. the modified-intranasal technique resulted in a similar and consistent distribution of vector to the upper and lower respiratory tract, including all lung lobes, with vector seldom reaching the most distal aspect of the lung. intratracheal and intubation methods resulted in less consistent vector transduction with a lack of localization to the right middle lung lobe; however, both methods resulted in vector transduction of the distal most part of the lung. the intranasal technique resulted in vector localization primarily within the upper respiratory tract and significantly less vector transduction in the lung compared to the other three methods. these results indicate that the method of vector administration should be chosen based on preferred target site and expertise of the researcher, and that appropriate technique selection may influence experimental outcomes. the skills and/or amount of training required to perform each technique varied considerably. the intratracheal injection method required basic surgical skills for closure of the skin incision. the intubation method was inconsistent, likely due to the challenging nature of the technique. both of these techniques required considerable practice in order to ensure the well-being of the animal following recovery, and efficient/consistent administration of the viral vector to the respiratory tract. in contrast, both intranasal delivery methods required minimal training and efficient and consistent transduction was observed in the respiratory tract following a relatively short time commitment to practicing the modified-intranasal technique. see table  for a summary of the advantages and disadvantages of the four different delivery methods evaluated.table summary of the advantages and disadvantages of the four delivery methods evaluated - tracheal epithelium - little skill/training required - can be performed by one person - inexpensive - best option if wanting to target the upper respiratory tract - inefficient delivery to the - tracheal epithelium - little skill/training required - inexpensive - least variable delivery method - tracheal epithelium consistently reaches the distal aspect of the lung lobes - requires more equipment than the other methods - variable - time consuming - possibility of losing vector if esophagus is penetrated - tracheal epithelium - proximal to middle aspect of lung lobes respiratory tract - limited delivery to the nasal epithelium - requires surgical skills - variable - invasive - requires analgesic previous studies have shown that depth of anesthesia can influence the amount of administered substance lost to the gastrointestinal tract. for both the intranasal and the modified-intranasal technique, the mouse was briefly removed from the source of isoflurane anesthesia prior to vector administration. therefore, quick recovery and a light plane of anesthesia may have resulted in an increased loss of vector to the gastrointestinal tract. according to our criteria, which were established based on previous experiments, swallowing was documented to have occurred if liquid was found pooling in the mouth of the mouse immediately following vector administration or if bubbles of liquid were released from the mouth during or immediately following vector administration. unfortunately, because we did not harvest the esophagus and stomach from the animals used in this study, we cannot substantiate these observations. nevertheless, pooling of liquid in the mouth and production of bubbles was commonly observed with the intranasal delivery method and may have contributed to significantly less vector particles being delivered to the lungs. however, modifying this technique such that the mouth was covered and the animal maintained upright at a 45 °c angle during vector administration resulted in no apparent loss of vector into the gastrointestinal tract. repeat-administration using the intratracheal injection technique may be challenging. the intratracheal injection technique requires a skin incision, which will begin to heal immediately following closure. if re-administration is required within a relatively short timeframe, the same incision site could be re-opened since healing would be minimal. greater than seven days following closure will result in significant fibrosis of the incision site, which may make re-incision challenging. the second incision may need to be made slightly lateral to the first; however, this could impede proper visualization of the trachea. for this reason, if repeat administration is required, an alternate method of administration is recommended. additional studies are required to evaluate the extent and character of inflammation resulting from each technique. control mice received saline in order to evaluate the effects of each technique at the tissue level. no changes consistent with trauma, inflammation or repair were observed. however, histopathology at several time points immediately following each technique would need to be evaluated in order to properly characterize whether tissue damage or local inflammation resulted. the intratracheal injection technique likely resulted in the most inflammation locally, at the site of incision; however, evaluation of an acute immune response may be confounded by a local immune response at the site of the incision. when performing the intubation technique, considerable swelling of the tongue and throat may occur if repeated attempts at insertion of the catheter are required. this is more likely to occur if the individual performing the procedure does not practice on a regular basis. if extensive swelling does occur, the procedure must be halted until the swelling has subsided, usually within to 48 hrs. the local and systemic effects of tissue inflammation should be taken into consideration when selection of method of administration is made. lastly, analgesia is required for the intratracheal injection technique. the use of certain analgesics may be contraindicated in a particular study. examples include opioids in behavioural research and nsaids in cancer research. however, this should not supersede the use of analgesics. if this is the case, it is recommended that an alternate mode of administration be chosen or a different class of analgesics be utilized.  in summary, we evaluated three methods of aav vector administration to the upper and lower respiratory tract in mice: intranasal, intubation, and intratracheal injection. in addition, we presented a modified-intranasal delivery technique. all methods of viral vector administration resulted in transduction of the trachea and nasal epithelium, albeit to a lesser extent with the intubation and intratracheal methods of delivery. intubation was the only method that resulted in transduction of alveoli in the distal most aspect of the lung. the modified-intranasal technique resulted in the most efficient distribution of vector to the upper and lower respiratory tract with the least amount of inter-animal variability and requiring the least amount of training. technique selection may greatly influence experimental outcomes and should be selected based on a variety of factors including time, skill, and desired target site. 
aav vector genome copy numbers in the lung were approximately four-fold lower in mice that received vector via intranasal administration in comparison to the other three methods of vector delivery. our results show that mode of aav vector administration to the murine respiratory tract should be selected based on desired target site and skill of the researcher, and that appropriate technique selection may greatly influence experimental outcomes. improving the accuracy and precision of lung delivery will not only reduce the number of animals required to detect statistically significant differences, but may reduce the variability of studies from different laboratories. targeted delivery of gene therapy vectors to the mouse respiratory tract is often performed via intranasal or intratracheal administration; however, there can be a great deal of variability between these methods, which could potentially influence experimental results. we also present a modified-intranasal delivery technique that is superior to passive administration of vector into the nares of anesthetized supine animals. here we evaluated three different methods of adeno-associated virus vector administration to the respiratory tract in mice and discuss the advantages, challenges, and shortcomings of each. the modified intranasal, intubation and intratracheal injection methods of vector administration did not yield statistical differences in aav vector genome copy numbers in the lung. transgene expression was consistently visible in the nasal cavity, trachea, and proximal to middle aspect of all lung lobes for all four methods, whereas transgene expression was consistently observed in the most distal aspect of lung lobes only with the intubation and intratracheal injection techniques. with regard to reproducibility of vector distribution within and between animals, the modified-intranasal technique was superior. adeno-associated virusvector instillationtransgene expressionintratrachealintranasalintubationrespiratory tractrespiratory epitheliumhttp://dx.doi.org/ /501100000082cystic fibrosis canadahttp://dx.doi.org/ /501100000221canadian lung associationissue-copyright-statement© the author 2017
 targeted delivery of gene therapy vectors to the mouse respiratory tract is often performed via intranasal or intratracheal administration; however, there can be a great deal of variability between these methods, which could potentially influence experimental results. improving the accuracy and precision of lung delivery will not only reduce the number of animals required to detect statistically significant differences, but may reduce the variability of studies from different laboratories.  here we evaluated three different methods of adeno-associated virus vector administration to the respiratory tract in mice and discuss the advantages, challenges, and shortcomings of each. we also present a modified-intranasal delivery technique that is superior to passive administration of vector into the nares of anesthetized supine animals. transgene expression was consistently visible in the nasal cavity, trachea, and proximal to middle aspect of all lung lobes for all four methods, whereas transgene expression was consistently observed in the most distal aspect of lung lobes only with the intubation and intratracheal injection techniques. aav vector genome copy numbers in the lung were approximately four-fold lower in mice that received vector via intranasal administration in comparison to the other three methods of vector delivery. the modified intranasal, intubation and intratracheal injection methods of vector administration did not yield statistical differences in aav vector genome copy numbers in the lung. with regard to reproducibility of vector distribution within and between animals, the modified-intranasal technique was superior.  our results show that mode of aav vector administration to the murine respiratory tract should be selected based on desired target site and skill of the researcher, and that appropriate technique selection may greatly influence experimental outcomes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords adeno-associated virusvector instillationtransgene expressionintratrachealintranasalintubationrespiratory tractrespiratory epitheliumhttp://dx.doi.org/ /501100000082cystic fibrosis canadahttp://dx.doi.org/ /501100000221canadian lung associationissue-copyright-statement© the author 2017 